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Reproductive Neuroendocrinology Judy Cameron, PhD.

Monday, February 9, 2009 8:30 am

Recommended Reading: Scientific Essentials of Reproductive Medicine, SG Hillier, HC Kitchener, JP Neilson eds. W.B. Saunders 1996. Assignment: Chapter 2.2. Essential Reproduction, MH Johnson and BJ Everitt eds. 5th edition. Blackwell Science, 2000. Assignment: Chapters 5 and 6.

Learning Objectives: Define: GnRH Pulsatile Secretion Negative Feedback Be able to: 1. 2. 3. 4. 5. 6. 7. Describe the anatomical location of GnRH neurons, where they project to, and what afferents they receive. Explain how various frequencies of pulsatile GnRH secretion lead to different patterns of LH and FSH release. Discuss the mechanisms controlling the frequency and magnitude of pulsatile LH secretory episodes. Explain the factors necessary to get positive feedback of estradiol on GnRH/LH/FSH secretion. Describe the two sites of action where estradiol can have negative feedback effects that lead to a decrease in LH and FSH secretion. Describe the ontogeny of the GnRH neuronal system. Describe the locus of action of stress on the reproductive axis. LH/FSH Positive Feedback Down Regulation

A. Hormones of the Reproductive Axis 1. Gonadotropin-Releasing Hormone (GnRH) The central nervous system governs the reproductive system in both males and females. This is accomplished by a population of neurons in the medial basal hypothalamus, which produce the neurotransmitter, GnRH, and release it into the portal bloodstream of the median eminence (Figure 1). GnRH travels via the portal capillaries to the anterior pituitary where it stimulates the synthesis and release of the pituitary hormones, LH and FSH. As GnRH neurons fire, pulses of GnRH are released into the portal bloodstream and the pattern as well as amplitude of GnRH pulses conveys important information to the pituitary. GnRH neurons are small, diffusely located cells that are not concentrated in a nucleus. Many neurotransmitter systems from the brainstem, limbic system, and other areas of the hypothalamus convey information to GnRH neurons. These afferent systems include neurons that contain norepinephrine, dopamine, serotonin, GABA, glutamate, endogenous opiate peptides, neuropeptide Y, and galanin, as well as a number of other peptide neurotransmitters (Figure 1).

Figure 1. GnRH neurons, diffusely located in the hypothalamus, project to the median eminence where they release GnRH into the hypothalamo-hypophyseal portal system. GnRH travels to the pituitary to stimulate synthesis and secretion of LH and FSH. A variety of neurotransmitters and neuropeptides modulate GnRH release. (Johnson and Everitt, Essential Reproduction, p 90, 2000.)

GnRH neurons originate from outside the central nervous system, coming originally from the epithelial tissue of the nasal placode. During embryonic development GnRH neurons migrate across the surface of the brain and into the hypothalamus, with the final hypothalamic location differing somewhat among various species. Failure of GnRH neurons to migrate properly leads to a clinical condition, Kallmans Syndrome, in which GnRH neurons do not reach their final destination and thus do not stimulate pituitary gonadotropin secretion. Patients with Kallmans Syndrome do not exhibit any spontaneous activity in the reproductive axis. Administration of exogenous GnRH effectively treats this form of hypothalamic hypogonadism. Patients with Kallmans Syndrome often have other congenital midline defects, including anosmia which results from hypoplasia of the olfactory bulb and tracts. Although cell bodies of GnRH neurons are comparatively diffusely located and occur in several loci of the hypothalamus, the terminal field of these neurons is largely in the portal capillary plexus of the median eminence (Figure 2).

Figure 2. Immunofluorescence of GnRH neurons in median eminence. GnRH is a 10 amino acid peptide that is synthesized as part of a larger precursor molecule of 92 amino acids, shown in figure 3 below.

At the pituitary, GnRH binds to a 7-transmembrane-domain receptor and stimulates LH and FSH secretion by increasing calcium transport into gonadotrophs and calcium release from internal stores via a diacylgycerol/protein kinase C pathway. A critical factor governing LH and FSH secretion and release is the rate of pulsatile GnRH stimulation of the gonadotrophs. At physiological rates of GnRH stimulation, each pulse of GnRH generally leads to a pulse of LH secretion from the pituitary. GnRH only reaches measurable levels in the hypothalamo-pituitary portal vasculature; thus, this nearly 1:1 secretory ratio of GnRH to LH secretion (Figure 4) is used clinically to make assumptions about GnRH secretion, using LH measured in peripheral serum.

Figure 4. Correlation between hypophyseal portal GnRH concentrations and LH measured in peripheral blood. Vertical arrows indicate statistically measurable pulses. The significance of the extremely small pulses is a not yet understood. One theory is that they may serve a priming function, increasing the LH response to the next pulse. While spontaneous GnRH is always pulsatile, continuous exposure to GnRH leads to down regulation of GnRH receptors and an accompanying decrease in LH and FSH synthesis and secretion (Figure 5). This concept has a number of clinical applications. For example the most common current therapy for precocious puberty of hypothalamic origin (i.e., precocious GnRH secretion) is to treat the child with a long-acting GnRH agonist, which will down regulate pituitary GnRH receptors and effectively turn off the reproductive axis. Long-acting GnRH agonists are also used in the treatment of forms of breast cancer that are estrogen dependent, because down regulation of LH and FSH secretion leads to a decline in hormonal support to the ovary and a subsequent decrease in ovarian production of estrogen.

Figure 5. Maintenance and inhibition of LH and FSH secretion in a rhesus macaque with an arcuate nucleus lesion and supported by either pulsatile or continuous GnRH administration. (Wildt, Haulser, Marshall, Hutchison, Plant, and Knobil, Endocrinology 109: 376-385, 1981). 2. The Gonadotropins, Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) LH and FSH are structurally similar glycoprotein hormones, produced within the same population of pituitary cells, the gonadotrophs. Each of these hormones is made up of an alpha and beta subunit. LH, FSH and TSH share a common alpha subunit, and each have a unique beta subunit which conveys tissue specificity to the intact hormone. Although gonadotrophs produce both LH and FSH, the production of these hormones can be independently regulated by hormones released from the gonads, as well as by the frequency of pulsatile GnRH stimulation. For example, the gonadal peptide hormone, inhibin, feeds back at the level of the pituitary to inhibit FSH, but not LH, secretion. In addition, relatively rapid pulses of GnRH lead to higher circulating levels of LH compared to FSH, but when GnRH pulse frequency slows LH levels decline, but FSH levels can actually increase (Figure 6, next page). Before secretion of gonadotropins, terminal sugars are attached to each gonadotropin molecule. The sugars include sialic acid, galactose, N-acetylglucosamine, and mannose, but the most important is sialic acid. The extent of glycosylation of LH and FSH are important for the physiological function of these hormones. Forms of gonadotropin with more sialic acid have a longer half-life because they are protected from degradation by the liver. Forms of gonadotropin with less sialic acid can have more potent effects at their biological receptors. Both the rate of GnRH stimulation, as

well as ovarian hormone feedback at the level of the pituitary, regulate the degree of LH and FSH glycosylation.

Figure 6. Changes in the secretion ratios of LH and FSH in a rhesus macaque with an arcuate nucleus lesion and supported by varying frequencies of pulsatile GnRH administration. (Wildt, Haulser, Marshall, Hutchison, Plant, and Knobil, Endocrinology 109: 376-385, 1981). LH and FSH travel through the peripheral bloodstream to the gonads, where they bind to membrane receptors within specific tissue compartments of the ovary and testes to stimulate cellular growth and synthesis of the gonadal steroid hormones, estradiol, progesterone and testosterone. 3. Gonadal Steroid Hormones LH and FSH act at the testes and the ovaries to stimulate the production and secretion of gonadal steroid hormones. Leydig cells in the testes produce primarily testosterone in response to circulating levels of LH. Tissues of the ovary produce primarily 17estradiol (stimulated primarily by FSH and later by FSH and LH) and progesterone (stimulated primarily by LH). However, the pathway for estradiol production involves an intermediate step of androgen production, and thus the ovary is a source of low levels of androgens. All steroid hormones in the body are produced from a common precursor, cholesterol, through a series of enzyme-regulated pathways which you vividly recall from your Endocrine course. The specific steroid hormones produced by a particular tissue are regulated by the level of activity of key enzymes in the synthetic pathway. For example, to produce progesterone the cells of the corpus luteum must have cholesterol side chain cleavage enzyme and 3 -hydroxysteroid dehydrogenase. However, to produce estradiol these same cells must also contain additional enzymes, including 17-hydroxysteroid dehydrogenase and aromatase, which act to convert

progesterone to estradiol. The amount of each hormone produced depends on the relative amount of each enzyme in the synthetic pathway. The gonadotropins regulate enzyme levels in the synthetic pathway. Gonadal steroid hormones have many actions on tissues of the reproductive axis and throughout the body, that will be discussed in later lectures. Gonadal steroid hormones also play an important role in "feedback" to the hypothalamus and pituitary to regulate GnRH and gonadotropin secretion, as discussed below. B. Control of GnRH and Gonadotropin Secretion 1. The Pulsatile Nature of GnRH The ensemble of GnRH neurons in the hypothalamus that send axons to the portal blood system in the median eminence, fire in a coordinated, repetitive, episodic manner, so as to produce distinct pulse of GnRH in the portal bloodstream (Figure 4). The pulsatile nature of GnRH stimulation to the pituitary leads to the release of distinct pulses of LH into the peripheral bloodstream (Figure 4). Because the portal bloodstream is generally inaccessible, it has only been possible in experimental animals to directly measure pulsatile GnRH release. However, clinically collection of frequent blood samples from the peripheral bloodstream can be used to define the pulsatile nature of LH secretion (i.e., frequency and amplitude of LH pulses), and pulsatile LH is used as an indirect measure of the activity of the GnRH secretory system. A central, unsolved question in the field of reproductive neuroendocrinology is: what causes GnRH neurons to fire in a coordinated manner? To date, this question remains unanswered. We do, however, know quite a bit about factors which modulate the firing pattern of GnRH neurons. These factors can be divided into two categories. The first category is other neural systems that project onto GnRH neurons and modulate their firing rate. These can be stimulatory to GnRH release, increasing the probability that GnRH neurons will fire. Examples of such systems are glutamate-containing neurons and noradrenergic neurons. Afferent input to GnRH neurons can also be inhibitory, decreasing the probability that GnRH neurons will fire. Examples of such systems are GABA-containing neurons and opioidergic neurons. Some appreciation of the complexity of this system can be illustrated by showing catecholamine immunostaining of the periventricular region (Figure 7; same region as shown in figure 2).

Figure 7. Immunocytochemical localization of tyrosine hydroxylase , an enzyme in the catecholamine neurotransmitter pathway, in the hypothalamus. The second category of modulation of GnRH neurons comes from feedback of steroid hormones, both negative and positive feedback (as discussed in the section below).

2. Steroid Hormone Feedback Control of GnRH and Gonadotropin Secretion Steroid hormones can dramatically alter the pattern of pulsatile release of GnRH and of the gonadotropins, via actions both at the hypothalamus and the pituitary (Figure 8). At the hypothalamus, estradiol, progesterone and testosterone can all act to slow the frequency of GnRH release into the portal bloodstream, an action referred to as "negative feedback." It is likely that the effects of steroid hormones on the firing rate of GnRH neurons are mediated by steroid hormone actions on a neural system(s) that provides afferent input to GnRH neurons, because GnRH neurons have been generally shown to lack steroid hormone receptors. For example, progesterone-mediated negative feedback on GnRH secretion in primates appears to be regulated by opioid neurons in the hypothalamus. If an opiate antagonist is administered along with progesterone, then the negative feedback action of progesterone on GnRH secretion can be blocked. Negative feedback of steroid hormones can also occur directly at the level of the pituitary. For example, estradiol has been shown to be capable of binding to the pituitary and decreasing LH and FSH synthesis and release and decreasing the sensitivity of pituitary gonadotrophs to the actions of GnRH, such that less LH and FSH are released when a pulse of GnRH stimulates the pituitary. Evidence for such a direct pituitary action of estradiol came from studies with rhesus monkeys which had been rendered deficient in endogenous GnRH by a lesion in the arcuate nucleus, and showed a decline in endogenous gonadotropin secretion. When these monkeys received a

pulsatile regimen of GnRH, gonadotropin secretion was restored, and then subsequent estradiol infusions were shown to dramatically suppress responsiveness of the pituitary to GnRH, and again lead to a suppression of gonadotropin secretion. Both estradiol and testosterone can have direct negative feedback actions at the pituitary, however, the extent of hypothalamic versus pituitary negative feedback actions are species specific. In humans, there is considerable feedback of estradiol at the pituitary, but most of the testosterone negative feedback occurs at the level of the hypothalamus. The negative feedback actions of progesterone are mediated solely at the level of the hypothalamus, and are greatly intensified by prior exposure to estradiol, which leads to an increase in progesterone receptors within hypothalamic neurons.

Figure 8. Negative feedback effects of estradiol on pulsatile LH secretion in an ovariectomized rhesus macaque. (Yamaji, Dierschke, and Knobil, Endocrinology 70: 771-777, 1972). In addition to negative feedback, estradiol can have a "positive feedback" action at the level of the hypothalamus and pituitary to lead to a massive release of LH and FSH from the pituitary. This massive release occurs once each menstrual cycle, and is referred to as the LH/FSH surge. The positive feedback action of estradiol occurs as a response to the rising tide of estradiol that is produced during the process of dominant follicle development in the late follicular phase of the menstrual cycle. [The ovarian mechanism resulting in this increase in estradiol is the subject of Dr. Zelezniks lecture.] In women, elevated estradiol levels are generally maintained at about 200 pg/ml for about 36 hours prior to stimulation of the gonadotropin surge. Experiments have shown that both a critical concentration of plasma estradiol and a critical duration of elevated estradiol are necessary to achieve positive feedback and a resulting gonadotropin surge. As with negative feedback in response to estradiol, the positive feedback actions of estradiol

occur both at the hypothalamus, to increase GnRH secretion, and at the pituitary to greatly enhance pituitary responsiveness to GnRH. The hormonal changes that occur during the human menstrual cycle are a direct expression of the interplay between central (CNS and hypothalamic) and peripheral (circulating concentrations of estradiol and progesterone) factors regulating GnRH and gonadotropin secretion. These patterns are summarized in figure 9.

Figure 9. Hormonal changes during the human menstrual cycle.

C. Many Changes in Activity of the Reproductive Axis Result from Changes in the Neural Drive to the Reproductive System The susceptibility of the reproductive system to disruption most likely stems from the importance of input from higher cortical areas. During adulthood, many forms of stress can lead to an impairment in secretion of GnRH neurons, including psychosocial stress, undernutrition, and strenuous exercise training. In each case, if the stress is great enough it can lead to a dramatic suppression of GnRH secretion, with an accompanying suppression of pituitary gonadotropin secretion. If the stressful condition is extended for a period of time, the decrease in gonadotropin support can lead to decreased gonadal

hormone production and eventually to infertility (i.e., a loss of menstrual cyclicity in females and decreased sperm production in males). The mechanism(s) whereby different stressors influence the reproductive system are not yet understood. However, inhibition of the GnRH pulse generator, often by activation of inhibitory neurotransmitter neurons is usually the final pathway of such endocrine disruption. Other, physiologic changes in activity of the reproductive neuroendocrine axis [puberty, pregnancy, menopause] will be discussed during the remainder of this course.

The Ovarian Cycle Gabriella Gosman, MD February 9, 2009 Recommended reading: Lange Endocrine Physiology. Chapter 9. Female Reproductive Physiology. o Beginning of Chapter through and including Ovarian Cycle Section. Fulltext online through HSLS health sciences ebooks. http://www.accessmedicine.com/resourceTOC.aspx?resourceID=81 This course breaks down the reproductive cycle into 3 components: neuroendocrine regulation, ovarian component, and endometrial response. Make sure you can describe how these all work together in a normal reproductive cycle, and in the pathologic situations that you encounter in PBLs, workshops, and CPCs. Watch this step-by step animation of the reproductive cycle. The animation describes each components changes at each stage of the cycle. http://www.aamc.org/meded/mededportal/downloads/MEP-ID-0012.htm [Molson Medical Informatics Project M, Morris D, The Menstrual Cycle and Ovarian Function. MedEdPORTAL; 2006. Available from: http://services.aamc.org/jsp/mededportal/retrieveSubmissionDetailById .do?subId=12 ] Watch this several times as you learn the material during the first week of the course. You should be able to do the following for a normal cycle: 1. describe the entire process from the perspective of each of the components (ovary, endometrium, hypothalamus/pituitary) 2. describe what is happening simultaneously at each time point in the cycle

Key considerations about the timing of the reproductive cycle: -Day 1 is the first day of flowing menses (not just spotting) -Number of days from day 1 to ovulation (follicular phase) varies from woman to woman, cycle to cycle. -Number of days from ovulation to day 1 (luteal phase) has a much more standard duration for all women, normal is 12-16 days (average is 14 days). OVARY The Follicle The key reproductive structure in the ovary is the follicle. The follicle consists of the primary oocyte (oocyte that is arrested in first meiotic division) and granulosa cells. -Primordial follicle: primary oocyte with single layer of flat granulosa cells. -Primary follicle: same with single layer of cuboidal granulosa cells. -Secondary follicle: oocyte with several layers of cuboidal granulosa cells.

-Antral follicle: oocyte with zona pellucida, many layers of granulosa cells, follicular fluid accumulated, theca cell layer visible outside the granulosa layer. This is the stage of follicle that is eligible for cyclic recruitment to compete to ovulate. This looks like a 2-9mm fluid-filled cyst on ultrasound. -Preovulatory follicle (dominant follicle close to time of ovulation): oocyte with zona pellucida and several layers of cumulus cells (granulosa cell crown around the oocyte), lots of follicular fluid making a large antrum, visible layer of theca cells outside the granulosa layer. Around the time of ovulation, this is 18-25mm in size and looks like a fluid-filled cyst. -Corpus luteum: follicle after ovulation. Granulosa cells become luteinized and transition to produce progesterone. On ultrasound, this looks like a blood and clot-filled cyst. -From the antral stage on, follicles look like cysts on ultrasound (transvaginal). FOLLICULAR PHASE Cyclic Recruitment of follicles Antral follicles eligible for cyclic recruitment (approximately 10 per cycle in younger women) respond to rising FSH levels in the late luteal and menstrual part of the cycle. Granulosa cells in these follicles have FSH receptors. FSH makes granulosa cells proliferate and induces the enzyme aromatase that can convert testosterone to estradiol within granulosa cells. Androgen substrate is primarily made in the adjacent layer of theca cells in response to LH stimulation of LH receptors. This theca-granulosa teamwork is the two cell, two gonadotropin model for estrogen secretion by the follicle. As estradiol and inhbin manufactured in granulosa cells of the developing follicles increases, the FSH signal decreases. Thus, follicular estrogen and inhibin exert negative feedback on the hypothalamus and pituitary re: FSH production. By days 5-7, the dominant follicle has established itself. This is the largest, the highest estrogen producer, and (usually) the only follicle to progress to ovulation. Possible mechanisms of dominance include the following: 1. increased sensitivity to FSH signalmore receptors, more efficient response; and 2. induced LH receptors on granulosa cells of the dominant follicle that allow it to grow based on LH stimulation also. There may be other mechanisms of dominance. OVULATION Toward the end of the follicular phase, sustained high-levels of estradiol exert positive feedback on the pituitary to stimulate the LH surge. A small increase in granulosa cell progesterone facilitates this process. What does the LH surge accomplish? -Resumption of meiosis of the primary oocyte so it is fertilizable. The first meiotic division is completed at ovulation and the first polar body is extruded. The resulting secondary oocyte and the first polar body each have 23 chromosomes. At the time of fertilization of the secondary oocyte, the second meiotic division is completed.

-Ovulation. This occurs about 36 hours after the onset of the LH surge. LH stimulates production of prostaglandins, progesterone, and the proteolytic enzymes that make the follicle rupture. -Luteinization of the granulosa cells. This ultimately turns the deflated follicle into a progesterone and estradiol factory, the corpus luteum. Angiogenesis in the corpus luteum allows delivery of a large amount of cholesterol substrate to fuel progesterone production. It also allows a large amount of progesterone to get into the circulation. The result is a very vascular cyst. LUTEAL PHASE Luteinized granulosa cells produce large amounts of progesterone. Substantial progesterone production will not occur without ovulation. No ovulation, no progesterone. LH stimulates progesterone production. It does not take very much LH as you can see quite low levels on the diagram in the luteal phase. These cells also continue to make estradiol. If pregnancy does not occur, the luteal phase lasts 12-16 days. Luteinized granulosa cells regress and produce diminishing amounts of progesterone and estradiol. If pregnancy occurs, hCG replaces LH as the stimulus for continued corpus luteum function. hCG binds to and signals via the LH receptor. The corpus luteum is required to support a developing pregnancy until the placenta can take over responsibility for progesterone and estrogen production. This occurs at about 7 weeks gestation. ENDOMETRIUM (BRIEFLY) This will be covered in depth in CPC2: Menstrual Cycle. Proliferative phase (ovary perspective=follicular phase): estradiol acts on the endometrium to produce proliferation of glandular cells. The lecture slideset shows the difference between endometrium in the early proliferative versus the pre-ovulatory timeframe (much thicker). This transformation is due to estrogen. Secretory phase (ovary perpective=luteal phase): estradiol continues to be present. Progesterone transforms the endometrium into its secretory morphology. Glands and the glandular cells transform their appearance during this phase to become tortuous, full of secretory material, and then actively secreting material. The lecture slideset shows secretory endometriumthe appearance becomes more echogenic (whiter) in this phase of the cycle. Late in the secretory phase, the decline in estradiol and progesterone result in disruption of the endometrial tissue. Subsequent shedding of the endometrium is called menstruation. (Keep in mind that any significant decline in estrogen and/or progesterone for any reasonnot just from the ovulatory process--can result in endometrial shedding experienced by the patient as bleeding or spotting). BACK TO THE OVARY How do follicles get into the cyclic recruitment pool? Most oocytes and follicles are destined to become atretic. Less than 1% of follicles ever ovulate. The signals that start a primordial follicle on the road to development (Initial recruitment) are not well understood. The first primordial follicles to start the maturation process begin doing so in the 5th to 6th month of

gestation. A primordial follicle starts this maturation process about 1 year prior to potential ovulation. Follicles acquire a blood supply at the secondary stage. At this point, they become potentially responsive to circulating hormones. Follicles begin to respond to FSH some time between the secondary to antral stage. The follicle is sensitive to FSH for approximately the last 50 days of its development. By menopause, all functional follicles have undergone atresia or ovulated. Thus, there are no follicles that can respond to the hypothalamic-pituitary reproductive signal. No ovarian estrogen can be made. No progesterone is made because ovulation has ceased. STEROIDS Sex steroids have 21 carbons (progesterone), 19 carbons (androgens), or 18 carbons (estrogens). The ovary requires more cholesterol than it can manufacture, so circulating cholesterol is a key source for sex steroid production. You should be able to describe the steroid synthetic pathways for the gonadal steroids and know the organ and cell types where these occur. The ovary does not make glucocorticoids or mineralocorticoids. Otherwise, the steroid pathway is the same.

REFERENCES Strauss JF, Williams CJ. The Ovarian Life Cycle in Yen and Jaffes Reproductive Endocrinology: Physiology, Pathophysiology, and Clinical Management. 5th Edition. Strauss JF and Barbieri RL eds. Elsevier Saunders 2004. McGee EA, Hsueh AJ. Initial and Cyclic Recruitment of Ovarian Follicles. Endocrine Reviews 2000 21(2): 200-14.

Clinical Pathology Correlation #1 Female Reproductive System: Introduction to the Basic Histology Monday, February 9, 2009 10:30-12:00 Georgia Duker, Ph.D. The objectives of this lecture are for the students: 1. To distinguish the primordial, preantral, antral and Graafian follicles by morphology, receptors and secretions. 2. To recognize the wall structure and cell types that characterize the Fallopian tube. 3. To histologically identify the uterine endometrium during menses, proliferative and secretory periods and be able to discuss the hormone effectors and secretions in each portion of the menstrual cycle. 4. To identify the vagina and cervix, and be able to explain physiological manifestations of the follicular and luteal phases of the ovary on these organs. 5. To recognize the structural alterations in the female breast during maturity, pregnancy and lactation. Female Reproductive System The female reproduction system consists of the ovaries, Fallopian tubes, uterus, cervix, vagina, external genitalia and breasts. We will not cover the external genitalia in this lecture; students should refer to their gross anatomy course and Dr. Lance-Jones lecture on development of the two systems. For the laboratory component, students should be able to identify examples of normal histology for the ovary, Fallopian tube, uterus, cervix, vagina and breast.

1. The ovary Grossly, the human ovaries are slightly flattened intrapelvic paired organs, each measuring between 2.5 to 5 cm in length, 1.5 to 3 cm in width and 0.6 to 1.5 cm in thickness. A cross section through the entire organ reveals a cortex and medulla. The cortex consists of a unique, swirled, dense connective tissue stroma, in which the follicles are embedded. The medulla is a loose connective tissue, richly supplied with blood and lymphatic vessels. The outermost layer is a simple cuboidal visceral mesothelium called the germinal epithelium. The name is a misnomer from an era when it was thought to contribute germ cells; it is, however, the most common site for ovarian malignancies (CPC#5).

Examination of the cortex of an ovary from a postpubescent woman can reveal all the classes of follicles: primordial, preantral, antral, Graafian and corpus luteum. A primordial follicle has existed in this state since birth. It contains a primary oocyte, which has been halted late in prophase I of meiosis. Cross-overs have occurred, but the points of contact are still held together by chiasmata. The oocyte is surrounded by a closely associated single squamous layer of pregranulosa cells. Primordial follicles can persist for years, until each is stimulated to develop and possibly to ovulate. A preantral follicle is stimulated by local growth factors. The primary oocyte enlarges and becomes surrounded by a extracellular layer, the zona pellucida. One of the proteins of the zona pellucida, ZP3, will later serve as the sperm

receptor. The associated layer of pregranulosal cells has divided to produce multiple layers of cubodial granulosa cells, enclosed by a basement membrane. Outside of this basement membrane, layers of stromal cells become associated with the follicle and differentiate into the theca. Maturation of a preantral follicle requires 85 days and occurs over a number of menstrual cycles. The antral follicle is distinguished by the appearance of an antrum, a fluid filled cavity within the granulosa cell layers. Antral development requires reproductive levels of FSH. The expanding antrum separates the granulosa cells into two populations: those surrounding the zona pellucida, and those forming layers of the follicular wall. Granulosa cells that immediately surround the zona pellucida and primary oocyte are called the corona radiata. This entire complex projects out into the antrum on a stalk of granulosal cells called the cumulus oophorus. The ball of the enlarging antral follicle is surrounded by the remaining granulosa cells and the theca. Together, these cells secrete rising levels of estrogen. LH stimulates the theca cells to synthesize precursor androgens; FSH stimulates the granulosa cells to convert these androgens into estrogens. The mature Graafian follicle measures 20 mm in diameter. A rise in pituitary LH, 36 hours prior to ovulation, results in an increase in antral fluid and the synthesis of collagenase that weakens the overlying ovarian wall. Just prior to ovulation, the cumulus oophorus stalk is severed, and the oocyte/zonula pellucida/corona radiate complex floats free in the antral fluid. The oocyte is stimulated to reenter meiosis and proceeds to metaphase of meiosis II, i.e. a secondary oocyte. At this point in development, the entire complex is expelled at ovulation. Meiosis will only be completed after ovulation if a sperm fertilizes the oocyte. After ovulation of the secondary oocyte/zona pellucida/corona radiata complex, the remaining stromal cells of the Graafian follicle develop into the corpus luteum. Granulosa cells are invaded by theca cells and blood vessels. Granulosa lutein cells secrete progesterone, while synthesis of estrogens again requires the cooperative efforts of both theca lutein and granulosa lutein cells. The corpus luteum has a life cycle of 14 days before involuting. If a pregnancy occurs, the corpus luteum of pregnancy secretes progesterone and estrogen for 9-10 weeks until the placenta can take over synthesis of the reproductive hormones. 2. The Fallopian Tubes The Fallopian tubes are open ended, muscular tubes that extend from the ovary to pierce the uterine wall. The tubes provide the appropriate environment for fertilization and facilitate transportation of a conceptus to the uterus. The flared open end of the Fallopian tube is the infundibulum, characterized by fimbria that help to capture the oocyte and sequester it into the tube. A cross section through the Fallopian tubes reveals two structures important for oocyte transport: cilia and smooth muscle. From the lumenal surface outward, the Fallopian tube is composed of mucosa, muscularis and serosa layers. The mucosa is a simple columnar epithelium with ciliated and secretory peg cells. Both cell types increase in height and function under the influence of estrogen.

The secretions are important for support of both sperm and egg, as fertilization typically occurs in the upper 1/3 of the tube. The muscularis contains an inner circular and an outer longitudinal smooth muscle layers. The peristaltic action of the muscle layers, as well as the beating action of the cilia move an ovum toward the uterus.

3. The Uterus The uterus is the organ that receives the embryo, provides for its attachment, establishes the vascular support for pregnancy and in the end, helps to expel the fetus. Grossly, it is a pear shaped organ approximately 6.5 cm long, 3.5 cm wide and 2.5 cm thick, in the nonpregnant state. The two major layers of the uterus are the myometrium, a thick mass of smooth muscle, and the endometrium, the epithelial lining. The endometrium consists of coiled tubular glands that proliferate and are sloughed off during each menstrual cycle. The endometrium is therefore divided into a stratum functionalis that regenerates every month only to be eliminated, and a stratum basalis that remains after menses to serve as the progenitor cells for the next round of regrowth. The lab slides include sections of menstrual, proliferative, secretory and ischemic endometrium. Menses (day 1-4) is the result of a degenerating corpus luteum from the prior cycle. The resulting decreases in estrogen and progesterone cause vascular spasms that produce ischemia and necrosis in the endometrial functionalis. This layer exfoliates and the necrotic stratum functionalis and hemorrhaged blood (50250 ml) are discharged as menstral flow. The stratum basalis remains, having an independent blood supply from that of functionalis. During the proliferative phase of the menstrual cycle (days 5-14) the endometrium grows rapidly. Mitoses are seen in all three components: the epithelium/glands, the stroma, and the blood vessels. Since all three are growing at the same rate, proliferation is characterized by long, straight glands in a loose connective tissue stroma. The high estrogen of this period induces progesterone receptors on all endometrial cell types. The secretory phase of the menstrual cycle (days 14-28) is influenced by progesterone from the corpus luteum. The stroma stops growing, but the blood vessels and glands continue to expand a few days further, forcing them to coil. The glands also accumulate lipid and glycogen secretions and become tall columnar cells. However, by late in the secretory phase, all of these nutrient stores are released into the tortuous lumens, and the cells appear quite flat. The ischemic phase of the menstrual cycle (days 27-28) is really just the last days of the secretory phase. With the regression of the corpus luteum, the support of progesterone and estrogen is lost. The spiral arteries undergo spasms of contraction and relaxation (due to prostaglandins) and fluid and blood begin to leak into the stroma.

4. Cervix The cervix is the distal, cylindrical end of the uterus that protrudes into the vagina. The cervical canal is called the endocervix and is lined by a simple columnar mucus secreting epithelium. Branched glands project from the epithelial surface and occasionally swell to form cysts. Cervical mucus fluxes under the influence of cycling estrogen: thinner, more copious and alkaline under the influence of estrogen, versus rather viscous and acidic under the influence of progesterone. The portio vaginalis is the transition to the stratified squamous epithelium of the ectocervix, which is continuous with the vagina. The ectocervix faces the harsh, acidic environment of the vagina. The cells of the ectocervix are those that are sampled for a PAP smear. In the myometrium of the cervix only 15% of the cells are smooth muscle; the predominant tissue is dense bundles of collagen I for strength. 5. Vagina The vagina is a muscular tube extending from the vestibule of the external genitalia up to the cervix. The vaginal mucosa is stratified squamous epithelium with no glands. The only lubrication for the vagina comes from cervical drainage. Under the influence of estrogen, the epithelium increases stores of glycogen, which serve as nutrition for lactobacilli. The bacteria flora secrete lactic acid, and lower the pH to a level critical for maintaining vaginal flora. A shift in pH can result in bacterial vaginosis. The muscular layer of the vagina contributes to sexual arousal and childbirth. 6. Breast The breast is a secondary sex organ of the female reproductive system. It is responsive to ovarian hormones and is a critical organ in pregnancy and the subsequent nutrition of a newborn. Each breast consists of 15-25 lobes, which in turn, are comprised of numerous lobules. The changes that occur in breast structure relevant to puberty, pregnancy and lactation are easy to differentiate in the laboratory slides. This topic will be covered in greater detail in Dr. Ryans lecture.

Female Reproductive System: Histology Laboratory Clinical Pathology Correlation #1 Georgia Duker, Ph.D. Histology Tray 7, Slots 1-88; Slides O1-O88. All slides are on the Navigator website for the Reproduction Course. Ovaries O1 (4X) Cat H&E O2 (10X) Cat H&E O3 (50X) Cat H&E O4 (50X) Cat H&E O5 (10X) Cat H&E O6 (50X) Cat H&E

O7 (100X) Cat H&E O8 (50X) Cat H&E O9 (10X) Cat Mallory O10 (25X) Cat Mallory O11 (100X) Cat Mallory O12 (100X) Cat Mallory

The ovaries are composed of a cortex and medulla (Slide O1). The cortex contains the developing oocytes and the medulla is composed of connective tissue and blood vessels. The mesovarium (Slide O2), a fold of peritoneum, suspends the ovary from the abdominal wall. Note the large blood vessels that gain access to the ovary through this fold (Slide O3). A simple cuboidal visceral peritoneum surrounds the ovary (Slide O4). This gives the ovary a dull surface in vivo, compared to the shiny appearance of the simple squamous mesothelium surrounding other peritoneal organs. From fetal life through the initiation of the reproductive years, folliculogenesis begins independent of any stimulation from gonadotropins. However, at puberty, preovulatory follicles are recruited to develop into antral follicles and finally into mature Graafian follicles that can result in the release of an oocyte. Antral folliclular development requires reproductive levels of follicle stimulating hormone, FSH; while Graafian follicle maturation requires the stimulation of FSH and luteinizing hormone, LH. Initially, each oocytes is enclosed by a single layer of flattened pregranulosa cells; the entire structure is referred to as a primordial follicle (Slide O4). As follicles develop, they shift from the outer cortex toward the medulla of the ovary (Slide O5). The primordial follicles are surrounded by connective tissue stroma with unique, multi-directional swirls (Slide O6). As development into a preantral follicle begins, the oocyte enlarges and is surrounded initially by a single layer of now cuboidal granulosa cells (Slide O6, right side), and finally by multiple layers of granulosa cells (Slide O7). The enlarged germ cell at this stage is still a primary oocyte, held in the final stage of prophase I of meiosis. In Slide O7 note the zona pellucida, a clear nutrient & receptor layer forming between the egg and the granulosa cells. Simultaneously, flattened, ovarian stromal cells differentiate into theca cells. Try to visualize the location of a basement membrane limiting the outer aspect of the granulosa cells; the theca cells surround the follicle immediately outside of this basement membrane. Although not clearly visible in the light microscope, arterioles vascularize the theca and arborize into a wreath of capillaries.

Next, several clefts appear in the granulosa cell mass and give rise to the beginnings of a fluid-filled cavity, the antrum (Slide O8). The entire structure is called an antral follicle. Follicular (antral) fluid is a transsudate of plasma that also contains secretory products of the granulosa cells. Antrum formation only occurs with FSH stimulation. It is of interest to note that the granulosa cells are the only cells in the body with FSH receptors. The theca becomes organized into two layers: a theca interna (synthesize androgens) and theca externa (fibroblast/smooth muscle-like). Slide O8 illustrates three histologic consequences of FHS stimulation: granulosa proliferation, oocyte growth, and antral formation. Biochemical consequences of FSH stimulation are that granulosa cells produce aromatase to metabolize androgens to estrogens, and they begin to express LH receptors. Finally, the antrum enlarges greatly (from 400m to 15mm), under the influence of FSH, to produce a mature Graafian follicle (Slide O9). Rapid granulosa mitoses result in the suspension of the oocyte on a stalk of cells called the cumulus oophorus. The granulosal cells that immediately surround the oocyte and zona pellucida are called the corona radiata (Slide O10). Slide O11 provides a high power view of a Graafian follicle illustrating an oocyte, zona pellucida, corona radiata, and cumulus oophorous. A surge of LH, 36 hours prior to ovulation, stimulates the oocyte to resume meiosis. A secondary oocyte has completed meiosis I by the separation of homologous chromosomes (reduction division), without a centromeric division, and halted at metaphase of meiosis II. Meiosis II is completed only after fertilization by a sperm. The preovulatory surge of LH will also result in the breaking of the cumulus oophorus stalk, allowing the 2o oocyte, zona pellucida and corona radiata to be expelled as one mass. Slide O12 shows a high power view of the layers of the follicular wall: the granulosa (innermost layers with rounded cells, limited by a basement membrane), the theca interna (second layer with spindle shaped modified stromal cells), and the theca externa (outermost layers of stromal cells mixed with blue stained collagen fibers). The vascularized theca interna are well differentiated into steroid producing cells; the theca externa contain actin and myosin and are innervated by autonomic nerves.

Ovary - Corpus Hemorhagicum O13 (5X) Human, H&E

O14 (10X) Human, H&E

Immediately following release of the oocyte-zona pellucida-corona complex, the cavity of the follicle becomes filled with blood from ruptured vessels. This is a transient stage since the cells of the granulosum and the theca interna will rapidly involute and develop into the corpus luteum.

Ovary - Corpus Luteum O15 (5X) Monkey, H&E O16 (10X) Monkey, H&E O17 (50X) Monkey, H&E

O18 (5X) Cat Mallory O19 (25X) Cat Mallory O20 (50X) Cat Mallory

After ovulation, the remaining granulosa and theca cells of the Graafian follicle undergo dramatic morphological changes under the influence of LH. The granulosa cells become granulosa lutein cells and the theca interna cells become thecal lutein cells. The gonadotropin surge stimulates the granulosa lutein cells to secrete progesterone. Estradiol synthesis also slightly rises, but not to the levels of the pre-LH surge. The granulosa lutein cells are large, enladdened with lipid, and centrally located in the folds of tissue. The theca lutein cells are smaller, more darkly stained and tend to follow infoldings of connective tissue and blood vessels that invade and contribute to the corpus luteum. Slides O1517 show a corpus luteum from a monkey stained with H&E. Slides O18-20 show a similar series from cat stained with Mallory to emphasize the septa of connective tissue. If pregnancy occurs, the corpus luteum remains functional through the third month of pregnancy. If the ovum is not fertilized, and therefore no signal of hCG (human chorionic gonadotropin) is received, the corpus luteum regresses two weeks after ovulation.

Ovary - Corps Albicans O21 (10X) Human, H&E O22 (25X) Human, H&E

O23 (5X) Cat Mallory O24 (10X) Cat Mallory

The regressing cellular components of the corpus luteum are replaced by fibrous tissue of the corpus albicans. This structure is resorbed over a period of months. Although not readily apparent in the light microscope, theca interna cells from atretic follicles and degenerated corpus leutea remain as interstitial cells. These cells can secrete in response to LH, similar to theca cells.

Fallopian Tubes Infundibulum O25 (5X) Human, H&E O26 (50X) Human, H&E

O27 (268X) Human, H&E O28 (268X) Human, H&E

The infundibulum of the Fallopian tube opens to surround the ovary. There is no actual attachment, rather finger-like extensions called fimbriae encompass the ovary (Slide O25). The folds are composed of simple columnar epithelium and lamina propria. The lamina propria is a loose connective tissue with abundant large fibroblasts and capillaries (Slide O26). The epithelium contains two types of cells, cilitated cells with round nuclei, and secretory peg cells with elongated densely staining nuclei and a protruding apical surface. The distribution of these two cell types varies along the length of the Fallopian tube, with a greater percentage of ciliated cells at the distal portions, and a greater percentage of peg cells proximal to the uterus. Metabolically, these cells also respond to cyclic changes in estrogen from the ovarian hormone cycle. Slide O27 shows a field of predominantly tall, ciliated cells typical seen during estrogen influence (ovarian follicular phase). The ciliated cells have increased in height to overshadow the peg cells. Slide O28 shows a mixture of ciliated and peg cells seen during the luteal phase. Both cell types regress in size, often with the ciliated cells shrinking more. Ciliated cells are most numerous in the infundibulum and ampulla regions of the Fallopian tube.

Fallopian Tubes - Ampulla O29 (5X) Human, H&E O30 (25X) Human, H&E

O31 (50X) Human, H&E O32 (100X) Human, H&E

The ampulla of the Fallopian tube is the outer third of the tube. The mucosa of the ampulla is thrown into extensive, branched folds. The muscularis is roughly organized into two indistinct layers, an inner circular and outer longitudinal (Slide O29-O30). The muscularis is surrounded by a serosa (upper right, Slide O29). The lamina propria core of the muscosal folds consists of well vascularized loose connective tissue (Slide O31). The large fibroblasts seen here are capable of undergoing a decidual reaction in the case of a tubal pregnancy. The simple columnar epithelium is composed of peg cells and ciliated cells, which hypertrophy and atrophy with the cycling of the ovarian hormone estrogen. Slide O32 shows an ampullar epithelium with numerous protruding peg cells, typical of the luteal ovarian phase.

Fallopian Tubes - Isthmus O33 (5X) Human, H&E O34 (25X) Human, H&E

O35 (268X) Human, H&E

The narrow isthmus of the Fallopian tube is the inner 1/3 of the tube. Slide O33 shows the mesosalpinx, the portion of the broad ligament which supports the Fallopian tube and transmits blood vessels to it. The muscularis of the isthmus is thicker than in the ampulla due to an increase in the circular layer (Slide O34). The epithelium surrounding the small lumen has very few muscosal folds and is composed predominantly of peg cells with scattered ciliated cells (Slide O35).

Fallopian Tubes - Interstitial or Intramural O36 (5X) Human, H&E O37 (25X) Human, H&E

O38 (100X) Human, H&E O39 (50X) Human, H&E

The interstitial or intramural Fallopian tube pierces the uterine wall (Slide O36). The mucosa is only moderately folded (in comparison to ampulla) (Slide O37). The lumen frequently contains debris, and lymphocytes can be seen underlying and penetrating the epithelium (Slide O38). The epithelium is predominantly peg cells with few ciliated cells (Slide O38). The organization of muscular layers is lost as the tube penetrates into the diversely oriented uterine smooth muscle. (Slide O39).

Uterus Menstrual (The uterus and the menstrual cycle are covered in their own CPCs; histology slides are included here as an extra reference material) O40 (5X) Human, H&E O43 (100X) Human, H&E O41 (25X) Human, H&E O44 (50X) Human, H&E O42 (100X) Human, H&E O45 (100X) Human, H&E

During menstruation, (days 1-4), the stratum functionalis of the endometrium is sloughed off. The endometrial portion that is retained is the stratum basalis (Slide O40, basalis is the upper portion of the slide, most of image is muscularis). The endometrium is very thin during menstruation; the glands are short and the endometrial stroma is quite dense (Slide O41). The basal portion of the endometrial glands persists during menses (Slide O42). The surface epithelium (functionalis) has been totally lost, and numerous red blood cells and small dark staining lymphocytes crowd the stroma. At the end of menstruation, resurfacing of the endometrium begins as cells in the basal glands proliferate to produce the new epithelium (Slide O43). The myometrium consists of compactly arranged smooth muscle bundles that run in many different directions (Slides O44, O45). The myometrium is well vascularized. Uterus - Proliferative O46 (10X) Human, H&E O47 (25X) Human, H&E

O48 (25X) Human, H&E

Proliferative (estrogenic, reparative, or follicular) phase: days 5-14. The epithelial, stromal and vascular components of the endometrium proliferate. Slide O46 shows a low power view of the uterine wall at this stage. The myometrium, basal endometrium and the functional or regenerating endometrium can be easily distinguished (bottom to top). The glands and stroma proliferate rapidly under the influence of rising estradiol. The endometrial glands are long and straight due to rapid mitoses in the regenerating functionalis (Slide O47). If cut in cross section the glands would appear like small dark doughnuts. The endometrial stroma is composed of fibroblasts held in a meshwork of fine collagenous fibers and fluid. In the functionalis, the stroma is very loose, clumping to yield a peppery appearance after fixation (Slide O47). The glands in the basalis tend to remain slightly coiled (Slide O48, basalis and myometrium). The stroma remains more compact and dense in the basalis (Slide O48). Although not readily apparent in these micrographs, the microvasculature is also regenerating from arteriolar stumps remaining in the basalis. Blood vessel proliferation is probably under the control of local tissue growth factors including angiogenic growth factor, epidermal growth factor, fibroblast growth factor, and transforming growth factor . Uterus - Secretory O49 (5X) Human, H&E O50 (50X) Human, H&E

O51 (10X) Human, H&E O52 (50X) Human, H&E

Secretory (progestational or progravid) phase: days 15-26. Few changes occur in the first two days after ovulation (days 15,16) as the corpus luteum is forming. With corpus luteum maturity and increased progesterone secretion, a secretory transformation of the uterine endometrium takes place. During the secretory phase, the endometrium achieves maximum thickness due to the secretory activity of the glands and the fluid edema in the stroma. The glands are highly coiled and tortuous arteries reach the entire width of the endometrium (Slide O49).

During days 17-19, early in the secretory phase, glycogen synthesis increases, and begins to accumulate in the basal portion of the glandular cells of the endometrium. Slide O50 shows a diagnostic sign of early secretory phase, subnuclear vacuolation (note the fixation artifact of the epithelial basement membrane pulling away from the stroma). Basal vacuoles push the nuclei toward the lumenal aspect of the glands. This appearance lasts for two or three days. Release of secretions (days 20-21) will stagger the nuclei to various heights in the cell, resulting in the nuclear stratification typical of mid-secretory phase. By day 21-22, all the secretions have shifted to the apical side of the endometrial cells; by day 22 the lumens of the glands become dilated and filled with secretions (not shown). Note that this would coincide with implantation of an arriving blastocyst. By day 23, the stromal cells enlarge, undergoing a predecidual transformation, characteristic of early pregnancy; stromal edema can increase markedly (Slide O51). Decidual stromal differentiation is also accompanied by extravasation of PMN leukocytes and granulocytes. Proliferating, coiled arteries also are found in the myometrium at this phase (Slide O52). The terminal portion of the secretory phase is referred to as the ischemic phase (days 26-28) and is characterized by constriction and then sudden dilation of arteries forcing blood to accumulate in the endometrial stroma (not shown). The vasospasms are most likely stimulated by local release of prostaglandin F2, which begins to accumulate in the endometrial stroma around day 26, reaching maximal concentrations at day 28, also the onset of menses. Cervix O53 (5X) Human, H&E O54 (25X) Human, H&E

O55 (50X) Human, H&E O56 (100X) Human, H&E

The cervix is the narrow inferior continuation of the uterus. The endocervix (nearest to the uterus) is characterized by branched tubular glands (Slide O53). This is in contrast to the straight coiled glands of the uterus. The glands extend deep into the lamina propria and are lined by mucous-secreting columnar cells with basal nuclei (Slides O54, O55). Cyclic changes occur in the endocervical glands that parallel the uterus. Late follicular phase delivers greater estradiol and produces abundant, watery mucus, with an alkaline pH. Progesterone of the luteal phase decreases the mucus secretions, and produces a viscous, acidic secretion. The portion of the cervix that projects into the vagina (ectocervix) is covered by stratified squamous epithelium (not shown). Occasionally the stratified squamous epithelium will grow backward from the portia vaginalis into the endocervical canal, covering the outlets of the cervical glands. The glands occasionally fill with mucous and form cysts.

The muscularis layer of the cervix is composed primarily of fibroblasts and thick collagen I, with very little smooth muscle (Slide O56). Note the wavy pink collagen bundles and intercellular spaces in this slide; contrast this appearance with the smooth muscle of the uterine myometrium (Slide O44). In the uterine myometrium, the myofilaments of smooth muscle cells contract upon fixation producing the crinolated or wavy nuclei. In the cervix, the fibroblast nuclei remain oval and are surrounded by large, extracellular collagen bundles.

Vagina O57 (25X) Human, H&E

O58 (100X) Human, H&E

The vagina is lined by nonkeratinized stratified squamous epithelium. Beneath this is a cellular lamina propria that projects into the epithelium (rete pegs) and below into the muscle layer (Slide O57). The muscle layer of the vagina wall is composed of intertwining circular and longitudinal bundles (Slide O57). The epithelial cells respond to the presence of estrogen by accumulating glycogen. During the follicular phase of the ovarian cycle, the cell cytoplasm will swell and appear vacuolated when visualized with H&E staining (Slide O58).

Umbilical Cord (Skip this section) O59 - O63

Mammary Gland O66 (5X) H&E O67 (50X) H&E

O68 (50X) H&E O69 (50X) H&E

This tissue is from a postpubertal female who has never had a child (nulliparous). Under low power, the mass of the breast is adipose tissue and areolar connective tissue; the ducts appear in septa of denser connective tissue as clear spaces lined by an epithelium of dark bluish-purple closely packed nuclei (Slide O66). The ducts are very few in number, and the epithelium varies from simple cuboidal or low columnar to a stratified cuboidal (Slide O67). Thickenings on the lateral surface of the ducts are the buds of alveolar growth, which will proliferate during pregnancy (due to prolonged, increased progesterone). Blood vessels can also be seen in the connective tissue septa (Slide O68). The majority of the organ is composed of adipose and areolar connective tissue (Slide O69).

Mammary Gland - Pregnancy O70 (5X) H&E

O71 (25X) H&E

During pregnancy, profound changes occur in the mammary gland. The ends of the ducts grow, and numerous alveoli (sacs) proliferate at the expense of the surrounding fat and connective tissue.

Mammary Gland - Lactating O72 (10X) H&E O73 (25X) H&E

O74 (100X) H&E

During lactation, the alveoli enlarge and the lumens expand, filling with secretory product. There only remain thin septa of dense connective tissue separating alveoli into lobules (Slide O72). In the active gland, the alveoli constitute the major mass of the breast. (Slide O73). Alveoli branch, much like the pattern of the lungs. The alveolar epithelia range from squamous to columnar, depending on the amount of stored secretory products. Lipid droplets are easily seen in the lumen and in the apical cytoplasm. (Slide O74).

CLINICAL PATHOLOGIC CORRELATION #2 THE MENSTRUAL CYCLE Tuesday, February 10, 2009 8:30 a.m. 10:20 a.m. I. OBJECTIVES: The general objectives for this laboratory are to: (1) recall the fundamental endocrine physiology of the menstrual cycle by briefly reviewing the hormonal regulation of the pituitary, ovary and endometrium by FSH, LH and the sex steroid hormones estrogen and progesterone. recognize the microanatomy of the major physiologic phases of the endometrium and understand their functional basis (proliferative, secretory, menstrual and gestational endometrium). begin to understand the physiologic, anatomic and molecular basis of reproductive medicine and gynecology and to apply this understanding to clinical situations.





This 90 minute laboratory period will begin with a twenty-minute review of menstrual cycle physiology and an introduction to the histology of the endometrium (lecture room 2). This will be followed by a seventy-minute interactive laboratory session based on three clinical scenarios (small group rooms 502-517). III. PREPARATION FOR THE SESSION / REFERENCES

Preparation for this laboratory will include reading the provided introduction and clinical scenarios and the pathology text reference described in the reference section. Review of menstrual cycle physiology from previous course work and endometrial microanatomy using any standard histology atlas may also be useful. IV. INTRODUCTION AND CASE SCENARIOS

The endometrium is one of the principal tissues involved in reproduction and a major anatomic component in the menstrual cycle. The other two major organs involved in the menstrual cycle are the pituitary gland and ovary. The interaction between the pituitary gland and ovary, which is controlled by two glycoprotein gonadotropic hormones produced by the pituitary gland (specifically FSH or follicle stimulating hormone and LH or luteinizing hormone), determines the cycle nature of the menstrual cycle. Although a number of organs such as breast and cervix are influenced by ovarian hormone production, the primary effector portion of the menstrual cycle is the interaction between the ovary and the endometrium, which is regulated by the sex steroid hormones produced by the ovary (estradiol and progesterone).

The menstrual cycle can be conveniently divided into two halves, which are defined by their relationship to ovulation. During the first half of the cycle (pre-ovulation, approximately 14 days), ovarian follicles produce estrogen which leads to a proliferation of endometrial glands and stroma. Not surprisingly, this portion of the menstrual cycle has been called the estrogenic phase, follicular phase, pre-ovulatory phase, or proliferative phase. The second half of the menstrual cycle (following ovulation, approximately 14 days) is characterized by the production of progesterone as well as estrogen by the ovarian corpus luteum resulting in the conversion of the endometrium from a proliferation and growth phase to a phase of secretory development. Not surprisingly, this portion of the menstrual cycle has been classified as the progestational phase, luteal phase, post-ovulatory phase, or secretory phase. Depending on whether or not pregnancy occurs in a given cycle, the cycle is completed by either a menstrual or gestational phase. If a pregnancy does not occur, the secretory endometrium is shed (menstruation) as a consequence of falling sex steroid hormone levels due to corpus luteum regression. If pregnancy does occur, the endometrium's secretory development continues under the influence of continued sex steroid hormone production by the corpus luteum of pregnancy, resulting in a gestational phase endometrium. Each of these four major hormonally dependent phases of the endometrium has a distinct microscopic appearance. The proliferative phase is characterized by a growing endometrium with tubular glands and a mitotically active endometrial epithelium (glands) and stroma. The secretory phase is characterized by dramatically decreased mitotic activity, glandular secretion, increasing tortuosity of glands and predecidual changes within the endometrial stroma. The menstrual phase of the endometrium is characterized by fragmentation of endometrial glands, hemorrhage, thrombosis, stromal condensation and degenerative changes with inflammatory infiltrate. The microanatomic appearance of gestational endometrium is characterized by dilated hypersecretory glands and extensive stromal decidualization. The hypersecretory response often leads to enlargement of glandular epithelial cells which protrude into gland lumens (Arias-Stella reaction). In addition to the four endometrial phases described above, which are present in reproductively active women, atrophy of the ovaries at menopause leads to a lack of estrogen and progesterone resulting in atrophy of the endometrium characterized by a marked decrease in glandular and stromal activity (atrophic endometrium).

CLINICAL CASE SCENARIO #1 Premise/Background: Infertility (the inability to successfully conceive, carry, and deliver a pregnancy) is a significant medical concern in reproductive gynecology. Causative and contributing factors in infertility include male infertility and impotence, structural anatomic lesions of the fallopian tubes and uterus, and functional menstrual cycle abnormalities. Clinical evaluation of infertility includes microscopic examination of endometrial biopsy tissue during luteal phase, as an assay of the general functional integrity of the menstrual cycle.

Clinical Summary: Chief Complaint: 23-year-old nulligravida female unable to become pregnant after trying for six months Patient has enjoyed general good health, had regular periods since age 15, had no previous gynecologic problems, and has not attempted to become pregnant until six months ago. Review of systems was noncontributory with pertinent negative findings including no history of STD (sexually-transmitted disease), PID (pelvic inflammatory disease), endometriosis, or ectopic pregnancy. Physical examination including speculum and manual pelvic exam was unremarkable except for an irregular, moderately enlarged uterus. Laboratory tests included an endometrial biopsy obtained on day 24 of her regular 28-day cycle, which showed an endometrium in appropriate phase.



Questions for assessment/discussion: 1. Identify the endometrial phase of each of the provided PowerPoint histology slides and select the one which represents the endometrial phase identified in this patient's biopsy. What does this biopsy suggest about the integrity of this patient's reproductive endocrine system? What would it suggest if the biopsy had shown a proliferative phase endometrium? What is the possible significance of the negative findings in the review of systems and the enlargement of the uterus identified on physical examination?


3. 4.

CLINICAL CASE SCENARIO #2 Premise/Background: Pregnancy interrupts the cyclic endometrial shedding and regrowth pattern of the menstrual cycle and leads to the development of its full secretory capacity. Although most pregnancies implant appropriately in the uterus, some implant outside of the uterus, most often in the fallopian tubes, and are classified as "ectopic." The diagnosis and treatment of ectopic pregnancy is potentially a medical emergency and requires accurate integration of clinical history, physical examination, and laboratory results.

Clinical Summary: Chief Complaint: 31-year-old, gravida 3 para 1 female with one missed menstrual period three weeks ago and recent onset of nonspecific lower abdominal pain. Patient has enjoyed general good health except for appendicitis seven years ago requiring surgical intervention. She has had regular periods since age 13, has been pregnant three times with one cesarean section and two elective terminations. Review of systems was not contributory with pertinent negative findings including no history of STD (sexually-transmitted disease), PID (pelvic inflammatory disease), or previous ectopic pregnancy. Physical examination showed a young woman in mild acute distress with pain and tenderness localized to the right lower quadrant. Remainder of the physical examination was noncontributory. Laboratory tests included a positive serum pregnancy test (b-hCG) and a pelvic sonogram showing an unremarkable uterus and a right adnexal mass. Endometrial curettings were obtained prior to laparoscopic surgery and showed a gestational endometrium without evidence of intrauterine pregnancy. Laparoscopic intervention confirmed the presence of a right ectopic pregnancy.



Questions for assessment/discussion: 1. 2. 3. Identify the example of gestational endometrium from among the PowerPoint slides provided. What is the functional/molecular basis for the histologic appearance of this endometrium? Which (if any) risk factors for ectopic pregnancy were present in this patient?

CLINICAL CASE SCENARIO #3 Premise/Background: Abnormal uterine bleeding is the passage of uterine blood and tissue other than physiologic menstruation. It is identified as abnormal because of abnormalities in timing and/or amount. Although there are many potential anatomic causes of abnormal uterine bleeding (primarily lesions of the endometrium and myometrium), some abnormal uterine bleeding has no underlying anatomic cause and is classified as dysfunctional uterine bleeding or DUB.

Clinical Summary: Chief Complaint: History/ROS: 46-year-old, gravida 3 para 3, female with irregular vaginal bleeding every ten to fifteen days for the past four months. Patient has enjoyed general good health, had regular periods since age 14, had three uneventful pregnancies and deliveries, and had no major gynecologic problems until four months ago when her periods began to become irregular with vaginal bleeding and spotting every ten to fifteen days. Review of systems shows a history of hypertensive cardiovascular disease treated pharmacologically and a breast biopsy in the remote past for benign breast disease. Physical examination including speculum and manual pelvic exam was unremarkable. Laboratory tests included a fractional D&C, which showed endometrial changes consistent with dysfunctional uterine bleeding.


Questions for assessment/discussion: 1. The provided PowerPoint slide shows an example of the endometrial histopathology frequently seen with dysfunctional uterine bleeding. How does this compare with the histologic features of proliferative endometrium? What does this histologic appearance suggest about the underlying pathophysiology of dysfunctional uterine bleeding? Which of the common anatomic causes of abnormal uterine bleeding could be identified on the basis of an endometrial curettage and which would require other methods of identification?

2. 3.

V. A.


The objectives and format of this two-hour laboratory on the menstrual cycle were described earlier. Briefly, the laboratory period will begin with a twenty-minute review of menstrual cycle physiology and an introduction to the histology of the endometrium. This introduction will be given to the class as a group in lecture room 2. Therefore, presentation of this introductory material and review is not the responsibility of the laboratory faculty. This introduction will be followed by a seventy-minute interactive laboratory session based on three brief clinical scenarios, each of which is divided into three segments: 1) background, 2) clinical summary, and 3) assessment/discussion. Recommended student preparation and references for this laboratory can be found in the student syllabus. Discussion of these clinical cases will take place in the small group rooms (502-517 Scaife) with each room being the responsibility of one faculty facilitator. Each case should be introduced by the facilitator by going over the premise/background section of the case. Following this, beginning with the chief complaint, a student should read aloud the clinical summary section by section. Finally, the exercises and questions in the assessment/discussion section should be examined (n.b., appropriate PowerPoint histology slides will be shown in each seminar room). Important points to consider in going over the exercises and questions in the assessment/discussion section are: 1. The students should be allowed to do the exercises or answer the questions as a group with the faculty member participating as a source of information rather than as the leader of the discussion. Because of the time limitation, each case scenario should be resolved in a reasonable time period (20-30 minutes). This permits each clinical case adequate time for discussion. It is important for each faculty facilitator to allow for a five or ten minute period at the end of the case studies to review the objectives of the laboratory, summarize critical concepts and facts, and for general student questions. Although this last period may seem repetitive, it is important to give the laboratory a sense of closure and completeness, and to assure that all necessary concepts and content have been discussed.




Clinical Case Discussion for Faculty (Cases 1-3)

Case #1 The assessment/discussion segment of clinical case scenario #1 requires the students to examine a number of PowerPoint histology slides of the different phases of the endometrium including examples of secretory endometrium, proliferative endometrium, and menstrual endometrium. They will also be required to recognize that the secretory endometrium is the pattern that would be present in the patient in this case (question #1). An effective method for carrying out the identification and description of these is to have one or two of the students run the PC projector as well as lead the discussion. Once secretory endometrium has been appropriately identified as the one belonging to this patient, question #2 should lead to discussion by the students of why this endometrium suggests that the pituitary-ovarian-endometrial axis is functioning appropriately. Question #3 is intended to generate a discussion of anovulation in infertility, as the persistence of a proliferative endometrium on day 24 would document the absence of ovulation and the resultant production of corpus luteal progesterone. Such a finding would suggest that the underlying basis of a patient's infertility might be endocrine in nature. Question #4 is intended to introduce a discussion of the anatomic causes of infertility including structural disease of the fallopian tubes resulting from pelvic inflammatory disease, endometriosis and structural disease of the uterus such as distorting leiomyomata. The finding on physical exam of an irregular moderately enlarged uterus suggests that leiomyomata may be an important causative factor in the case presented here. Case #2 The assessment/discussion segment for clinical case scenario #2 begins with the identification of the histology of gestational endometrium from among PowerPoint histology slides of gestational endometrium, secretory endometrium, proliferative endometrium and atrophic endometrium (Question #1). As in clinical scenario #1, an efficient method for carrying out this exercise is to allow the students to operate the PC projector as well as to lead the discussion. A brief discussion of which tissue was absent from this patient's endometrial specimen indicating the lack of an intrauterine pregnancy (i.e., chorionic villi and trophoblasts) might be appropriate at this time. Question #2 is intended to permit a discussion of the effects of extended progesterone exposure in pregnancy on the endometrium, as well as an opportunity for discussion of the endocrine physiology of the placenta and the importance of the placental-ovarian-endometrial axis in the endocrine physiology of pregnancy. For example, students could discuss the effect of placental hCG on sustaining the corpus luteum of pregnancy in the first trimester of pregnancy and its production of progesterone to maintain the endometrium in the second and third trimesters. Question #3 is intended to develop a discussion of ectopic pregnancy and specifically a discussion of risk factors such as pelvic inflammatory disease and previous abdominal surgery. The students should be encouraged to review the history, review of systems and physical exam in order to identify the potential significance of this patient's history of surgery for appendicitis.

Case #3 In the assessment/discussion section for clinical case scenario #3, a PowerPoint histology slide f endometrial tissue from this patient with dysfunctional uterine bleeding will be shown to the students. The students should be asked to compare the histologic features of this slide the example of proliferative endometrium they saw in case #1 (Question #1). Significant points in this comparison should include that the glands in this dysfunctional endometrium show the proliferative characteristics seen in proliferative endometrium and have a somewhat "hyperproliferative appearance" with glandular crowding and dilatation, while at the same time the endometrial stroma shows the degenerative changes characteristic of the menstrual endometrium they also saw in case #1. Question #2 is intended to lead to a discussion of the underlying pathophysiology of dysfunctional cyclic progesterone. By understanding the proliferative influence of estrogen, the "hyperproliferative" characteristic of the histopathology just examined should be obvious. It should be discussed that at no point in the normal cycle would glands of this proliferative pattern be present at the same time as degenerating stroma, which in the current case is not the result of a withdrawal of progesterone as in normal menstrual endometrium but rather the result of continued unopposed estrogen or possibly the withdrawal of this unopposed estrogen. Question #3 is intended to lead to a discussion of some of the anatomic causes of abnormal uterine bleeding including endometrial polyps, endometrial hyperplasia, endometrial carcinoma, and leiomyomata. It should be pointed out that all of these except for the last (leiomyomata) could be identified by endometrial curettage and that all of these anatomic causes of abnormal bleeding might be found in a 46-year-old patient. The clinical significance of these anatomic lesions, especially endometrial hyperplasia and endometrial carcinoma, can be briefly discussed if time permits. REFERENCES 1. Cotran RS, Kumar V, Robbins SL. Robbins Pathological Basis of Disease, WB Saunders, Philadelphia, Endometrial Histology and Menstrual cycle; Functional Menstrual Disorders; Pelvic Inflammatory Disease; Endometriosis; Ectopic Pregnancy or: Similar topics in any other standard pathology textbook. 2. Appropriate sections in Comprehensive Gynecology, Herbst AL (editor) and The Menstrual Cycle, Ferin M (editor).

PROBLEM BASED LEARNING SESSIONS The material for each PBL will be handed out in the small group rooms on the day of the initial case presentation. At that time, each small group will appoint both a reader and a scribe, who will write the salient features, possible diagnosis/hypotheses, and learning objectives on the board. Ideally, each member of the group will research all of the learning objectives and be prepared for meaningful participation at the time of the case resolution. At the end of the resolution, after the case has been dissected, the students will be given a list of what the PBL author considered to be important learning objectives. PBL sessions are intended to be group activities. Therefore, absenteeism is detrimental to both the group dynamics and the learning process. Although participation in PBLs is not quantified by a point value in this course, attendance at PBLs will be assigned a point value. It is possible for an individual to pass the course without attending any PBL; however, it will not be possible to attain an honors distinction without such attendance.

Problem Based Learning, Case # 1 Tuesday, February 10, 2009, 10:30 am 12:00 pm Menstrual Cycle Physiology and Endocrinology

Pharmacology of Infertility therapy Gabriella Gosman, MD Wednesday, February 11, 2009 1:00 pm
Objectives Describe the endocrine signaling and reproductive tissue response of a normal menstrual cycle. Describe how fertility drugs can mimic the normal menstrual cycle. Describe how fertility drugs can alter the normal menstrual cycle. Describe the complications of fertility drugs, including multiple gestation and ovarian hyperstimulation syndrome. Introduction
There are many clinical reasons to manipulate the menstrual cycle. One of the most common reasons is to treat infertility. Drugs are used for female infertility patients to achieve 4 goals: 1) establish ovulation (in patients that have ovulatory disorders), 2) to stimulate multiple ovulations to improve the chances for pregnancy, 3) to produce multiple eggs to be fertilized in vitro (in vitro fertilizationIVF), or 4) to make the endometrium a hospitable place for an embryo to implant. To understand how these drugs work, you must have a solid understanding of hormonal, ovarian, and endometrial events that occur during the normal menstrual cycle. Please also refer to Dr. Camerons and my previous lecture and the CPC on the endometrial aspects of the menstrual cycle. Figure 1. Normal menstrual cycle.

Ovulation Induction and Superovulation

Ovulation induction (OI) uses fertility therapy to establish ovulation in a patient with an ovulatory disorder, such as polycystic ovary syndrome (PCOSyou will cover this in PBL1). The goal of OI is to mimic a normal menstrual cycle and optimally produce a single ovulation. Superovulation (SO) uses fertility drugs to achieve multiple ovulations. Patients who undergo this therapy tend to have normal ovulation and no other detectable fertility problem despite problems conceiving (unexplained infertility). In couples in which the male partner has sperm problems, this treatment may also be indicated as a way to boost the chances for conception each month. To accomplish either OI or SO, the physician needs to increase the FSH signal and needs to ensure that an LH surge occurs (or if it doesnt occur normally, to give a drug that mimics the LH surge). The FSH signal can be increased directly by giving

injectable FSH. The FSH signal can be increased indirectly by targeting the amount of estradiol that the hypothalamus senses. If estradiol negative feedback to the hypothalamus is diminished using drugs, the hypothalamus will increase the GnRH signal to the pituitary. The pituitary will then produce and secrete more FSH and LH. Clomiphene citrate Clomiphene citrate (CC) is a selective estrogen receptor modulator (SERM). For fertility uses, it is primarily an estrogen antagonist in the tissues of the reproductive axis. The molecule is a triphenylethylene derivative; it is not a steroid. CC shares structural similarity with Tamoxifen. Like Tamoxifen, it was originally developed as an estrogen antagonist for the treatment of breast cancer. CC is a mix of 2 isomers: enclomiphene and zuclomiphene. The cis isomer (zuclomiphene) has a long half life, but its clinically relevant effect is only during administration. CC produces ovulation by acting as a competitive inhibitor of the estrogen receptor at the level of the hypothalamus. The hypothalamus senses a change from an estrogenic environment to a hypoestrogenic environment. The hypothalamus responds by increased output of GnRH. This prompts the pituitary to secrete 50-60% more FSH. The amplified FSH signal prompts the ovary to develop one or more follicles. CC only works in women with adequate estrogen. If a woman has an ovulatory disorder that renders her hypoestrogenic, this is not the right drug. The ovulatory patient takes CC daily by mouth for five days from days 3-7 (first day of menses=day 1) or days 5-9. Patients take this early in the follicular phase before follicle dominance has been established, to enhance the chance that more than one follicle may be recruited and go on to ovulation. After these five days, the rest of the menstrual

cycle looks similar to figure 1. Most patients will have an endogenous LH surge, ovulate, make progesterone and either conceive or get their period. In anovulatory patients (such as PCOS women), clomid can be given at any time (same starting regimenone pill by mouth daily for five days) as long as the woman has not had a spontaneous ovulation. Many practitioners initiate a withdrawal bleed using a progestin and then administer the drug as described above once bleeding has started. CC has antiestrogen effects that manifest as side effects and a possible negative effect on the chances for conception. Some women on the drug feel hot flashes. CC hinders proliferation of the endometrium, and may make ovulatory cervical mucus less hospitable to penetration and survival by sperm. The most significant risk of CC is multiple gestation. Approximately 10% of pregnancies are twins; approximately 1% are triplets. High order multiple gestation is extremely rare, but possible on this drug. For all of the ovulation stimulation drugs discussed in this class, there is a controversial association with a longterm increased risk of ovarian cancer. The existing literature is not conclusive about whether this is a definite risk. Aromatase inhibitors Aromatase inhibitors also enhance pituitary secretion of FSH by diminishing estradiol negative feedback to the hypothalamus. Aromatase inhibitors decrease circulating estrogen levels by blocking androgen conversion to estrogen. Similar to CC, the hypothalamic response to this change is enhanced GnRH output leading to enhanced FSH and LH output. It is administered according to a similar regimen and timing as CC. This is not currently commonly used as a first line medication. More data on safety and

efficacy remains to be collected. These medications are most commonly used for estrogen-sensitive malignancies. Gonadotropins (injectable FSH or FSH + LH) Injectable FSH can be used for OI and SO. The drug is derived from 2 sources: 1) human menopausal urine purified for LH and FSH (hMG) or just FSH (uFSH); or 2) recombinant production by transfecting Chinese hamster ovary cell lines with the genes for the FSH alpha and beta subunit (recFSH). In ovulatory patients, FSH is administered early in the follicular phase before follicle dominance is established. Patients usually give themselves one subcutaneous injection daily for the duration of the follicular phase. This amplified and prolonged FSH signal usually results in multiple follicular recruitment and maturation, and increased estrogen levels. This hormonal milieu impairs the brains ability to generate a normal LH surge. All patients who take injectable gonadotropins take a medication to mimic or initiate the LH surge. The most common drug for this use is pharmacologic preparation of hCG. Human Chorionic Gonadotropins for injection Patients administer hCG when their follicles reach maturity. LH is not used for this indication because its half-life is too short (about 45 minutes). hCGs half life is about 23 hours. Preparations are made from pregnant womens urine or via the recombinant procedure described for FSH. The drug is injected intramuscularly or subcutaneously. hCG makes the mechanical process of ovulation occur. The drug also stimulates the egg to resume meiosis and go through its final maturation to become fertilizable. Ovulation happens between 34-36 hours after the patient takes hCG. hCG

serum levels can remain elevated and measurable for up to about 9 days after the injection. Short term Risks of OI and SO with injectable gonadotropins Multiple Gestation Approximately 20% of women who conceive on this therapy have twin pregnancies, approximately 5% have triplet pregnancies. Injectable gonadotropin therapy is the treatment that places patients at highest risk for high order multiples (quadruplets and more). Practitioners cannot perfectly predict how many eggs will ovulate during any given cycle. Patients undergo rigorous monitoring on gonadotropins that entails having a transvaginal ultrasound and blood drawn every several days during the 1-2 weeks that they take the gonadotropin injections. Ultrasound allows the provider to count the developing follicles, to predict when they will be mature, and to modify the patients dose if needed. In patients who have too many follicles, the provider will cancel the cycle (the patient stops taking gonadotropins and does not take hCG) because the risk of high order multiple gestation (triplets or more) is unacceptably high. This monitoring technique also helps to predict which patients are at particularly high risk for ovarian hyperstimulation syndrome (OHSS). Ovarian Hyperstimulation Syndrome Patients at high risk for OHSS are those who develop many follicles and those with very high estrogen levels at the time that hCG is given. The pathophysiology of OHSS requires the follicle to go through the luteal transition, so OHSS cannot happen if the patient does not take hCG or other ovulation trigger. This is an iatrogenic medical condition related to ovulation stimulating drugs. Excessive vasoactive substances are

secreted during the follicular-luteal transition, particularly vascular endothelial growth factor (VEGF). This is an exaggeration of the natural process of neovascularization that occurs after ovulation in order to supply the luteinized granulosa cells with a direct blood supply to get the substrate for progesterone production. The excessive vasoactive substances lead to increased capillary permeability in the peritoneal cavity. Patients develop ascites and this leads to hypovolemia and hemoconcentration. The time course for developing this disorder is 5-10 days after ovulation. Pregnancy exacerbates the disorder because rising hCG drives the corpus luteum to make more vasoactive substances and to do this for a longer period of time. Abdominal fluid accumulation leads to patient discomfort and inability to take food/fluid by mouth. Hemoconcentration and hypovolemia can lead to organ perfusion problems based on low blood pressure and/or hypercoagulability. As a consequence, patients may experience oliguria, renal failure, venous thromboembolism, stroke, and adult respiratory distress syndrome (ARDS). Approximately 20% of patients taking this therapy develop mild OHSS, 6% develop mild disease, and 1% develop severe disease that requires hospitalization.

Drugs for In Vitro Fertilization (IVF)

IVF requires fertilization in a petrie dish. In order to have enough eggs to maximize success for this procedure, ovarian stimulation tends to be more aggressive than that described above for OI and SO. This means that most patients take a higher dose of injectable gonadotropins. When this higher dose is given, 20-25% of patients will initiate their own LH surge. IVF practitioners use a medication to eliminate this from happening. More eggs of higher quality can be obtained when the LH surge is scheduled

and controlled (not endogenous). To achieve this control, patients take a GnRH analogue medication that prevents LH from rising, but still allows them to conceive. Endogenous GnRH has an extremely short half life (about 4 minutes). Two groups of pharmacologic GnRH analogues have been developed by manipulating the 10 peptide structure of the endogenous hormone. GnRH agonists The GnRH agonists (leuprolide acetate, Lupron is the most common in the US) deliver a tonic signal. This initially stimulates the pituitary to secrete the LH and FSH it has stored (flare effect). After several days, the constant GnRH signal causes the pituitary to down-regulate its GnRH receptors and LH/FSH production. This results in a profound decrease in LH and FSH. In order to minimize the flare effect (which for most indications is undesirable), GnRH agonists are administered during the luteal phase of the cycle. During this time, there is the least amount of stored LH and FSH that can be secreted during the flare. The flare is maximal if the drug is administered in the early follicular phase. Leuprolide comes in two basic dosing forms: 1) depot given intramuscularly. 3.75mg is the monthly dose, which maintains steady serum levels for 4-5 weeks; and 2) non-depot form (fertility) given by subcutaneous injection daily .25-0.5mg per day. Leuprolide is used for 2 general categories of clinical problems: 1) fertility; and 2) conditions for which a hypoestrogenic state achieves a therapeutic goal such as endometriosis, preoperative preparation of fibroids, precocious puberty, and estrogenresponsive malignancies. For endometriosis and fibroids, practitioners limit the duration

of GnRH use because prolonged hypoestrogenism during the reproductive years leads to an increased risk of osteoporosis and fracture later in life. GnRH antagonists These compounds bind the GnRH receptor but do not activate it. They function by competitive inhibition. Administration of a GnRH antagonist immediately shuts down production and release of pituitary LH and FSH. At approximately 6 hours after a daily dose, patients achieve maximal suppression of LH. LH and FSH return to baseline within about 24 hours. GnRH antagonists do not have the flare effect. Nearly all dosing forms (cetrorelix and ganirelix) are short acting, with either daily or 4-day formulations. The most common use of these drugs is for fertility. The FDA approved abarelix (a longacting formulation) for advanced prostate cancer. Long acting formulations of these drugs may ultimately be used for all the clinical conditions listed under GnRH agonists, should studies prove them to be tolerable, effective, and affordable. Stimulation for IVF using injectable FSH, hCG, and GnRH antagonist On day 3, a patient using this drug regimen begins daily dosing of injectable FSH (total duration about 7-14 days). In the mid follicular phase, she begins taking the GnRH antagonist to eliminate the possibility of an endogenous LH surge. When the follicles are mature, she takes hCG to mimic the LH surge and force the eggs to resume meiosis I and become fertilizable. Egg retrieval occurs before the eggs can be ovulated into the abdomen. This stimulation has similar risks to those described above for injectable gonadotropins. High order multiple gestations (quadruplets and more) are less common with IVF than gonadotropins because US practitioners generally adhere to professional society guidelines for the number of embryos to transfer for particular groups of patients.

(American Society for Reproductive Medicine is currently making this available to the public for a limited time at http://www.asrm.org/Media/Practice/NoEmbryosTransferred.pdf) Among IVF pregnancies in 2005 (n=33,101), 28.5% were twins, 4.4% were triplets. (http://www.cdc.gov/ART/ART2005/index.htm) Luteal phase support The hormonal milieu produced by ovarian stimulation for IVF produces an abnormal luteal phase characterized by shortened duration. Part of this problem is the LH suppression due to the GnRH analogues (agonists or antagonists). Part of this is because of the heightened ovarian response. Unless this abnormality is corrected, patients do not achieve optimal pregnancy rates. Practitioners may take two approaches to correcting the luteal phase problem: 1) amplify the signal to the corpus luteum (CL) by using hCG. This also drives more vasoactive substance production and an increased risk of OHSS; 2) give progesterone. Progesterone Progesterone is one of many progestins. This is the progestin of choice for fertility treatment because it facilitates conception and poses no risk to the fetus. Three formulations are available. 1) oral micronized, which has a rapid first-pass effect and low absolute bioavailability. Dose is 100-200mg 2) intravaginal gel, which probably produces high uterine tissue levels. Dose is 90-100mg 3) intramuscular progesterone in oil. This produces the highest serum levels. Dose is 25-50mg. This is obviously the most uncomfortable and risky for patients, as they must administer an intramuscular injection daily. Vaginal and intramuscular preparations confer the best chance for pregnancy.

Progesterone support is administered daily until the pregnancy test is negative. If the patient conceives, she gives herself progesterone until the placenta takes over progesterone production at 7 weeks (many practitioners continue therapy through the first trimester). Oral and vaginal progesterone are used for other clinical problems. Both can be used as the progestin component of menopausal hormone replacement. Both can be used to assist in diagnosis and/or treatment of ovulatory disorders in reproductive age women.

Male Reproduction Kyle E. Orwig February 11, 2009 Overall Objectives: 1. Understand the anatomy and physiology of the testis a. morphophysiology b. spermatogenic lineage development 2. Understand the somatic cell and endocrine environment of the testis a. somatic regulation of spermatogenesis b. endocrine signaling in the hypothalamic-pituitary-testicular axis c. paracrine signaling between testicular somatic cells and germ cells 3. Understand sperm transport from the testis to the female reproductive tract a. Epididymis and vas deferens b. accessory sex glands c. ejaculation 4. Understand sperm structure and function a. structure b. capacitation/acrosome reaction c. fertilization 5. Think about how functions and dysfunctions of the male reproductive system effect fertility and infertility a. Understand normal and abnormal sperm parameters b. Causes and treatments of male infertility The male reproductive system is comprised of the organs of germ cell formation (testes), the conduit system for the delivery of germ cells (epididymis, vas deferens and urethra), the accessory organs (seminal vesicles, prostate and Cowpers gland) and the organ of copulation (penis). Spermatogenesis is regulated by local signals in the testis as well as endocrine signals from the hypothalamus and pituitary. Testicular Anatomy and Physiology The testis is located extraabdominally in the scrotum where the temperature is 2-3 C lower than core body temperature. The lower temperature is required for normal sperm production in men. Testicular

Figure 1. Testis descent

descent during fetal development (Fig. 1) is associated with the concomitant regression of the cranial suspensory ligament and thickening of the gubernaculum, and occurs in two phases. The transabdominal phase is completed by 15 weeks of gestation and involves migration from a location near the kidneys to the inguinal region of the lower abdomen. This phase is dependent on insulin-like hormone 3 (INSL3) from Leydig cells. In the second phase (inguinoscrotal), the testis is guided by the gubernaculum from the inguinal area to the base of the scrotum. The inguinoscrotal phase is androgen dependent and usually completed by birth (2). Maldescent of the testis, or cryptorchidism, occurs in 2-9% of newborn boys (3). The mechanisms of cryptorchidism are probably multifactorial and can be associated with premature birth and genetic or endocrine lesions. In most cases, the testis descends spontaneously by about 3 months of age, so the eventual incidence after 1 year is 0.8%. Left uncorrected, the cryptorchid condition can result in infertility and increased risk of testicular cancer (4). Early correction is important because damage to the sperm-making ability of the testis can begin as early as 12 months of age. The testis is contained in a tough fibrous sheath called the tunica albuginea. Invaginations of the tunica albuginea create septa, which divide the testis parenchyma into 250-400 lobules, each containing 2-4 convoluted seminiferous tubules. Each tubule is approximately 2 meters in length and thus, there are between 1000 and 3200 meters of seminiferous tubule in the adult human Figure 2. Human testis gross anatomy testis. Each end of U-shaped convoluted tubule connects to a common straight seminiferous tubule that connects to the rete testis. The rete testis connects to the head of the epididymis via the efferent ductules (10-15 small ducts (5)). The testis is comprised of two functional compartments. The spermatogenic compartment islocated inside the seminiferous tubules and androgen production occurs in Leydig cells located in the interstitial space between seminiferous tubules (Fig. 2). Spermatogenesis Spermatogenesis is a highly organized process that generates millions of sperm each day. Spermatogenesis in the postnatal testis arises from spermatogonial stem cells (SSCs). These adult tissue stem cells balance self-renewing and differentiating divisions to maintain the stem cell pool and also meet the biological demand to produce spermatogenesis. The mammalian testis produces millions of sperm every day. This level of productivity is possible because once a SSC commits to differentiate; it

undergoes several Sperm mitotic divisions, two mouse/rat /clone /g testis meiotic divisions and 4096 40x106 spermiogenesis to give rise to mature rhesus monkey spermatozoa. When 41x106 256 male germ cells divide, they exhibit incomplete man cytokenesis and 32 5.5x106 daughter cells remain connected by a thin Figure 3. Kinetics of premeiotic germ cell expansion in rodents, monkeys cytoplasmic bridge. and man. Mitotic population doublings are shown. The spermatogenic lineage Therefore, all cells in for each species also undergoes two meiotic divisions and spermiogenesis to the resulting clone are produce mature sperm. The total number of population doubling divisions for rodents, rhesus monkey and man are 12, 8 and 5, respectively. Modified from exposed to the same Ehmcke and et al., 2006 (1). signals and develop in unison. In mice and rats, there are 12 amplifying divisions between the stem cell and the spermatozoa. Thus, a single stem cell that commits to differentiate can theoretically give rise through clonal expansion to 4096 spermatozoa (6). The kinetics of germ cell expansion in primates is not so well understood and the identity of the stem cell is subject to debate. The most undifferentiated germ cells in primates are designated type Adark and Apale spermatogonia based histological evaluation of nuclear staining intensity (Fig. 3). Whether stem cell activity correlates with either of these descriptions of nuclear morphology remains to be determined. However, it is interesting that there are only 8 population doubling divisions between the Adark spermatogonium and sperm in rhesus macaques. Yet, macaques produce about the same number of sperm per gram of testis parenchyma as mice (~40 x 106 sperm/g/day, (7, 8)). To meet this biological demand, the stem cell population in macaques must be significantly larger than rodents, or the stem cells must divide more frequently. The latter explanation is unlikely because stem cells tend to divide very slowly, thus reducing the risk of mutagenesis. The risk of mutagenesis is particularly onerous in the germ line because accumulated mutations are passed to subsequent generations. There are only 5 population doubling divisions between the Adark spermatogonium and sperm in human testes. Accordingly, men produce about 8 times fewer sperm per gram of testis parenchyma (5.5 x Figure 4. Spermatogenic wave 106 sperm/g/day) than their rhesus counterparts (9). in the seminiferous epithelium.
In the rat, 14 distinct stages can be recognized in histological section. In humans, 6 stages have been described, but they are more difficult to recognize because clone size is smaller and stages are mixed within a segment of tubule.

Due to clonal expansion, whole regions of seminiferous tubule develop in unison. However, each segment of seminiferous tubules is in a different stage of spermatogenic development relative to the neighboring segments and this phenomenon sets up the spermatogenic wave [Fig. 4, (10)]. While approximately

74 days are required to produce a mature sperm in humans (11), the asynchronous spermatogenic wave means that there are always segments of the seminiferous epithelium that are shedding sperm into the lumen of the seminiferous tubule. Therefore, men continuously produce sperm (approximately 1000 sperm per second) throughout postpubertal life from a seemingly inexhaustible pool of stem cells. This is in contrast to the situation in women, where one oocyte is released per menstrual cycle from a finite pool of follicles that is established by the time of birth and is exhausted by about 53 years of age. Somatic and endocrine environment of the testis Male germ cell development is dependent on the somatic cell and endocrine/paracrine environment of the testis. The principle somatic cell types of the testis are the Sertoli cells and Leydig cells and these cells mediate pituitary endocrine signals (Figs. 5 and 6). Sertoli cells line the basement membrane inside the seminiferous tubules, are in direct contact with germ cells and, like granulosa cells in the ovary, are targets for pituitary follicle stimulating homone (FSH). Leydig cells are located in the interstitial space outside seminiferous tubules, are the major source of testosterone and, like thecal cells of the ovary, are targets of pituitary luteinizing hormone (LH). These testicular somatic cells, in turn, produce inhibin and testosterone, which feed back to regulate GnRH from the hypothalamus and gonadotropins (FSH, LH) from the pituitary (Fig. 5). Defects in hypothalamic or pituitary regulation result in hypogonadotropic hypogonadism, which is associated with absent or decreased testicular function, absence of secondary sexual characteristics (e.g, pubic, facial and underarm hair) and short stature (in some cases). Hypothalamic failure most commonly results from Kallmanns syndrome, which usually also involves a disorder with the sense of smell (12). The KAL-1 gene (X-chromosome) encodes the anosmin protein, a neural adhesion molecule that may be Figure 5. Hypothalamic-pituitaryrequired during fetal development for the migration of testis axis. GnRH neurons from the olfactory placode to the brain. Sertoli cells are essential for the formation of seminiferous tubules during fetal development and this process is dependent on specific genes (e.g., SRY) on the Y chromosome. In the postnatal testis, Sertoli cells proliferate until puberty, at which time they differentiate and mature. FSH drives Sertoli cell proliferation and consequently seminiferous tubule length, during the prepubertal period. The number of Sertoli cells determines the total spermatogenic output. While FSH stimulates prepubertal Sertoli cell proliferation, it is not required for spermatogenesis. A null mutation of the FSH receptor in mice and men results in decreased testis size and germ cell number, but these males are fertile [reviewed in (13)].

Sertoli cells are essential for spermatogenesis; they are in direct contact with germ cells and mediate the biochemical environment of germ cells. In addition, Sertoli cells divide the seminiferous epithelium into basal and adluminal compartments and these compartments each comprise unique microenvironments. Undifferentiated and differentiating spermatogonia are located on the basement membrane and are exposed to factors in the general circulation. In contrast, differentiating spermatocytes and spermatids that are undergoing meiosis and spermiogenesis are on the adluminal side of the blood-testis barrier that is formed by tight junctions between Sertoli cells (indicated by bold arrowheads in Fig. 6). The testis is considered an immune-privileged site because the majority of germ cells are isolated from circulating immune cells. This may be important because autoantigens develop on the surface of differentiating germ cells and may elicit an immune response (orchitis). High levels of Fas ligand, produced by Sertoli cells, may also contribute to the immune privilege of the testis. In addition to maintaining the blood-testis barrier, Sertoli cells must mediate the migration of germ cells from the basal compartment, across the blood-testis barrier to the adluminal compartment. Communication among Sertoli cells and Figure 6. Testis somatic between Sertoli and germ cells is mediated in part by the environment. formation of gap junctions, which probably helps coordinate the cycle of the seminiferous epithelium. Sertoli cells mediate endocrine signals that cannot access the majority of germ cells in the testis. One way that these signals are mediated is through the production of paracrine factors that are essential for spermatogonesis. Two examples are glial cellline derived neurotropnic factor (Gdnf) and Kit ligand (Kitl, also known as Steel factor or stem cell factor). Both factors are produced by Sertoli cells and bind specific receptors on the surface of germ cells (14, 15). Animal models deficient for Kitl or Gdnf are infertile and their testes contain only undifferentiated spermatogonia. In addition, mutations in the KIT/KITL system are associated with human male infertility (16).

Leydig cells Androgen biosynthesis, metabolism and action Testosterone biosythesis is the primary function of Leydig cells. Testosterone is synthesized from cholesterol that is derived from circulating lipoproteins (LDL, HDL), de novo synthesis from acetate or cholesterol esters stored in cellular lipid droplets (Fig. 7). In response to LH-stimulated cyclic AMP, testosterone is synthesized from cholesterol and released into the blood. The rate limiting step of testosterone biosynthesis is the delivery of cholesterol to the inner mitochondrial membrane, where it is converted by the P450 side-chain cleavage (P450scc) enzyme to pregnenolone. The hydrophobic nature

of cholesterol prevents it from easily crossing the aqueous space between the outer and inner mitochondrial membrane. Acute stimulation of testosterone synthesis and secretion is facilitated by LH-simulated production of the steroidogenic acute regulatory (StAR) protein, which transports cholesterol to the inner mitochondrial membrane (Fig. 7). Long-term maintenance of testosterone biosynthesis is also dependent on trophic hormone-induced synthesis of enzymes in the steroidogenic pathway. Leydig cells of the testis produce 95% of circulating testosterone in males. Testosterone concentration in the testis is 100-600 ng/ml, while the concentration in the general circulation is about 4 ng/ml. Only about 1-3% of circulating testosterone is unbound and biologically active. The remainder is bound to sex hormone binding globulin and albumin. Albuminbound testosterone may dissociate readily in capillaries and become biologically active (17). The classical mechanism of androgen action is binding to the intracellular androgen receptor that acts as a Figure 7. Leydig cell testosterone production. ligand-dependent transcription factor. When testosterone binds to the androgen receptor, it causes a conformational change and translocation to the nucleus where it binds to androgen response elements in the promoters of target genes, stimulating expression and consequent changes in cell function. High intratesticular testosterone is required for initiation and maintenance of spermatogenesis and cellular targets of testosterone include Sertoli cells, peritubular myoid cells and some Leydig cells. There is controversy about whether androgen receptors are present on germ cells, but testosterone effects on spermatogenesis are generally considered to be mediated indirectly through Sertoli cells. However, the targets of testosterone action in Sertoli cells are not well understood. In some tissues, testosterone is converted by 5-reductase to the more potent dihydrotestosterone (DHT). During embryonic development, testosterone controls the differentiation of the Wolffian ducts into epididymis, vas deferens and seminal vesicles while DHT is required for the development of external male sex characteristics, including the scrotum, penis and penile urethra. In addition, androgen effects on the prostate and sebaceous glands of the skin require the 5-reduction of testosterone to DHT. Similarly, testosterone effects on bone development and sexual differentiation of the brain require its conversion to estradiol by aromatase (17). Estrogen is also required for spermatogenesis because mutation or deletion of estrogen receptor

(ER) causes infertility in mice and humans (18, 19). Mouse studies suggest that this is a defect of the somatic environment and not germ cells because when ER-/- germ cells were transplanted to the testes of normal mice (with ER+/+ somatic cells), they produced fertilization competent sperm (20). Further analysis of the mouse model indicated that estrogen is required to maintain the architecture of the efferent ducts and for fluid resorption and concentration of sperm in the head of the epididymis (21). Disruption of androgen signaling-male pseudohermaphriditism Male pseudohermaphroditism is diagnosed if the external genitalia are ambiguous or if an individual with known 46XY karyotype has female external genitalia. The most common causes of male pseudohermaphroditism are androgen insensitivity syndrome (also known as testicular feminization, TFM) and 5-reductase deficiency. Androgen insensitivity (AIS) is a collection of genetic disorders/syndromes that all have some abnormality of the androgen receptor (Table 1). Depending on the severity, the mutation may result in complete or partial insensitivity to androgens in 46XY fetuses. Therefore, the resulting phenotypic characteristics range from normal-appearing females (Complete androgen Table 1. Classification of AIS Phenotypes Type External Genitalia Findings insensitivity, Testicular feminization (synonyms) syndrome) to normal appearing men Female (testicular Absent or rudimentary Wolfian duct derivatives CAIS feminization) Absence or presence of epididymides and/or with infertility (mild androgen vas deferens Inguinal or labial testes insensitivity, undervirilized male Short blind-ending vagina Scant or absent pubic and /or axillary hair syndrome) as the only evidence of Gynecomastia in puberty Predominatly female Inguinal or labial testes androgen receptor deficiency (22, (incomplete AIS) Clitoromegaly and labial fusion PAIS Distinct urethral and vaginal openings or a 23). 46XY individuals with complete urogenital sinus Gynecomastia in puberty androgen insensitivity are also Ambiguous Microphallus (< 1cm) with clitoris-like underdeveloped glans; labia majora-like bifid characterized by the absence of scrotum pubic or axillary hair and Descended or undescended testes Perineoscrotal hypospadias or urogenital gynecomastia. Individuals with 5sinus Gynecomastia (development of breasts) in reductase deficiency do not develop puberty Predominately male Simple (glandular or penile) or severe gynecomastia and usually undergo (perineal) isolated hypospadias with a normal-sized penis and descended testes or some masculinization at puberty severe hypospadias with micropenis, bifid scrotum, and either descended testes when testosterone levels increase Gynecomastia in puberty Male (underivirilized Impaired spermatogenesis and/ or impaired MAIS (also known as penis at 12 male syndrome) pubertal virilization Gynecomastia in puberty syndrome) (23).

Transport and Delivery of Spermatozoa Sertoli cells secrete fluid into the seminiferous tubule lumen that carries the released spermatozoa. Included in this fluid are nutrients for the sperm and sex hormone binding protein (SHBP) that carries testosterone. Both ends of the seminiferous tubules open into a branched resevoir called the rete testis. From the rete testis the sperm travel via the efferent ducts into the epididymis. The epididymis contains a single highly convoluted duct closely adjacent to the testis. Spermatozoa are concentrated 100-fold in the epididymis by reabsorption of fluid. Sperm mature during the 15 day transit from the caput (head) to the cauda (tail) epididymis and acquire forward motility.

Figure 8. Sperm transport and accessory sex glands.

From the epididymis the sperm are moved as a very dense mass (too dense to swim) to the vas deferens by the muscular activity of the epididymis and vas deferens. The vas deferens is approximately 25 cm long in humans. In the absence of ejaculation spermatozoa move through the terminal ampulla into the urethra and are removed with the urine. Sperm that are carried to the female reproductive tract do so in seminal fluid that is produced by the accessory sex glands (seminal vesicles, prostate, ampulla, and Cowpers gland), which secrete the constituents of seminal fluid. Seminal fluid contains nutritional factors (fructose, sorbitol), buffering agents to protect against the acid pH of vaginal fluids and reducing agents to protect from oxidation (Fig. 8).

The seminal vesicles are paired pouches located directly on the posterior side of the bladder in humans and produce 60% of seminal fluid. Their secretions are particularly rich in fructose, which is an essential nutrient for spermatozoa. They also produce a substance that causes the semen to clot after ejaculation, thought useful for keeping the semen in the cervix. The prostate lies immediately below the base of the bladder surrounding the proximal portion of the urethra and produces 30% of the seminal fluid. Prostrate secretions contain several chemicals, including prostate specific antigen (PSA), a coagulase enzyme that liquefies clotted semen in the female reproductive tract, releasing the sperm to fertilize the egg. Men with prostate cancer can have high levels of PSA in their general circulation. The seminal vesicles join the ampulla to form the beginning of the ejaculatory duct. The Cowpers gland is a paired pea-sized tubular gland located directly below the prostate and produce secretions that lubricate the reproductive tract, but may also contain anti-sperm antibodies that can affect motility and prevent fertilization (Fig. 8). Transmission of sperm Erection results from increased blood flow to the penis and reduced blood outflow from the organ (Fig. 9). These alterations in blood flow are caused by the release of nitric oxide from parasympathetic nerve endings in the

Figure 9. Erectile function and dysfunction

walls of precapillary arterioles and sinusoids of the corpus cavernosum. The released nitric oxide activates guanylate cyclase, which converts GTP to cGMP, causing relaxation and dilation of arterial smooth muscle and increased blood flow. The expanded corpus cavernosum presses against the inflexible tunica albuginea and reduce the outflow of venous blood causing an increase in blood pressure in the corpus cavernosum and penile erection. The cGFP second messenger is degraded by the action of the phosphodiesterase, PDE5. The erectile dysfunction drugs, Sildenafil (Viagra), tadalafil (Cialis) and vardenafil (Levitra) are competitive inhibitors of PDE5 and thus, prevent the degradation of the cGFP signal.

Sperm Structure and Function Unlike other mammalian cells, sperm must leave the body in order to carry out their physiological role of fertilizing the egg and form the zygote. Sperm have a unique morphology that is optimized to 1) carry and protect the haploid genome and 2) transport that genome through the male and female reproductive tracts and 3) Figure 10. Sperm structure fertilize the oocyte. The sperm head carries a haploid genome (one-half of the genetic DNA complement) that is highly condensed and transcriptionally inactive. The acrosomal compartment is located on the anterior aspect of the sperm head and is filled with hydrolytic enzymes (primarily hyaluronidase and acrosin). The mid-piece contains the mitochondrial sheath and is the powerplant for tail locomotion (Fig. 10). Ejaculated sperm are not able to fertilize the egg immediately. They must first be activated in the female reproductive tract by a process called capacitation. Capacitation destabilizes the sperm head membrane, which prepares it for the acrosome reaction (see below) and makes it more fusogenic. The destabilized membrane of capacitated sperm has increased fluidity and permeability to Ca2+, which activates cAMP and causes hyperactive motility. Fertilization occurs in the fallopian tube (oviduct) of the female reproductive tract (Fig. 11). The oocyte is surrounded by an extracellular matrix called the zona pellucida (yellow vitelline layer in Fig. 11), which is composed of three major glycoproteins (ZP1, ZP2 and ZP3). ZP3 is thought to be important for sperm binding and also cause the acrosome reaction. The acrosome releases enzymes that drill through the zona pellucida and this process is aided by the driving force of the sperm tail. Sperm that lose their

Figure 11. Fertilization

acrosomes before encountering the oocyte are unable to bind the zona pellucida and fertilize. Once the sperm penetrates through the zona pellucida, it fuses with the plasma membrane of the oocyte. This binding activates the egg to continue meiosis and also stimulates release of cortical granule contents from the egg into the zona pellucida. Proteases from the cortical granules alter the structure of the zona pellucida (including destruction of ZP3), block further penetration by runner-up sperm and prevent polyspermy.

Male Infertility Infertility affects 10-15% of couples in the Unites States and is defined as the inability to conceive after 12 months of unprotected intercourse. In about 40% of cases, the cause of infertility is attributed to a male factor. Male infertility is diagnosed by analysis of two semen samples (separated by several weeks) following 2-5 days of abstinence. Normal sperm count parameters are defined by the World Health Organization (WHO) as >20 million sperm/ml of ejaculate (ejaculate volume 2-5 ml) with 75% viability, 50% forward mobility and at least 30% normal shape and form. Men with less than 20 million sperm per milliliter of ejaculate are oligospermic and those with no sperm in the ejaculate are azoospermic. The etiology of azoospermia can be subdivided into obstructive (plumbing problem) and nonobstructive (e.g., spermatogenic defects). Approximately 50% of male infertility has no known cause (idiopathic) and there are no treatments to improve the defect in 75% of cases. Causes of male factor infertility include, varicocele, infectious diseases or inflammatory conditions (eg., mumps), endocrine disorders (e.g., Kallmanns syndrome), immunological disorders, environmental factors and genetic diseases. Several genetic causes of male infertility are listed in Table 2.
Table 2. Some genetic causes of male factor infertility* Cystic Fibrosis: 6-10% of men with obstructive Sex reversal syndrome: XX male resulting in azoopermia have congenital bilateral absence of the vas azoospermia and other characteristics deferens. 70% of these may carry the cystic fibrosis mutation. Noonan Syndrome: Occurs in males and females. Male pseudohermaphroditism: XY male with androgen Causes abnormal gonadal function in males. insensitivity. Myotonic dystrophy: Multisystem inherited disease that Klinefelters Syndrome: XXY males often do not produce sperm or produce very low quantities of sperm. can cause underdeveloped testes and abnormal sperm production. Deletions in the Y Chromosome: 3-30% of infertile Hemachromatosis: Condition affecting iron storage. men have deletions in the Y chromosome. 80% of men with the condition have testicular dysfunction. *50% of male infertility is idiopathic in nature and most of these are likely due to undiscovered genetic defects.

It is important to note that infertility does not mean unable to conceive. Dramatic advances in assisted reproductive technologies (ART) make it possible for infertile men to conceive, even with poor quality, immotile sperm. Oligospermia can be overcome with intrauterine insemination (IUI). Ideally, 10-20 million sperm are needed for IUI, but this method can be successful even with 5-10 million sperm (24). Severe male factor infertility can be managed with in vitro fertilization (IVF), which requires 50,000-100,000 sperm per oocyte (Speroff, 7th edition). IVF in combination with intracytoplasmic sperm injection (ICSI) requires only one sperm per oocyte. Even poor quality, immotile sperm

can fertilize an egg when facilitated by ICSI (25). Sperm does not have to traverse the male reproductive tract to fertilize an egg. Testicular sperm can be obtained by biopsy or testicular sperm extraction (TESE). Even azoospermic men with a Sertoli cell only pattern, can have focal areas of seminiferous tubule that contain sperm. TESE followed by IVF with ICSI enable these infertile men to father normal children with pregnancy rates greater than 20% (26).

Reference List 1. J. Ehmcke, J. Wistuba, S. Schlatt, Hum Reprod Update 12, 275 (2006). 2. J. Toppari, M. Kaleva, H. E. Virtanen, K. M. Main, N. E. Skakkebaek, Molecular and Cellular Endocrinology 269, 34 (2007). 3. K. A. Boisen et al., The Lancet 363, 1264 (2004). 4. I. M. McAleer, G. W. Kaplan, in Encyclopedia of Reproduction, E. Knobil, J. D. Neill, Eds. (Academic Press, San Diego, 1999), vol. 1, pp. 784-791. 5. J. F. Redman, in Encyclopedia of Reproduction, E. Knobil, J. D. Neill, Eds. (Academic Press, San Diego, 1999), vol. 3, pp. 30-41. 6. L. D. Russell, R. A. Ettlin, A. P. SinhaHikim, E. D. Clegg, Histological and histopathological evaluation of the testis (Cache River Press, Clearwater, FL, ed. 1st, 1990), pp. 1-286. 7. K. A. Thayer et al., Hum. Reprod. 16, 988 (2001). 8. G. Gupta, J. P. Maikhuri, B. S. Setty, J. D. Dhar, J Med. Primatol. 29, 411 (2000). 9. L. Johnson, C. S. Petty, W. B. Neaves, Biol Reprod 29, 207 (1983). 10. R. A. Hess, in Encyclopedia of Reproduction, E. Knobil, J. D. Neill, Eds. (Academic Press, San Diego, 1999), vol. 4, pp. 539-545. 11. L. Johnson, T. A. McGowen, G. E. Keillor, in Encyclopedia of Reproduction, E. Knobil, J. D. Neill, Eds. (Academic Press, San Diego, 1999), vol. 4, pp. 769-784. 12. L. M. Halvorson, in Encyclopedia of Reproduction, E. Knobil, J. D. Neill, Eds. (Academic Press, San Diego, 2008), vol. 2, pp. 921-924. 13. M. D. Griswold, in Encyclopedia of Reproduction, E. Knobil, J. D. Neill, Eds. (Academic Press, San Diego, 1999), vol. 4, pp. 212-215. 14. The coat colors of mice (Springer-Verlag, New York, ed. 1st, 1979), pp. 1-379. 15. X. Meng et al., Science 287, 1489 (2000). 16. J. J. Galan et al., Hum. Reprod. 21, 3185 (2006). 17. S. Bhasin, in Encyclopedia of Reproduction, E. Knobil, J. D. Neill, Eds. (Academic Press, San Diego, 1999), vol. 1, pp. 197-206. 18. E. M. Eddy et al., Endocrinology 137, 4796 (1996). 19. Y. Suzuki et al., Fertility and Sterility 78, 1341 (2002). 20. D. Mahato, E. H. Goulding, K. S. Korach, E. M. Eddy, Mol. Cell Endocrinol. 178, 57 (2001). 21. Q. Zhou et al., PNAS 98, 14132 (2001). 22. G. H. G. Sinnecker, O. Hiort, E. M. Nitsche, P. M. Holterhus, K. Kruse, European Journal of Pediatrics 156, 7 (1996). 23. J. Aiman, in Encyclopedia of Reproduction, E. Knobil, J. D. Neill, Eds. (Academic Press, San Diego, 2008), vol. 1, pp. 174-179.

24. B. J. Van Voorhis et al., Fertility and Sterility 75, 661 (2001). 25. L. van der Westerlaken, N. Naaktgeboren, H. Verburg, S. Dieben, F. M. Helmerhorst, Fertility and Sterility 85, 395 (2006). 26. J. D. Raman, P. N. Schlegel, J Urol. 170, 1287 (2003).

PHARMACOLOGY OF ANDROGENS AND ANTI-ANDROGENS Don DeFranco, Ph.D. Wednesday, February 11, 2009 3:30 p.m.

I. LEARNING OBJECTIVES 1. To understand the mechanism of action of androgens and anti-androgens 2. To understand the basis for clinical use of androgens and anti-androgens 3. To understanding the pharmacological parameters of clinically used androgens and anti-androgens 4. To understand the sides effects of androgen and anti-androgen treatment II. OUTLINE A. B. C. D. E. F. G. H. I. Androgen Synthesis Androgen Receptor Structure, Function & Mechanism of Action Clinical Use of Androgens in Men: Prepubertal, Postpubertal Other Uses of Androgens (non-reproductive) Clinical Use of Androgens in Women Therapeutic Androgen Preparations Side Effects of Androgen Therapy Inhibitors of Androgen Action: General Uses Inhibitors of Androgen Action: Treatment of Prostate Cancer

RECOMMENDED READINGS 1. Goodman & Gilmans The Pharmacological Basis of Therapeutics, 11th Edition, (Brunton, Lazo & Porter, Eds.), McGraw Hill, 2006. Chapter 58, Androgens, pp 1573-1586. 2. Williams Textbook of Endocrinology, 10th Edition (Larsen et al., Eds.), Saunders, 2003. Chapter 4, Mechanism of Action of Hormones that Act on Nuclear Receptors, pages 35-44. 3. Modern Pharmacology with Clinical Applications, 5th Edition (Craig & Stitzel, Eds.), Little, Brown & Co., 1997. Chapter 65, Androgens and Anabolic Steroids, pages 757-768. 4. Human Pharmacology: Molecular to Clinical, 3rd Edition (Brody, Larner & Minneman, Eds.), Mosby, 1998. Chapter 39, Androgens and Antiandrogens, pages 519-532. 5. Rhoden EL & Morgentaler A (2004). Risks of testosterone-replacement therapy and recommendations for monitoring. New Engl. J. Medicine 350, pp. 482-492.

A. Androgen Synthesis Like all steroid hormones, testosterone is synthesized from cholesterol. Leydig cells in the testes are the major sites of testosterone synthesis and also secret androgen precursors such as androsterone, androstenedione in small amounts (i.e. ~ 1 % of testosterone). In some cells, Testosterone is converted to the more potent androgen, dihydrotestosterone (DHT), by the enzyme 5Reductase. Testosterone and some of its metabolites are also synthesized in the adrenal cortex and ovary. The adrenal cortex secretes much more dehydroepiandrosterone (DHEA) (4 mg/day) and dehydroepiandrosterone Sulfate (DHEAS) (7-15 mg/day) than testosterone (0.05 mg/day). Adrenal androgens are responsible for ~50% of the circulating androgens in premenopausal women but of little consequence in normal males. The excess secretion of adrenal androgens in congenital adrenal hyperplasia (CAH) contributes to disease pathophysiology. IMPORTANT CONCEPT 1: The testes are the major sites of androgen synthesis although androgens produced in the adrenal cortex can be relevant under both normal physiological conditions (e.g. in females) and in some disease states (e.g. in CAH). B. Androgen Receptor The receptor for androgens is a member of a large superfamily of nuclear receptors that have a common structural organization. The androgen receptor (AR) is present in most tissues but in highest amounts in accessory organs of the male reproductive track and some areas of the brain. These receptors contain a centrally localized, highly related DNA-binding domain, a less conserved carboxyl-terminal ligand binding domain and a divergent amino terminal domain that participates in transcriptional activation. There is only one AR in the human genome, which is localized on the X-chromosome. Androgen binding triggers the nuclear accumulation of AR and its regulation of the transcription of specific target genes. Androgen regulation of gene transcription most often requires the specific binding of AR to select DNA sequences (termed androgen response elements or AREs) contained usually within the promoter region of target genes. A number of other proteins (e.g. transcriptional coactivators) that either directly or indirectly interact with AR or AR-regulated genes are involved in androgen regulated gene transcription. Many mutations have been identified in the AR gene in patients with complete or partial androgen-insensitivity syndrome. These mutations can occur within the DNA-binding domain, the ligand-binding domain or the amino-terminal domain and all primarily affect the effectiveness of AR as a transcriptional regulator.

IMPORTANT CONCEPT 2: Androgens act in target tissue predominantly to activate the nuclear AR, which affects the transcriptional activity of select target genes in cooperation with other proteins recruited directly or indirectly to the androgen-regulated gene promoter. C. Clinical Use of Androgens in Males Prenatal hypogonadism results in a deficiency in development of male sex accessory organs. This can result from deficient testicular steroidogenesis, 5reductase deficiency, AR defects, deficient pituitary gonadotropin secretion and deficient hypothalamic GnRH secretion. As long as end point organs contain functional AR, androgen replacement therapy can be an effective treatment. Although controversial, androgen replacement is also used to treat boys with delayed puberty (e.g. Constitutive (Idiopathic) Delay of Growth and Puberty or CDGP). The clinical features of CDPG are short stature (2-3 standard deviations below the mean), delayed bone age (i.e. 2-4 years behind chronological age) and delayed pubertal development (typically assessed by testicular volume) in the absence of other medical or endocrinological abnormalities. Children with CDGP will attain adult height (i.e. low normal range) and complete pubertal maturation at significantly later ages (i.e. late teens, early 20s). While CDGP does not typically require treatment, monthly injections of a long acting androgen for one to two year periods can hasten pubertal growth due in part to increased production of growth hormone. However the effectiveness of androgens in promoting growth in children is counterbalanced by the acceleration of epiphyseal closure, which can result in reduced height. Postpubertal hypogonadism can result from testicular destruction (i.e. in hypergonadotropic hypogonadism) or from hypopituitarism (i.e. in hypogonadotropic secondary hypogonadism). Androgen replacement can be used to restore secondary sex characteristics, libido and fertility. D. Other Uses of Androgens (non-reproductive) Androgens are used for some anemias (e.g. aplastic or Fanconi anemia, which can lead to severe bone marrow failure) since they stimulate erythropoesis by increasing renal erythropoietin production. This effect may not be due to classical AR action since 5-androgens, which have a low affinity for AR, are more effective than higher affinity 5-androgens.This treatment may be effective for many years, but most patients eventually fail to respond. Nandrolone (an anabolic steroid) is used to manage anemia associated with chronic renal failure. Androgens have also been to treat diseases with associated muscle wasting (e.g. cancer, HIV-AIDS). IMPORTANT CONCEPT 3: Androgens can be used in males both prior to and following puberty to correct for growth defects or development of

secondary sex characteristics that result from hypogonadism. In addition, muscle-wasting associated with various diseases or aggressive chemotherapy can be reversed to some extent by anabolic effects of androgens. E. Clinical Use of Androgens in Women The use of androgens in women is very limited given their virilizing effects. In females with hypopituitarism, androgen (i.e. typically methyltestosterone) in combination with estrogen replacement is used to facilitate long bone growth and development of axillary and pubic hair. However, an Endocrine Society Task Force headed by Dr. Maggie Wierman, made the following recommendations regarding androgen treatment of women (see Wierman et al., (2006) J. Clin. Endo. Metab. Vol. 91, pp 3697-3710). Although there is evidence for short-term efficacy of testosterone in selected populations, such as surgically menopausal women, we recommend against the generalized use of testosterone by women because the indications are inadequate and evidence of safety in long-term studies is lacking. IMPORTANT CONCEPT 4: Androgen treatment of sexual dysfunction in postmenopausal women remains controversial due to uncertainty regarding potential side effects of long-term use. F. Therapeutic Preparations of Androgens The most effective compounds used to bring about masculinization are the longlasting enanthate, cypionate or propionate esters of testosterone. These esters are more lipidophilic than testosterone and are hydrolyzed in vivo. They are typically delivered (dissolved in oil) intramuscularly every two weeks. Deep IM delivery slows hydrolysis of the ester group. Poorer therapeutic outcomes and wider fluctuations in serum testosterone results if high doses are given less frequently. Treatment is continued for 2-3 years or until adequate masculinization has occurred. Lower maintenance levels of these drugs can then be used. Oral preparations of alkylated testosterone such as methyltestosterone or fluoxymesterone are effective for treatment of postpubertal hypogonadism, since they are relatively short acting and can be taken daily. The alkylated derivatives of testosterone have reduced hepatic metabolism when administered orally but they are less androgenic than testosterone and can cause hepatotoxicty. Oral methyltestosterone use is less popular since it is not easily detectable by immunoassays and therefore difficult to monitor. IMPORTANT CONCECPT 5: Testosterone derivatives have been developed that are therapeutically useful given their reduced metabolism in liver (or muscle) or increased lipid solubility.

Transdermal delivery systems for androgens have become popular in recent years. Chemicals called excipients are used to facilitate the absorption of testosterone across the skin in a controlled manner. The use of testosterone patches such as androderm or gels such as androgel provides the most effective means to generate stable serum testosterone levels. Transdermal methods can result in daily delivery of 5mg/day of testosterone. Testosterone gels are often preferred since patches can cause rashes and are visible. IMPORTANT CONCEPT 6: Transdermal delivery of testosterone is the most effective means for attaining stable circulating hormone levels. Typically, anabolic steroids are used to promote linear growth in children. These androgens (e.g. 19-nortestosterone, oxandrolone, oxymetholone, and stanozolol) have the greatest ratio of protein anabolic effects (e.g. increase in muscle mass) to virilizing effects. Newer versions of anabolic steroids (e.g. tetrahydrogestrinone [THG] or The Clear) are metabolized quickly and difficult to detect. Androstenedione (Andro) is a dietary supplement marketed for its anabolic androgenic activity. Andro is a weak androgen but is converted to testosterone by the enzyme 17-dehydrogenase, which is present in most tissues. Andro can also be aromatized to estrogen. IMPORTANT CONCEPT 7: Anabolic steroids have gained popularity due to their enhanced anabolic effects (i.e. increased muscle mass) and limited virilizing effects. Based upon the flexibility of the ligand-binding domain of AR, recent efforts have been made to develop new ligands that induce different conformational changes within the receptor. Theoretically, ligand-bound AR with distinct conformations may have selective action within individual tissue or cell types. Drugs of this type called Selective Androgen Receptor Modulators (SARMs) would be expected to activate the AR in some but not all tissues. A number of SARMs have been found in rodent models to bind AR with affinities comparable to testosterone, exhibit full agonist activity in levator ani muscle but only partial agonist activity in prostate and seminal vesicles. They also have exhibited minimal effects on pituitary gonadotropin secretion. The most effective SARMs to date in rodent models are modified forms of the non-steroidal anti-androgen bicalutamide. Phase II clinical trials are currently underway (Merck) for the use of a SARM in cancer patients to reduce muscle loss. IMPORTANT CONCEPT 8: SARMs exert androgenic effects in select tissues due to their ability to induce distinct conformation changes in AR. G. Side Effects of Androgen Therapy In sexually mature males, administration of androgens inhibits secretion of pituitary gonadotropins (FSH, LH) due to feedback inhibition. This inhibits the

endogenous testicular production of testosterone and may reduce spermatogenesis and fertility. High doses of androgens can also cause erythrocytosis. Androgens that can be aromatized to estrogen can also, when given in high doses, cause gynecomastia (benign enlargement of the male breast resulting from a proliferation of the glandular component of the breast). Other side effects of excess androgen include polycythemia (excess production of red blood cells) and acne. Androgens are also likely to increase the risk of benign prostate hyperplasia or prostate cancer. In women and children, androgens can lead to virilization (including facial hair and body hirsutism). Children who have not yet had epiphysial closure can exhibit premature closure and stunted growth if treated with androgens. One side effect that may occur with the abuse of anabolic steroids is the imbalance in blood lipids. For example, excess Andro use has been found to reduce HDL-cholesterol and increase LDL-cholesterol (probably due to an increase in hepatic triglycerol lipase activity). This may increase the risk of atherosclerosis. Some recent studies also suggest that long-term abuse of anabolic steroids may increase left ventricular mass and contribute to dilated cardiomyopathy. Finally, some researchers suggest that anabolic steroid abuse may lead to increased aggressiveness and the potential for violent behavior. IMPORTANT CONCEPT 9: Side effects of excess androgens can result from loss of gonadotropin feedback inhibition, aromatization to estrogen or increased action of AR in target tissue. H. Inhibitors of Androgen Action: General Uses Inhibition of androgen action is used to treat female hirsutism, alopecia, precocious puberty in boys and prostate cancer. Three different types of antiandrogen treatment are used. Both agonists and antagonists of the GnRH receptor are used to block testosterone secretion. Superactive GnRH analogs (Lupron, Zoladex) lead to downregulation of the GnRH receptor while GnRH antagonists (e.g. Plenaxis, Abarelix) block the action of the GnRH receptor. GnRH analogs may be somewhat slower in their ability to reduce testosterone secretion and initially cause a surge in testosterone secretion. Long acting GnRH analogs (e.g. triptorelin) have also been considered for treatment of men with paraphilia (tendency for deviant sexual behavior). 5- reductase inhibitors (e.g. Finasteride [Proscar, Propecia] or dutasteride [Avodart]) block the conversion of testosterone to dihydrotestosterone. These drugs are used to treat benign prostate hyperplasia or male patterned baldness (Propecia). Impotence is a rare side effect of these drugs via some unknown mechanism.

IMPORTANT CONCEPT 10: Androgen action can be inhibited by disruptions in GnRH action in the pituitary or by reduced conversion of testosterone to dihydrotestosterone. I. Inhibitors of Androgen Action: Prostate Cancer AR antagonists (both steroidal and nonsteroidal) have been developed, particularly for treatment of prostate cancer. Most often AR antagonists are given with GnRH agonists for Combined Androgen Blockade (CAB). Flutamide (Eulexin) was the first non-steroidal AR antagonist used for prostate cancer therapy but has increased incidence of hepatotoxicity and gynecomastia. The effectiveness of Bicalutamide (Casodex) for prostate cancer treatment has been extensively studied. Bicalutamide is preferred over Flutamide due to its reduced hepatotoxicity. In addition, bicalutamide has a longer elimination half-life (6 days) as opposed to flutamide (6 hours) and can be given once daily. IMPORTANT CONCEPT 11: AR antagonists, typically in combination with blockers of testosterone synthesis, are most often used for the treatment of prostate cancer. Androgen deprivation therapy rarely cures prostate cancer due to the development of hormone-independent or hormone refractory tumors. A number of mechanisms have been postulated to account for hormone-refractory prostate tumors, which most often possess AR and may be AR-dependent. Many proposed mechanisms hypothesize that AR in hormone refractory tumors is hypersensitive. This hypersensitivity of AR may result from increased expression of AR protein, mutations within the AR that increase its sensitivity to low levels of circulating androgens or make it promiscuous and responsive to other steroids, ligand-independent activation of the AR by other signaling pathways perhaps activated by growth factors, and alterations in the levels, structure or activity of AR interacting proteins (e.g. coactivators). IMPORTANT CONCEPT 12: The effectiveness of androgen ablation therapy for the treatment of prostate cancer is limited by the eventual development of hormone-refractory tumors.

Pharmacology: Drugs affecting sexual function

Ferruccio Galbiati, Ph.D. (feg5@pitt.edu) Thursday, February 12, 2009 9:00 am Learning Objectives I. To understand male sexual dysfunction and relative treatment. II. To understand female sexual dysfunction and relative treatment. III. To understand how medications may interfere with sexual function/desire.

I. To understand male sexual dysfunction and relative treatment. Male sexual function is a complex, multicomponent biological process guided by central mechanisms for regulation of libido and arousability in addition to local mechanisms that regulate penile erection, rigidity, orgasm, and ejaculation. Male sexual dysfunction includes (i) libidinal, (ii) ejaculatory, and (iii) erectile dysfunction. Libidinal dysfunction Inhibited desire or loss of libido: decreased interest in sexual activity.

CAUSES: a) b) c) d) Psychological problems such as anxiety and depression. Physical factors such as low levels of testosterone. Illnesses such as diabetes and high blood pressure. Medications. Ejaculatory dysfunction Premature ejaculation: before or soon after penetration. Inhibited or retarded ejaculation: slow to occur. Retrograde ejaculation: the ejaculate is forced back into the bladder rather than through the urethra and out the end of the penis.

CAUSES: a) Lack of attraction for a partner and past traumatic events (premature and inhibited ejaculation). b) Nervousness over how well he will perform during sex (premature ejaculation). c) Nerve damage to the spinal cord or back. d) Medications. Erectile dysfunction Inability of the male to attain or maintain a penile erection sufficient for satisfactory sexual intercourse.

The prevalence of ED increases with age: National Health and Social Life Survey (NHSLS): Age 18-29 30-39 40-45 50-59 % w/ ED 7 9 11 18

Massachusetts Male Aging Study (MMAS) Age 40-70 CAUSES: a) b) c) d) e) f) g) Hypertension. Diabetes. Cardiovascular disease. End-stage renal disease. Nerve disorder. Psychological factors (stress, depression, performance anxiety). Medications. % w/ ED 52

Libido appears to be testosterone-dependent while penile erection androgenindependent. However, there are evidences suggesting that testosterone is a regulator of nitric oxide synthase (NOS) activity in the cavernosal smooth muscle cells. Thus, it is possible that physiological levels of testosterone might be necessary to achieve optimal penile rigidity.

ED and testosterone. Of all men with ED, only 8% to 10% have low testosterone levels. The prevalence of low testosterone levels is not statistically different in men with ED and in age-matched population. Thus, it appears that ED and androgen deficiency are two common but independently distributed disorders. Testosterone administration in men with ED and normal testosterone levels does not to improve sexual function. Thus, indiscriminate administration of testosterone in all older men with ED is not recommended. However, it remains important to rule out androgen deficiency in ED patients. In fact, it may be a manifestation of an underlying disease such as a pituitary tumor. In addition, if left untreated, androgen deficiency may have negative effects on bone, muscle, energy level, and sense of well-being. Some patients with ED and low testosterone levels respond to testosterone replacement, as demonstrated by increased libido and overall sexual activity. However, the response to testosterone replacement is variable, probably because of the coexistence of other disorders (diabetes mellitus, hypertension, medications, peripheral vascular disease, psychogenic factors, and end-stage renal disease). In fact, in middle-aged and older men, ED is often a multifactorial disorder. Thus, testosterone replacement alone may be insufficient for the treatment of sexual dysfunction. MECHANISMS OF PENILE ERECTION: Normal penile erection requires coordinated involvement of: 1) Intact central and peripheral nervous system. 2) Intact corpora cavernosa and spongiosa. 3) Normal arterial blood supply and venous drainage. The erectile state of the penis is determined by the tone of the corporal smooth muscle cells. When the cavernosal smooth muscle cells are relaxed, the tone is low and the penis is engorged with blood and erect. When the cavernosal smooth muscle tone is high, the penis is flaccid. The smooth muscle tone in the corpora cavernosa is maintained by agonist-stimulated release of intracellular calcium into the cytoplasm and influx of calcium through voltagedependent membrane channels. An increase in intracellular calcium activates myosin light chain kinase which phosphorylates myosin light chain. As a consequence, actin interacts with myosin and muscle contracts. Calcium influx in the cavernosal smooth muscle cells is regulated by: 1) potassium ion (K+) flux through potassium channels. 2) connexin 43-derived gap junctions. 3) norepinephrine.

4) prostaglandin E1 (PGE1). 5) nitric oxide (NO). K+ flux and connexin 43-derived gap junctions Movement of K+ across the membrane (through at least four subtypes of potassium channels) determines the membrane potential of the cavernosal smooth muscle cells. Opening of K+ channels results in exit of the ion from the cell and hyperpolarization of the plasma membrane with the consequent reduction of calcium influx due to the inhibition of voltage-dependent calcium channels. Because adjacent smooth muscle cells are interconnected through connexin 43-derived gap junctions, changes in K+ channel activity in one cell affect the membrane potential of adjacent cells. As a consequence, there is rapid transmission of electrical and biochemical signaling among smooth muscle cells. Norepinephrin Norepinephrin stimuates the production of diacylglycerol and inositol triphosphate (IP3) through the binding to adrenergic receptors. Diacylglycerol activates protein kinase C (PKC), which inhibits K+ channels, whereas IP3 increases intracellular calcium and calcium influx through the membrane. The net increase in intracellular calcium promotes smooth muscle contraction. Prostaglandin E1 Through activation of its receptor, PGE1 promotes the production of cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA). Activated PKA promotes potassium efflux from the cells by stimulating K+ channels. In addition, PKA negatively regulates calcium channels. The net decrease in intracellular calcium promotes smooth muscle relaxation. Nitric oxide Nitric oxide is derived from the nerve terminals innervating the corpora cavernosa (noradrenergic, noncholinergic neurons), the endothelial lining of penile arteries and cavernosal sinues. NO stimulates the synthesis of intracellular cyclic guanosine monophosphate (cGMP) thorough activation of guanyl cyclase. cGMP promotes cavernosal smooth muscle relaxation and arterial dilatation by inhibiting calcium channels and stimulating K+ efflux. Cyclic nucleotide phosphodiesterases are a class of enzymes that degrades cGMP into an inactive form, 5 GMP. Among at least 11 different isoforms widely distributed throughout the body, phosphodiesterase type 5 (PDE5) is the predominant isoform expressed in the cavernosal smooth muscle cells. Hydrolysis of cGMP to 5GMP by this enzyme reduces the intracellular levels of cGMP with the consequent reversal of the smooth muscle relaxation and penile erection.

TREATMENT: The selection of the therapeutic approach is based on the underlying etiology, the patients preference, the presence or absence of underlying diseases. A stepwise approach is commonly used for the treatment of ED: 1. Patient and sexual partner can benefit from psychosexual counseling. 2. First-line therapies: a) Sildenafil citrate b) Vacuum constriction devices 3. Second-line therapies: a) Intracavernosal injection of alprostadil b) Intracavernosal injection of other vasoactive amines 4. Third-line therapies: a) Penile prosthesis b) Vascular surgery Sildenafil Sildenafil (Viagra) was the first effective oral agent for the treatment of ED. It was introduced into the US in 1998. Its a selective TYPE 5 PDE INHIBITOR. Mechanism of action: Sildenafil inhibits the hydrolysis of cGMP. Its action requires an intact NO response as well as constitutive synthesis of cGMP by the smooth muscle cells of the corpora cavernosa. By inhibiting cGMP catabolism in the cavernosal smooth muscle cells, sildenafil restores the natural erectile response to sexual stimulation. It does not produce erection in the absence of sexual stimulation. Therefore, both normal NO pathway and sexual stimulation are necessary for sildenafil to work. Efficacy: The efficacy of sildenafil was demonstrated in independent randomized clinical trials. In general, baseline sexual function correlated positively with response to sildenafil. Since there is no baseline characteristic that predicts the likelihood of failure to respond to sildenafil therapy, a therapeutic trial is indicated for all patients except for those in whom it is contraindicated Adverse effects:

Adverse effects of sildenafil reported in clinical trials include headache, flushing, dyspepsia, respiratory tract disorders, and visual disturbance. Sildenafil does not affect semen characteristics. Sildenafil and coronary artery disease: Hypertension or symptomatic coronary artery disease need to be addressed first. Sildenafil is CONTROINDICATED in patients taking nitrates; sildenafil should NOT be used within 24 hours of the use of nitrates because the vasodilator effects of nitrates are augmented by sildenafil. Concomitant administration of the two drugs can cause a potentially fetal decrease in blood pressure. In patients with congestive heart failure, patients receiving vasodilator drugs/antihypertensive drugs, blood pressure should be monitored after initial administration of sildenafil. Drug-drug interactions: Sildenafil is metabolized mainly by the P450 2C9 and P450 3A4 pathways. Inhibitors of P450 3A4 (i.e. cimetidine and erythromycin) increase the plasma concentrations of sildenafil. Protease inhibitors can also affect the activity of the P450 3A4 pathway and alter the clearance of the drug. Sildenafil is an inhibitor of the P450 2C9 pathway. Thus, it can affect the metabolism of drugs metabolized by this pathway, such as warfarin and tolbutamide. The most serious drug-drug interaction occurs with nitrates. Therapeutic regimens: Sildenafil can be started at an initial dose of 50 mg (25mg in patients with significant coronary artery disease). Move to 100mg if no adverse effects are reported. Therapeutic response and adverse effects should guide further dose adjustments. Sildenafil should be taken at least 1 hour prior sexual intercourse and not more than once every 24 hours. Alternatives to sildenafil: In addition to sildenafil (Viagra), Vardenafil hydrochloride (Levitra), and Tadalafil (Cialis) are the only two oral drugs approved by the FDA to treat erectile dysfunction. They increase the flow of blood into the penis by inhibiting type 5 PDE. They all have similar side effects (back pain specific for cialis). 1) Vardenafil hydrochloride (Levitra). Viagra and Levitra are very similar in the rate of uptake, in that the majority of men will have the effect of the medication between 30 and 60 minutes and the medication is metabolized after 8 hours. 2) Tadalafil (Cialis). Cialis is slightly slower in uptake, approximately 60 minutes, but has a duration lasting 36 hours. The choice of which medication to take depends on how the man and his partner typically engage in sexual activity. Some men prefer having a medication that works on

demand and is out of their bodies within 8 hours. Some men enjoy the spontaneity allowed by a medication with a long half-life and they do not need to time when the medication is taken relative to sexual activity.

II. To understand female sexual dysfunction and relative treatment. The female sexual response consists of three successive phases: desire, arousal, and orgasm. Female sexual arousal results in a combination of vasocongestive and neuromuscular events, which include increased clitorial, labial, and vaginal wall engorgement with blood as well as increased vaginal and clitoral length and diameter and lubrication. Female sexual dysfunction is a problem that adversely affects physical health as well as emotional well-being. Female sexual dysfunction can be classified into several categories: (i) Hypoactive sexual desire disorder, (ii) Sexual aversion disorder, (iii) Sexual arousal disorder, (iv) Orgasmic disorder, (v) Sexual pain disorders (a. dyspareunia; b. vaginismus; and c. other sexual pain disorders). Hypoactive sexual desire disorder Persistent or recurring deficiency of sexual fantasies or thoughts and/or desire for sexual activities that causes personal distress.

CAUSES: a) Psychological/emotional problems such as depression, stress, anxiety, and fatigue. b) Physiological problems such as hormone deficiency (natural/induced menopause, endocrine disorders). c) Medical or surgical intervention (for example cancer and chemotherapy). Sexual aversion disorder Persistent or recurrent phobic aversion to sexual contact that causes personal distress.

CAUSES: a) Usually a psychological/emotional problem that can result from physical or sexual abuse as well as childhood trauma. Sexual pain disorders a) Dispareunia.

Persistent or recurrent genital pain during intercourse that causes personal distress.

CAUSES: a) Psychological/emotional. b) Medical/physiological problems such as vestibulitis, vaginal atrophy, and vaginal infections. b) Vaginismus. Persistent or recurrent involuntary spasms of the musculature of the outer third of the vagina that interferes with penetration and causes personal distress.

CAUSES: a) Psychological/emotional. b) Conditioned response to painful penetration. c) Other sexual pain disorders. Persistent or recurrent genital pain induced by noncoital sexual stimulation that causes sexual distress.

CAUSES: a) Medical/physiological problems such as infections (i.e. HSV), vestibulitis, trauma, and endometriosis. Orgasmic disorder Persistent or recurrent failure, difficulty, or delay in attaining orgasm that causes personal distress.

CAUSES: a) Psychological/emotional problems including emotional trauma or sexual abuse. b) Medical/physiological problems such as hormone deficiency or the result of surgery, trauma. c) Medications. Sexual arousal disorder Persistent or recurring inability to attain or maintain sexual excitement that causes personal distress. They include diminished vaginal lubrification, decreased

clitorial and labial sensation and/or engorgement, and lack of vaginal smooth muscle relaxation. CAUSES: a) Psychological/emotional problems such as depression, anxiety, self-esteem, body image, quality of relationship with partner. b) Medical/physiological problems such as diminished vaginal or clitoral blood flow, previous pelvic trauma, pelvic surgery. c) Medications. Each of these categories can be further divided according to whether the disorder has been lifelong or acquired; generalized or situational; and organic or psychogenic. The causes of sexual dysfunctions in women are complex and multifactorial and often the disorders overlap. It is often difficult to clinically determine causal roles in individual patients. In addition, hormone deficiency may secondarily lead to anatomic, vascular, and neural changes that may cause sexual dysfunction. TREATMENT: The ideal approach to treating sexual dysfunction in women involves a team effort between the woman, doctors, and trained therapists. Sexual problems can be corrected by treating the underlying physical and/or psychological problems. Currently, no medications are approved by the U.S. Food and Drug Administration (FDA) to treat female sexual dysfunction. Most treatments are based on empirical observations and are not supported by clinical trial data. Thus, it is premature to make general evidence-based recommendations for the treatment of female sexual dysfunction. However, it is important to recognize that not all female sexual problems are psychological. Potential therapeutic options do exist. Estrogen therapy Estrogen deprivation causes a significant decrease in the clitorial intracavernosal, vaginal, and urethral blood flow. Thus, adverse effects on structure and function of the vaginal and clitoral tissues (including clitoral fibrosis, thinned vaginal epithelial layers, and decreased vaginal vasculature) can be the result of reduced circulating estrogen levels. These changes can affect female sexual function. Estrogen administration in postmenopausal women and in women after oophorectomy (surgical removal of the ovaries) improves the integrity of vaginal mucosa tissue. Estrogen therapy increases vaginal tone and lubrication, which will decrease vulval dryness and irritation. In addition, estrogen administration can increase the blood flow in the vagina. Thus, estrogen administration can enhance female sexual arousal. Estrogen therapy is available as a systemic or local treatment. Systemic estrogen and estrogen-progestin therapy are associated with benefits which are not limited to sexual

function. These include relief of hot flashes and night sweats, improved sleep, and prevention of bone loss. The womens Health Initiative (WHI) showed that the use of estrogen-progestin therapy in older postmenopausal women is associated with an increased risk of venous thrombosis, stroke, and breast cancer. An increased risk of coronary artery disease was associated to women who were more then 20 years post menopause. As a result of these findings, the use of lower estrogen doses or the use of locally administered therapies has been advocated in postmenopausal women who have only urogenital symptoms. The randomized controlled Womens health Osteoporosis Progestin and Estrogen (Womens HOPE) trial showed that lower doses of estrogen, with or without progestin, improved vaginal atrophy. In addition, local or topical estrogen therapy (cream, vaginal ring, or vaginal tablet) have been shown to similarly restore vaginal epithelium and improve vaginal atrophy. Androgen formulations In surgically menopausal women, supraphysiologic testosterone when given alone or in combination with estrogen produced a greater increase in sexual desire, fantasies, and arousal than women who received estrogen alone. In another study, administration of supraphysiologic testosterone and estradiol increased sexual activity, satisfaction, pleasure, and frequency of orgasm more than estrogen alone. In a later study, physiologic testosterone replacement was investigated in women who had undergone hysterectomy (surgical removal of the uterus) and oophorectomy and who were receiving estrogen replacement. Only women receiving the highest dose of testosterone (slightly above the physiological level of testosterone) showed significant increased frequency of sexual activity, pleasure, and orgasm. In addition, a study of perimenopausal women did not show correlation between sexual function and androgen levels. Testosterone formulations are now available by prescription in combination with estrogen. 17-Methyltestosterone is used in combination with estrogen in menopausal women for sexual dysfunction. However, conflicting reports exist on the benefit of methyl-testosterone for treatment of inhibited desire or vaginismus in premenopausal women. The suggested dose ranges from 0.25 to 1.25 mg/day and should be adjusted according to symptoms, free testosterone levels, cholesterol and HDL levels, as well as liver function. Transdermal matrix testosterone delivery system for women currently is being developed. A multicenter study was conducted on 533 surgically menopausal (hysterectomy to remove the uterus) women with hypoactive sexual desire disorder who were all taking oral or transdermal estrogen. The patients, who were around 49 and in stable relationships, were randomized to receive either a placebo patch or the female testosterone patch for 24 weeks. Researchers measured the change in total satisfying sexual activity and found that the women who received the testosterone patch experienced a 51% increase in the frequency of total satisfying sexual activity and a 49% increase in sexual desire. Women wearing the placibo patch also reported improvements,

but not as much as with the testosterone patches. Benefits were seen with the mid-level dose (300 g/day) but not with a lower dose. A higher dose was not more beneficial. The FDA did not approve transdermal testosterone because of concerns regarding its longterm safety and requested more safety data on the product. A testosterone gel for women is in initial stages of development. In conclusion, although supraphysiological doses of testosterone improve sexual functions in oophorectomized and hysterectomized women with sexual dysfunction, it remains unclear whether physiologic testosterone levels can produce improvements in sexual function in postmenopausal women. In addition, androgen supplementation may induce side effects like acne, facial hair, liver damage, loss of hair, menstrual irregularities, and deepening of the voice. Moreover, supraphysiologic testosterone decreases high-density-lipoprotein (HDL). However, the adverse event profile reported in clinical trials did not raise concern. Finally, women with history of breast cancer should not take testosterone because testosterone is converted to estrogen by the aromatase enzyme. Sildenafil Sildenafil decreases the catabolism of cGMP, the second messenger in nitric oxidemediated relaxation of clitoral and vaginal smooth muscle. Studies show an increase in blood engorgement in the female genitals after sexual stimulation. Results are mixed on any changes in sexual arousal. Other studies have shown that sildenafil successfully treated female sexual dysfunction associated with aging and menopause. Thus, sildenafil, alone or in combination with other vasoactive substances, may be useful for treatment of female sexual dysfunction. Phase II clinical studies are now in progress. Tibolone Tibolone is a synthetic steroid with weak estrogenic, progestogenic, and androgenic properties. Tibolone increases libido and is effective in treating the symptoms of vaginal atrophy in menopausal women. It has not received FDA approval for use in the U.S. Therapeutic agents under development L-Arginine. L-arginine is a precursor of nitric oxide. L-arginine has not been tested in clinical trials in women. However, preliminary studies in men look promising. A combination of L-arginine and yohimbine, an 2-adrenergic blocker, is currently under investigation for use in women. PGE1. An intraurethral formulation of PGE1, which is absorbed by the mucosa, is available for male patients. Intravaginally delivered PGE1 is currently under investigation for use in women.

Phentolamine. Phentolamine is a non-specific -adrenergic blocker that causes vascular smooth muscle relaxation. It is available as an oral preparation. Phentolamine enhanced vaginal blood flow and arousal in a pilot study of menopausal women with sexual dysfunction. Apomorphine. Apomorphine is a short-acting dopamine agonist that facilitates erectile responses in both normal men and men with erectile dysfunction. Apomorphine is currently under investigations in women with sexual dysfunction. Dopamine/noradrenaline agonists. The dopaminergic system is believed to affect sexual behavior through its activation of the drive to pursue pleasure. The antidepressant bupropion, a reuptake inhibitor of dopamine and noradrenaline, is associated with fewer sexual side effects compared to other antidepressant and has been shown to increase sexual arousability/responsivity, but nor desire, in a randomized controlled trial of premenopausal women with sexual desire disorder who were not depressed. Additional trials of bupropion need to be completed in postmenopausal women.

III. To understand how medications may interfere with sexual function/desire. A persons sexual function and desire, as well as the sexual organs themselves, may be affected by a number of medications. These include: 1) Antidepressants. 2) Antihypertensive drugs. 3) Antipsychotic agents. 4) Anticonvulsants. 5) Antiulcer drugs. 6) Anticancer drugs. Antidepressant A variety of antidepressants have been shown to induce sexual dysfunction. Sexual dysfunction includes reduced/loss of libido, inability to achieve an erection, and inability to achieve an orgasm. Studies indicated that the overall incidence of sexual dysfunction among patients taking antidepressants was 37%. The treatment of patients with depression and sexual problems may be challenging because sexual problems can be both a symptom of the depression or a side effect of medication that treats the depression. Some patients are so happy to have their depression under control that they are willing to pay a price (sexual life becomes secondary). However, sexual dysfunction may represent one of the major factors leading to noncompliance with antidepressant therapy and may also contribute to or complicate the patients depression. Antidepressants that will be discussed in class include:

Tricyclics: Imipramine (TOFRANIL); Amitriptyline (ELAVIL); Amoxapine (ASENDIN). Monoamine oxidase (MAO) inhibitors: ISOCARBOXAZID (marplan); Phenelzine (NARDIL). Selective serotonin reuptake inhibitors (SSRIs): Fluoxetine (PROZAC); Sertraline (ZOLOFT); Paroxetine (PAXIL); Venlafaxine (EFFEXOR).

Clinical Pathology Correlation #1 Male Reproductive System: Introduction to the Basic Histology Thursday, February 12, 2009 10:30 am -12:00 pm Georgia Duker, Ph.D. The objectives of this lecture are for the students to be able to: 1. Describe the basic histology of the testis, including the seminiferous tubules and the Leydig cells. 2. Describe the stages of spermatogenesis 3. Compare the stages of meiosis in the female and male systems. 4. Know the histological structure and general functions of the prostate gland. Male Reproductive System The male reproductive system includes the testes, genital duct system, accessory sex glands and the penis. For the purposes of this course, you will need to know only the basic histology of the testis and prostate gland.

1. The Testis The products of the testis are sperm and the hormones testosterone, inhibin and activin. Sperm are formed in the seminiferous tubules; many of these coiled tubules are packed into the testis. A cross section of a seminiferous tubule displays cells in various stages of the mitosis and meiosis of spermatogenesis. The spermatogonia are the most basal cells within the seminiferous tubule, adjacent to the basement membrane. Spermatogonia are stem cell in nature and multiply by mitosis. Spermatocytes, in contrast, divide by meiosis. The spermatocytes fill the middle of the seminiferous tubule wall. Spermatids are the final products of meiosis, small round cells with a condensed nucleus. Finally, spermatozoa are the fully differentiated cells at the luminal face. Spermatozoa have morphologically remodeled into a sperm with a head and tail, and are ready to be released into the ducts of the male reproductive tract. Cell Type Spermatogonia A dark Spermatogonia A pale Spematogonia B 1o Spermatocyte 2o Spermatocyte Spermatid Spermatozoa DNA 2N 2N 2N 4N 2N 1N 1N

Stem cell, nonmitotic reserve Stem cell, mitosis Mitotic ~ every 16 days, incomplete cytokinesis Meiosis I Meiosis II Final product of meiosis, round cell Maturation of sperm shape

Thus, three similar sounding periods are defined: Spermatogenesis nuclear manipulation to produce a round haploid spermatid. Spermiogenesis cytoplasmic remodeling into a condensed nucleus, acrosomal cap and flagellar tail; produces a haploid spermatozoa. Spermiation release of spermatozoa into the lumen of the seminiferous tubule.

Erlandsen & Magney, Color Atlas of Histology m = myoepithelial cells scy = spermatocytes s = spermatogonia spd = spermatid sc = Sertoli cells spz = spermatozoa

Weiss, Cell & Tissue Biology Spermaotgonia MP = midpachytene spermatocyte AD = A dark (quiescent) ES = early spermatid Ap = A pale (mitotic) LS = late spermatid (almost spermatozoa) B = mitotic, incomplete cytokinesis LM = limiting membrane of basal lamina & myoepithelial cells

In addition to the spermatogenic cells, the tubular epithelium also contains Sertoli cells. The Sertoli cells support and nourish the maturing spermatogonia, spermatocytes and spermatids, all of which are embedded within folds of the Sertoli cells. Sertoli cells extend the full width of the seminiferous epithelium, from the basement membrane to the lumen. FSH binds to Sertoli cells to stimulate their functions. Tight junctions between adjacent Sertoli cells create the blood-testes barrier, which protects the spermatogenic cells from exposure to immune cells, toxins or pathogens. Outside the basement membrane of the seminiferous tubules, interstitial tissue fills the space. This stromal compartment contains blood vessels, lymphatics, nerves, connective tissue and the major endocrine component the Leydig cells. Identified as large, eosinophilic cells, the Leydig cells respond to LH to secrete testosterone. 2. The Prostate Gland The prostate gland is the largest of the male accessory glands. It is located at the base of the urinary bladder and surrounds the ejaculatory duct and the urethra. Growth and secretions from the prostate are dependent on testosterone. The acidic prostate secretions contribute a variety of substances to the semen, including: Fibrinolysin - a protease for semen liquifaction. Citric acid Ca+2 chelator to limit coagulation of stones Prostatic acid phosphatase (PAP) phospholipid metabolism Prostate specific antigen (PSA) while normally produced by the prostate, PSA, is increased with prostatic cancer, and is used as a clinical measure. The prostate is composed of 30-50 branched tubuloalveloar glands immersed in a fibromuscular stroma. Multiple ducts (15-30) empty into the prostatic portion of the urethra. The prostate has 3 zones of secretory glands: Central zone - directly surrounds the ejaculatory ducts Transition zone surrounds proximal urethra; anterior-most glands; site of 34% of prostate cancers and of benign prostatic hypertrophy Peripheral zone posterior wall, surrounds distal urethra; site of 64% of prostatic cancer

Prostatic secretions, along with those of the seminal vesicles, serve as both a dilutent and a vehicle for sperm transport. The epithelium lining the highly folded lumen is varied and can appear pseudostratified or simple columnar. Developmentally, the prostate forms within the smooth muscle mass of the bladder wall. Therefore, the connective tissue stroma within the epithelial folds contains smooth muscle cells scattered throughout even the smallest trabeculae. The prostate is highly convoluted; histological sections often look disoriented because of the many different tangential cuts. The prostate is of obvious medical importance because of the common appearance of both benign and malignant tumors (see CPC#3). Layered prostatic concretions form increasingly with age.

Clinical Pathology Correlation #1B Male Reproductive System: Histology Laboratory Georgia Duker, Ph.D. Histology Tray 7, Slots 90-131; Slides P1 42. All slides are on the Navigator website for the Reproduction Course. Note that students are not responsible for all of these slides; exceptions are indicated.

At this time, review meiosis and compare meiotic events and stages with those of mitosis. The male reproductive system consists of the testis where sperm and testosterone are produced, together with the accessory glands that modify sperm activity and the ducts by which sperm leave the testis.

Testis P1 (5X) H&E, Testis, Cross section P2 (5X) H&E, Testis, Longitudinal Section P3 (25X) H&E, Testis, Tunica Albuginea P4 (50X) H&E, Seminiferous Tubules P5 (100X) H&E, Seminiferous Tubules P6 (268X) H&E, Tubules, Sertoli Cells, Spermatocytes P7 (268X) H&E, Seminiferous Tubules, Leydig Cells Connective tissue septa extend from the central mediastinum (Slides P1, cross-section; P2 longitudinal section) to the tunica albuginea (Slide P3) to divide the testis into pyramidal lobes. The highly coiled seminiferous tubules are found in lobules, and in testis sections appear as randomly curved lumens. The various stages of spermatogenesis can be identified in the seminiferous epithelium (Slide P4). From periphery to lumen (Slide P5, P6):

spermatogonia are found at the periphery, resting on the basement membrane (only a few in these particular sections) primary spermatocytes are the largest rounded cells with meitotic figures. secondary spermatocytes are smaller, with meitotic figures, but very rare; they are a transient stage that is only present for a few hours. (not seen in these sections) spermatids are small, round and have dark nuclei; they are abundant in Slides P5 &P6. spermatozoa can be identified at the luminal border by their tails and condensing heads.

The Sertoli cells (Slides P4-7) are tall, irregular cells that extend from the basement membrane to the lumen. Their large nuclei are euchromatic and have an easily distinguishable nucleolus. Sertoli cell nuclei are located between the layers of spematogonia and the primary spermatocytes. Sertoli tight junctions seal off the adluminal population of meiotic cells from the basal compartment of stem cells and mitotic cells, which faces the blood supply. Sertoli cells therefore, create the blood/testis barrier. Sertoli cells have receptors for FSH and for testosterone. The interstitial cells of Leydig (Slide P7) usually occur in compact groups in the stroma between the seminiferous tubules. They are fairly large ovoid or polygonal cells with a eosinophilic cytoplasm and a large, often eccentric nucleus. Leydig cells bind LH and secrete testosterone.

Students are not responsible for these structures: Tubuli Recti and Rete Testes, Slides: P8 - P11 Ductus Epididymidis, Slides: P12 P16 Spermatic Cord, Slides: P17 Vas Deferens, Slides: P18 P21 Seminal Vesicle, Slides: P22 P25 Penis, Slides: P35 P42

Prostate P26 (5X) H&E, Prostate P27 (10X) H&E, Prostate P28 (100X) H&E, Prostate P31 (10X) H&E, Prostatic Ducts P32 (100X) H&E, Prostatic Ducts P33 (5X) H&E, Prostatic Concretions P34 (100X) H&E, Prostatic Concretions

These slides are sections of the prostate gland. All the prostatic glands are of the compound tubuloalveolar type and embedded within a fibromuscular stroma. The glands may appear in one of many morphologic forms, ranging from large, irregular, saclike structures to small, tube-like structures. Thin folds of glandular epithelium with cores of smooth muscle stromal tissue extend deeply into the lumina of many glands (Slides P26, P27). Depending on the functional state of the prostate gland, the epithelium may be simple squamous, cuboidal, columnar, or pseudostratified columnar (Slides P28). The cells may contain secretory granules and droplets in their cytoplasm. The ducts resemble small, tube-like secretory glands (Slides P31, P32) except near the urethra where their epithelium is transitional. In the prostate gland of older males, lamellar prostatic concretions (corpora amylacea ) may be present in the lumina of the glands (Slides P33, P34).

Pathology Lecture Thursday, February 12, 2009 - 1:00 2:20 p.m. Prostate and Testes Rajiv Dhir, M.D. I. OBJECTIVES The general objectives of this module segment are to: A. B. Review the gross and microscopic anatomy of the normal prostate and testis. Understand the taxonomy, pathogenesis and natural history of the major neoplastic and non-neoplastic diseases of the prostate and testis and to apply this understanding to relevant clinical situations. Recognize the macroscopic and microscopic morbid anatomy of the major diseases of the prostate and testis.

C. II.

FORMAT This afternoon period will be divided into three time segments. The first hour will be a lecture (Lecture Room 2). The second two hours will be an interactive laboratory session based on clinical scenarios and recognition of normal and morbid anatomy. The final hour will be an optional review period in LR2. Items to be discussed include: A. B. Diseases of the prostate, including prostatic adenocarcinoma and nodular hyperplasia. Germ cell tumors of the testis including seminoma and non-seminomatous germ cell tumors and non-neoplastic diseases of the testes including atrophy and torsion.


RECOMMENDED PREPARATION/REFERENCES Preparation should include review of Sections IV (Lecture outline) and V (Laboratory materials) of the student syllabus and review of the required reading detailed at the end of this section. Also, a brief review of the normal microscopic anatomy of the prostate and testis in an appropriate histology text is recommended. In addition, a pathology text reference is provided at the end of this section for background.


LECTURE OUTLINE A. Diseases of the Prostate 1. Carcinoma a. b. c. Prostatic cancer is common in men over age 50. The etiology of prostatic cancer is unknown. Diagnosis of prostatic cancer is based on clinical symptoms, digital rectal examination, and serum analysis. Most prostatic cancers are histologically adenocarcinomas. Treatment of prostatic cancer includes surgery, irradiation, and hormonal therapy. Prognosis in prostatic carcinoma is predominantly a function of stage.

d. e.



Nodular Hyperplasia a. Nodular hyperplasia of the prostate is extremely common in older men. The etiology of nodular hyperplasia appears to be related to androgen stimulation. Nodular hyperplasia of the prostate is a major cause of urethral obstruction in older men and is characterized histologically by a benign proliferation of glands and stroma.




Diseases of the Testis a. Testicular germ cell neoplasms are clinically significant cancers in young men. The etiology of testicular germ cell tumors is unknown. Pure seminoma is the most common testicular germ cell tumor and has a distinct histologic appearance. Approximately sixty percent of testicular germ cell tumors are classified as non-seminomatous germ cell tumors and include a number of pure and mixed histologic types.

b. c. d.


The most common clinical presentation of germ cell tumors of the testis is that of painless testicular enlargement. Prognosis and therapy are predominantly functions of clinical stage and histologic type. Significant non-neoplastic disease of the testis include cryptorchidism, inflammations, atrophy, and torsion.


DISEASES OF THE PROSTATE 1. Prostatic cancer is common in men over age 50. Carcinoma of the prostate is the most common cancer and the second leading cause of death from cancer in men, with approximately 300,000 new cases diagnosed annually in the United States. Prostatic cancer is predominantly a disease of men over the age of 50 and most especially over the age of 70. The etiology of prostatic cancer is unknown. Risk factors in the development of prostatic cancer include age, race, family history, hormone level, and environmental factors. The potential importance of environmental factors and race is exemplified by the relatively rare incidence of prostatic cancer in Asians, the high incidence in Whites in the United States, and the even higher incidence among U.S. Blacks, as well as a tendency for the incidence of prostatic cancer to rise in populations moving from low incidence areas to higher incidence locations. Specific environmental factors however have not been identified. The significance of hormones and genetic factors in the etiology of prostatic cancers remains to be determined. Diagnosis of prostatic cancer is based on clinical symptoms, digital rectal examination, and serum analysis. Prostatic carcinoma is grossly a diffuse, firm and gritty lesion more easily palpated than visualized. In approximately 70% of cases these tumors arise in the peripheral zone of the prostate, often in a posterior location. Because of this posterior peripheral location, rectal digital examination is useful for the detection of prostatic carcinoma (especially in early stages). In addition, because most prostatic cancers arise in a peripheral subcapsular location, urinary symptoms are uncommon forms of presentation for early lesions. In more advanced stage prostatic carcinomas however, the most common clinical presentation is that of urinary symptomology including difficulty in starting and stopping urination, dysuria, frequency, and hematuria. Pain, reflecting perineural invasion or back pain caused by vertebral metastases, is an uncommon presentation except in far advanced disease. Finally, two biochemical markers, prostatic acid phosphatase and especially prostatic specific antigen (PSA) are produced by prostatic epithelium and are of value in the diagnosis and management of prostatic cancer.




Most prostatic cancers are histologically adenocarcinomas. Prostatic carcinomas are most often characterized histologically by diffusely infiltrating well differentiated neoplastic glandular epithelium, often with capsular and perineural invasion. Tumors may also be moderately or poorly differentiated histologically. They are graded using the system described by Gleason. Treatment of prostatic cancer includes surgery, irradiation, and hormonal therapy. Surgery and radiotherapy are the most common treatment of prostatic cancers which are confined to the prostate (Stage A and B), while hormonal therapy is the principal method of treatment for advanced metastatic carcinoma. Hormonal manipulation of the androgen sensitive prostatic cells may include orchiectomy, the administration of estrogens or the administration of synthetic luteinizing hormone releasing hormone agonists. Although hormonal manipulation often induces remission, it should be noted that, in time, hormone insensitive tumor clones develop. Prognosis in prostatic carcinoma is predominantly a function of stage. Stage A prostatic carcinomas are microscopic tumors confined to the prostate. These latent prostatic carcinomas have an incidence as high as 70% in men over the age of 80 but cause significant clinical consequences in only approximately 10-20% of affected patients. A good prognosis is also reported in Stage B tumors (macroscopic tumors confined to the prostate) when treated by surgery and radiotherapy. With combined clinical and pathological staging, more than fifty % of patients with prostatic carcinoma however present at Stage C or D (extension beyond the prostate capsule or metastatic disease), and have only a 10-40% ten-year survival. Local extension in prostate cancer most commonly involves the seminal vesicles, urinary bladder, and ureters. Hematogenous spread is most commonly to the bones of the axial skeleton and produces both osteolytic and most notably osteoblastic bony metastases. Lymphatic spread to lymph nodes also occurs frequently and may preceed spread to bones. Other factors which are important in prognosis include tumor grade and DNA ploidy. Nodular hyperplasia of the prostate is extremely common in older men. Nodular hyperplasia of the prostate has been reported to be histologicaly present in 70% of men by age 60 and 90% of men by age 80. The etiology of nodular hyperplasia appears to be related to androgen stimulation. Although the etiology of nodular hyperplasia is unknown, it appears to be related to the androgen dihydrotestosterone (DHT), which is a major mediator of prostatic growth and may be especially potent in older men in whom increased levels of estradiol may sensitize the prostate to the growth promoting effects of DHT. It is important to note that current evidence suggests that there is no relationship between nodular hyperplasia and prostatic cancer. Nodular hyperplasia of the prostate is a major cause of urethral obstruction in older men and is characterized histologically be a benign proliferation of glands and stroma. Nodular hyperplasia increases the weight of the prostate from the normal 20 grams to up to 200 grams or more. It most commonly effects






the periurethral central region of the prostate with a macroscopic appearance on cut surface of multiple well-defined nodules. Because these involve the periurethral portion of the prostate, the symptoms produced are often secondary to urethral compression and urinary retention. These include frequency, nocturia, difficulty in starting and stopping urination, overflow dribbling, dysuria, urinary tract infection, and secondary changes in the bladder. Microscopically, nodular hyperplasia is characterized by benign proliferation and dilatation or prostatic glands and/or stromal proliferation. Squamous metaplasia and small areas of infarction are also commonly seen.

DISEASES OF THE TESTIS 1. Testicular germ cell neoplasms are clinically significant cancers in young men. Germ cell cancers account for 95% of testicular neoplasms (5% are stromal). Although germ cell tumors of the testis have an incidence in the United States of only approximately 6 per 100,000, they are the most common malignant tumor of men in the 15-34 year old age group. The etiology of testicular germ cell tumors is unknown. Predisposing factors in the development of testicular germ cell neoplasia include cryptorchidism, genetic factors, and testicular dysgenesis (e.g. testicular feminization, Klinefelter's Syndrome, etc.). Although the pathogenesis of these tumors remains uncertain, cytogenetic changes involving chromosome 12 appear to be important in their development. Pure seminoma is the most common testicular germ cell tumor and has a distinct histologic appearance. Seminomas account for 30% - 50% of all testicular germ cell tumors. They are macroscopically characterized on cut surface by a homogeneous gray-white lobulated appearance, usually without extensive hemorrhage or necrosis. They are microscopically most often characterized by sheets of uniform, large cells with distinct cell membranes, clear cytoplasm, and large hyperchromatic nuclei with prominent nucleoli. Less common variants of seminoma include anaplastic and spermatocytic types. The spermatocytic type of seminoma is remarkable in that it most often affects men over the age of 65. Approximately sixty percent of testicular germ cell tumors are classified as non-seminomatous germ cell tumors and include a number of pure and mixed histologic types. Non-seminomatous germ cell tumors of the testis include embryonal carcinoma, yolk sac tumor, polyembryoma, choriocarcinoma, and teratoma. These may be pure but often show a mixed histologic appearance. Although these different non-seminomatous germ cell tumors have variable macroscopic and histologic appearances, their clinical behavior is similar and they can be considered as a group distinct only from seminomas. The most common clinical presentation of germ cell tumors of the testis is that of painless testicular enlargement. Prognosis and therapy are





predominantly functions of clinical stage and histologic type. Seminomas have the best prognosis of all germ cell tumors of the testis (with the exception of mature benign teratomas in children), tend to present at low stage, and are extremely radiosensitive. In contrast, a majority of patients with nonseminomatous germ cell tumors of the testis present with tumors spread beyond the confines of the testes (Stage 2 and 3) and these tumors are relatively radioresistant. Metastatic tumor spread is both lymphatic and hematogenous, with frequent sites of metastases including lung, liver, brain, and bone. Although non-seminomatous tumors are more aggressive than seminomatous tumors, with aggressive therapy complete remission is achieved in approximately 90% of cases. Finally, non-seminomatous germ cell tumors may secrete polypeptide hormones and enzymes including human gonadotropin (hCG) and alpha fetoprotein (AFP). These enzymes are useful in evaluating, staging and monitoring the germ cell tumors of the testis, most especially the nonseminomatous germ cell tumors. 6. Significant non-neoplastic disease of the testis include cryptorchidism, inflammations, atrophy, and torsion. Cryptorchidism (undescended testes) is found in approximately 0.5% of the adult male population in the United States and is clinically significant as a cause of infertility and as a predisposing factor in the development of testicular tumors. Significant causes of testicular atrophy include atherosclerotic vascular disease, inflammatory orchitis, cryptorchidism, hypopituitarism, malnutrition, semen outflow obstruction, irradiation, prolonged exposure to female sex hormones, and Klinefelter's Syndrome. When bilateral, testicular atrophy results in sterility. Significant inflammatory and infectious diseases of the testis include non-specific orchitis, autoimmune orchitis, gonorrhea, mumps, tuberculosis, and syphilis. Testicular torsion results from a twisting of the spermatic cord with a subsequent obstruction of venous but often not arterial vascular beds. In most cases, torsion is result of violent movement or physical trauma in patients predisposed to torsion by anatomic abnormalities.

Clinical Pathologic Correlation #3 Thursday, February 12, 2009, 2:20 4:00 pm DISEASES OF PROSTATE AND TESTES PROSTATE AND TESTES Clinical Scenario C1 Premise/Background: Problems with urination are clinically significant symptoms in older men with important prognostic and therapeutic implications depending on the underlying anatomic cause. The clinical evaluation of these patients may include physical examination, biopsy or aspiration, surgical resection and serum laboratory tests.

Clinical Summary: Chief Complaint: 62-year-old male with dysuria and difficulty starting and stopping urination. Patient enjoys general good health. Past medical history is remarkable for a ten year history of angina responsive to nitroglycerin, a 35 pack year history of cigarette use and the surgical removal of two benign polyps of the colon three years ago. Medical and family history and review of systems are otherwise non-contributory. Physical examination is unremarkable except for a diffuse firmness of the prostate on rectal examination. Routine laboratory results are pending or within normal limits. Needle biopsy of the prostate is reported to show carcinoma.



Assessment/Discussion: 1. Identify the representation of this patient's biopsy from among the provided photomicrographs and discuss the histologic type of this tumor and the concept of tumor grading in prostate carcinoma. What significant aspects of this patient's history and physical examination support the diagnosis of prostate carcinoma? What additional laboratory and imaging studies might be useful in the management of this patient's tumor? Given this patient's presentation, discuss the concept of stage in prostatic carcinoma and how this may be reflected in presenting symptoms. What other significant disease of the prostate might have been the cause of this patient's urinary tract symptoms. How is this disease related to the development of prostate cancer? What is the significance of this diagnosis to this patient? Despite aggressive hormonal therapy, this patient's tumor proved fatal 3 1/2 years after diagnosis. Identify these macroscopic and microscopic pictures taken at autopsy and discuss these with respect to this patient's disease process and treatment. Discuss other diseases that can cause similar results.


3. 4. 5.

6. 7.

Clinical Scenario C2 Premise/Background: Clinically significant diseases of the testis include atrophy, inflammation, torsion, and testicular neoplasms. Clinical symptoms resulting from testicular disease include empty scrotum, pain, and masses.

Clinical Summary: Chief Complaint: 33-year-old male with painless enlargement of the left testis. Patient enjoys excellent health and has had no significant medical problems except floppy mitral valve syndrome and an inguinal hernia repaired at the age of 6. Medical, family and social history are unremarkable. Review of systems is noncontributory. Physical examination, including rectal exam, is unremarkable except for an enlarged left testis measuring up to 7 cm. The mass is mobile and nontender. Routine laboratory work including serum human gonadotropin and alpha fetoprotein are within normal limits. All imaging procedures are noncontributory.



Assessment/Discussion: 1. List a practical differential diagnosis for this left testicular mass. Are torsion and inflammatory disease of the testes reasonable considerations in this differential diagnosis? What is the significance of the patient's age in predicting the nature of this lesion? This is a macroscopic specimen picture which shows the cut surface of the left testicle after surgical removal. Does this macroscopic picture confirm or contradict the original clinical impression? Microscopic examination of two sections taken from this testicular lesion reveal a seminoma. Identify the photomicrograph of this seminoma from among the photomicrographs provided. Discuss the classification and frequency of testicular neoplasia with respect to germ cell and stromal origin and seminomatous and non-seminomatous types. Discuss the adequacy of examination of two microscopic sections in testicular germ cell tumors with respect to the natural history and biologic behavior of these tumors. What is the significance of the clinical laboratory findings in the initial workup of this patient? What is the significance of this diagnosis to this patient with respect to therapy and prognosis?








Normal and Morbid Anatomy This portion of the laboratory will examine normal microscopic anatomy of the prostate and testis and the microscopic and gross morbid anatomy of benign prostatic hypertrophy, prostatic carcinoma, testicular germ cell neoplasia, testicular atrophy, and testicular torsion. Microscopic and gross specimens necessary for the exercises below are available at stations within the laboratory. Exercises can be done in any convenient order. (Microscopes will be needed.) 1. Microscopic Set I - Identify the examples of normal prostate and testis. Normal prostate Normal testis 2. A A B B C C D D

Microscopic Set II - Identify the examples of the following prostatic lesions. Benign prostatic hypertrophy Prostatic carcinoma A A B B C C D D


Microscopic Set III - Identify the examples of the following testicular lesions. Seminoma Testicular Atrophy Testicular Torsion A A A B B B C C C D D D


Macroscopic Set I - Identify the examples of the following lesions of the prostate and testis. Prostatic carcinoma Benign prostatic hypertrophy Seminoma A A A B B B C C C


REVIEW Prostate 1. How common is carcinoma of the prostate and what age group is most affected? Most carcinomas of the prostate are of what histologic type? Discuss the clinical significance of occult (Stage A) carcinoma of the prostate. What are the most common stages at diagnosis for prostate carcinoma and how does this affect prognosis? Discuss the clinical importance of digital rectal examination in men and relate this to the anatomic location of prostatic carcinoma. Describe the character of the bone metastases in prostatic carcinoma. What is the clinical value of prostatic acid phosphatase and prostatic specific antigen in prostatic carcinoma? What three therapies are used to treat prostatic cancer and how are these related to stage? Discuss age, race, and environment as risk factors in the development of prostatic carcinoma. What age group is most often affected by prostatic nodular hyperplasia? How common is prostatic nodular hyperplasia? What are the most common clinical presentations of prostatic nodular hyperplasia and how is this related to its anatomic location? What hormone appears to be related to the development of prostatic nodular hyperplasia? What is the relationship of prostatic nodular hyperplasia to carcinoma?

2. 3.



6. 7.




11. 12.



Testis 1. 2. 3. 4. 5. What is the cell of origin of most neoplasms of the testis? How common is testicular cancer? What age group is most often affected by testicular cancer? What is the most common type of pure testicular germ cell tumor? List three other types of malignant germ cell tumors of the testis and discuss why they are clinically classified together as non-seminomatous germ cell tumors. What is the difference in the biologic behavior of teratoma of the ovary and testis? What is the most common clinical presentation of germ cell tumors of the testis? Discuss the distinction of seminoma and non-seminomatous germ cell tumors of the testis with respect to stage, radiosensitivity, prognosis, and the secretion of polypeptide hormones and enzymes. Define cryptorchidism and testicular torsion. List five significant causes of testicular atrophy.




9. 10.

REFERENCES 1. 2. Naus G. Diseases of the prostate and testis. Cotran RS, Kumar V, Robbins, SL, Robbins Pathologic Basis of Disease, Diseases of the prostate and testis. WB Saunders, Philadelphia. or: Any suitable pathology textbook.

Lecture: Development of the reproductive system February 13, 2009 Cynthia Lance-Jones

LEARNING OBJECTIVES: 1. To describe the embryonic origins of male and female gonads, ducts, and external genitalia. 2. To identify genes with recognized roles in gonadal development. 3. To identify the cellular source and functions of androgens and anti-Mullerian hormone in the early development of genital ducts and external genitalia. 4. Given a common structural abnormality or complex of abnormalities, the student should be able to discuss likely causes.

RESOURCES: Chapter on reproductive system development in a medical embryology text like Sadler or Moore and Persaud. DiNapoli, L. and Capel, B. 2008. SRY and the standoff in sex determination. Molecular Endocrinology 22:1-9. MacLaughlin, D. T., and Donahoe, P. K. 2004. Sex determination and differentiation. New England Journal of Medicine 350:367-378. Sekido, R. and Lovell-Badge, R. 2008. Sex determination and SRY: down to a wink and a nudge? Trends Genetics epub

KEYWORDS/PHRASES: reproductive system development, gonad development, primary germ cells, primary sex determination, secondary sex determination, testis descent, AMH, testosterone, WT1, SF1, SRY, Sox9, Wnt4, disorders of sexual development, sex reversal, hypospadius, cryptorchism

LECTURE OUTLINE I. II. III. IV. V. VI. VII. VIII. IX. Early tissue origins Development of the gonads Development of the genital duct systems Development of the external genitalia Hox genes and the normal development of the genital ducts and external genitalia Genes involved in the development of the indifferent gonad Primary sex determination Secondary sex determination Summary schematic

LECTURE NOTES I. Early tissue origins A. The developing reproductive system is unique because of the early bipotential or indifferent nature of its components. In both males and females, the gonads, internal duct systems, and external genitalia appear morphologically similar at early stages (5-7 weeks post fertilization). Indifferent or bipotential precursors include: B. Genital ridges: thickenings of intermediate mesoderm along the medial border of the mesonephros. These structures will give rise to the gonads. In both male and female embryos, epithelial cells lining the genital ridges proliferate and move into the underlying mesenchyme to form primitive sex cords. C. Two pairs of genital ducts originating from intermediate mesoderm. The Wolffian or mesonephric ducts will give rise to the epididymis, vas deferens, and seminal vesicles in males. The Mullerian or paramesonephric ducts appear slightly later and will give rise to the uterus, cervix, and part of the vagina in females. D. Mesodermal swellings surround the cranial part of the cloaca (the urogenital sinus or UGS). These swellings will give rise to the external genitalia.

Wolffian duct

Mesonephric tubule

Lateral view: 5 weeks

Genital ridge Transverse views: 5 and 6 weeks

UGS Mullerian duct Genital swelling Anterior view: 6-7 weeks

after Moore and Persaud, 2008 and Gilbert, 2006

Primitive sex cords

E. Primordial germ cells (PGCs) arise outside of gonadal tissues. They originate from cells that pass through the primitive streak and become recognizable in the endoderm of the yolk sac. At approximately 5-6 weeks, PGCs migrate dorsally into the hindgut, through the dorsal mesentery and into the genital ridges. Many PGCs do not reach the genital ridges. Most die, but a few may survive in ectopic locations and can later form tumors called teratomas. Teratomas contain irregularly arranged masses of many tissues, including hair, teeth, cartilage, muscle, gut, and kidney tissue. These represent derivatives of all germ layers and it is believed (based on mouse models) that these ectopic diploid PGCs develop to the gastrulation stage but then lose spatial organization. Teratomas are usually benign.

after Moore and Persaud. 2008 and Gilbert, 2006

Primordial germ cells

Primordial germ cells

II. Development of the gonads A. Testes 1. Sex-specific differentiation within the indifferent gonad occurs earlier in males than in females. During the 6th week, the sex cords in a male embryo extend into deep parts of the developing gonad and fuse with mesonephric tubules. These cords will undergo a remodeling to form the seminiferous cords and rete testes, while the remnants of mesonephric tubules will form the efferent ducts (vas efferens). The seminiferous cords lose contact with surface epithelium and become separated from it by a thick matrix coat, the tunica albuginea. The seminiferous cords hollow out to form seminiferous tubules at puberty.

6, 7, and 20 weeks
after Gilbert, 2006 2. The first testes-unique cells to differentiate are the Sertoli cells that make up the seminiferous cords. In addition to supporting PGCs, embryonic/fetal Sertoli cells: a. Secrete anti-Mullerian hormone (AMH), a hormone required for the regression of the Mullerian duct in males. b. Secrete Desert hedgehog, a signaling molecule that regulates Leydig cell and peritubular myoid cell development. 3. Fetal Leydig cells are thought to originate from mesenchymal cells surrounding the seminiferous cords. Functions of the fetal Leydig cells include: a. Secretion of androgens during the period of male duct development (8-14 weeks) b. Secretion of Insulin-like factor 3 (INSL3), a growth factor required for transabdominal descent of the testis. 4. Cells from the mesonephros are uniquely attracted to testicular tissue and give rise to peritubular myoid cells as well as endothelial cells that give rise to male-specific vasculature. 5. PGCs divide mitotically as they migrate towards the gonads and continue to do so after entering the testis. They do not enter meiosis until puberty.

B. Ovaries 1. Within a developing ovary, the sex cords are less prominent than they are in a developing testis. During the fetal period in females (approx. 16 weeks), the primitive sex cords break up into isolated clusters of epithelial cells surrounding PGCs. These clusters or follicles form at the margins of the gonad. 2. The granulosa cells of the follicles are support cells, like the Sertoli cells of the testis but they are also the main sites of estrogen synthesis. Fetal granulose cells convert androstenedione (an androgen) into estradiol (an abundant estrogen) using a CYP aromatase. 3. Thecal cells can be considered the female equivalent of the male Leydig cells. Theca cells produce androstenedione from cholesterol. Androstenedione then diffuses into the granulosa cells. 4. Estrogens are not required for initial ovary formation but are required for the maintenance of normal structure in the adult. 5. PGCs initiate meiosis in the ovary and by 14 weeks, primordial follicles consisting of granulose cells and oocytes are evident. Meiosis appears to be stimulated by retinoic acid (RA) produced in the adjacent mesonephros. (Meiosis is not initiated in testis because developing Sertoli cells appear to produce degradative enzymes that break down RA.) In the developing ovary, signals from granulosa cells may arrest PGCs in prophase of meiosis.

6,12, and 20 weeks

after Gilbert, 2006

6. Germ cells are not required for the initial differentiation of the gonads. However, The terminal differentiation of gonads in females is compromised by the absence of germ cells. If PGCs do not reach the genital ridge or if they are abnormal and degenerate (as in XO females with Turner Syndrome), the ovaries fail to develop normally, remaining as streak ovaries. Streak gonads are small and fibrotic gonads that lack the typical germ cell or supporting cell morphology of testes or ovaries.

C. Descent of the gonads 1. Both testes and ovaries initiate development in the lumbar region and are attached to the posterior body wall by caudal and cranial mesenteries or ligaments. 2. In males, the cranial ligament regresses and the caudal ligament develops into a thick fibrous structure called the gubernaculum. In a manner that is incompletely understood, these events are associated with the descent of the testis through the posterior abdominal wall, over the pelvic rim, and into the inguinal canal and scrotum. Both peptide (insulin-like factor 3) and steroid (androgens) hormones have been implicated in gubernaculum development and testes descent. A failure of complete descent of the testis into the scrotum is called cryptorchism (3/100 male births). Insulin and androgen deficiencies as well as exposure to excess estrogens may be responsible. 3. In females, the ovaries move laterally and caudally but this movement is much less dramatic than that of the testes. The cranial ligament persists as the suspensory ligament of the ovary. The caudal ligament persists as the round ligaments of the ovary and uterus.

Adrenal Kidney Cranial suspensory ligament

Caudal suspensory ligament

After Netter, 2002


Site of original cranial suspensory ligament

Ductus deferens

Wolffian duct
7 and 28 weeks

QuickTime and a Photo - JPEG decompressor are needed to see this picture.

after Carlson, 1999 and MacLaughlin and Donahoe, 2004

Gubernaculum After Carlson, 1999 Vaginal process

Mullerian duct gonad

Wolffian duct Bladder

Caudal suspensory ligament Cranial part of cloaca (UGS)

after Patten

Indifferent stage



testis before descent Mullerian duct Wolffian duct gubernaculum


ovary before descent

Wolffian duct oviduct and ovary after descent

testis after descent



III. Development of the genital duct systems A. In both male and female embryos, two sets of paired ducts are associated with the mesonephros. Like the gonads, these ducts are of intermediate mesoderm origin. The Wolffian or mesonephric ducts are positioned medially. The Mullerian or paramesonephric ducts are positioned more laterally. Both ducts empty into the cloaca. The cranial portion of the cloaca is called the urogenital sinus. This sinus ultimately contributes to the bladder, urethra, prostate and perhaps also the lower part of the vagina. (See notes from Renal course). B. If a Y chromosome is present, testes form and produce two hormones. Anti-Mullerian Hormone (AMH) is produced by Sertoli cells and stimulates regression of the Mullerian ducts. This hormone binds to receptors on mesenchymal cells surrounding the Mullerian duct. These mesenchymal cells are thought to produce a factor that initiates apoptosis of the duct epithelial cells. Testosterone is produced by the fetal Leydig cells and stimulates the masculinization of the Wolffian ducts. The Wolffian ducts become the epididymis, vas deferens and seminal vesicles. C. In a female embryo, the Wolffian ducts spontaneously regress. The Mullerian ducts remain and become the oviducts, uterus, cervix and at least the upper part of the vagina. (The lower part of the vagina may originate from cells associated with the urogenital sinus.) The formation of the uterus, cervix and vagina requires that the two Mullerian ducts meet and fuse in the midline. Occasionally this does not happen correctly. One of the more common anomalies is bicornuate uterus, a uterus with two separate horns.

Bicornuate uterus

IV. Development of the external genitalia A. Bipotential stage: Three swellings or folds are involved in the formation of the external genitalia. 1. Urethral folds surround the cranial part of the cloaca (the opening of the urogenital sinus or UGS). 2. The genital tubercle lies cranial to the folds. Like the limb, the genital tubercle is initially present as a small bud and mechanisms involved in tubercle development

appear to parallel mechanisms involved in limb bud outgrowth. Both processes require Sonic and FGF signaling. 3. Lateral swellings are termed the genital swellings. B. In males: The genital tubercle expands to form the glans of penis. With expansion of the genital tubercle, urethral folds extend cranially and medially to form the shaft of the penis. The urethral folds meet and fuse ventrally, enclosing the urethra. The genital swellings fuse to form the scrotum. In approximately 1/300 newborn males the urethral folds do not fuse appropriately and the urethral opening may be located ventrally. This condition is termed hypospadias. Masculinization of the external genitalia is androgen-dependent. Within these tissues, the enzyme 5-alpha reductase converts testosterone to dihydrotestosterone (DHT), which is the essential activator for growth and differentiation of the penis and scrotum. DHT is also produced by the cells of the urogenital sinus and is required for normal development of the urethra and the prostate. C. In females: The urethral folds and genital swellings remain separate and become labia majora and minor. The genital tubercle becomes the clitoris.

indifferent stage
urethral fold genital swelling genital tubercle urogenital sinus


anus glans penis urethral groove

female 7 weeks
labia majora labia minora vaginal oriface clitoris urethral oriface


12 weeks

after Carlson, 1999

late fetus

V. Hox genes and the normal development of genital ducts and external genitalia A. Hox proteins are expressed in restricted patterns in the developing genital duct systems and in the genital tubercle. These patterns appear to be critical for the correct development of different duct regions (i.e. uterus vs. cervix vs. vagina) and for the proliferation of genital tubercle cells. B. Evidence of the importance of Hox genes in human reproductive system development. 1. Mutations in Hoxa13 are associated with uterine and vaginal atresia and bicornuate uterus in females and cryptorchism and hypospadius in males. 2. Exposure of the fetus to the synthetic estrogen DES (diethylstilbestrol), can lead to changes in Hox gene expression patterns. Individuals born to mothers who were given DES during pregnancy can show reproductive system abnormalities similar to those found with Hox mutation.

VI. Genes involved in the development of the indifferent gonad A. WT1 (Wilms tumor 1) is expressed early in kidney tissue and the indifferent gonads of both males and females. Studies of mice mutants suggest that WT1 is required for appropriate genital ridge development. At later stages, WT1 may regulate SRY and AMH. Patients with mutations in WT1 (Frasier Syndrome and Denys-Drash Syndromes) can show abnormalities in gonadal differentiation, the development of secondary sexual characteristics and a predisposition to childhood kidney tumors and nephropathy. B. SF1 (Steroidogenic factor 1) is expressed early in multiple steroidogenic tissues and appears to be required for the early formation of the gonad in both males and females. It encodes an orphan nuclear receptor. SF1 plays additional roles including the regulation of genes critical for testis development and for testosterone and AMH receptor synthesis.

VII. Primary sex determination A. Gonadal development regulates sexual development. Evidence: Jost removed undifferentiated gonads from fetal rabbits and found that all embryos developed with female anatomy. In contrast, exposure to a transplanted testis led to male duct development (see diagram below). These findings gave rise to the idea that female development represents a default state and that male development is an active process governed by testis products.

B. Factors on the Y chromosome determine maleness. Evidence: Humans with Turners Syndrome (XO) are phenotypically female. Humans with Klinefelters Syndrome (XXY) are phenotypically male. Thus, what matters is the presence of a Y chromosome not the number of X chromosomes. This conclusion led to an extensive search for a testes determining gene on the Y chromosome.

C. The SRY gene encodes a testis-determining factor. The SRY gene was identified through genetic analyses of patients showing sex reversal. Sex reversal refers to conditions in which there is a disparity between chromosomal sex and phenotypic characteristics. One way this can occur is by the translocation of Y-specific DNA to the X chromosome during meiosis. Through analyses of 4 human XX female-to-male sex reversal cases, the testes determining region was localized to a small (60kb) piece of Y DNA and, ultimately the SRY gene was identified. In turn, SRY deletion is associated with XY male-to-female sex reversal. The SRY protein is a transcription factor specifically expressed by Sertoli cells during initial stages of testis differentiation. Definitive evidence of its role came from the finding that transgenic XX mice carrying a copy of Sry developed testes and male secondary sex characteristics.

D. Sox9 is a downstream target of SRY. Sox9 encodes a transcription factor that is initially expressed at low levels in both male and female gonads. It is upregulated in Sertoli cells shortly after SRY is expressed and maintained throughout life. It is required for testis development and deletion of Sox9 leads to XY male-to-female sex reversal in humans. Sox9 is also important for cartilage development and patients with Sox9 deletion (Campomelic dysplasia) show cartilage abnormalities as well as sex reversal. (Both Sox9 and SRY expression appear to be upregulated by SF1, but Sox9 is unique in its ability to also activate SF1, i.e positive feedback loop).

E. Recent data suggest that female development is not a default or passive process and that the bipotential gonad is the battleground between two active and opposing signaling pathways(see DiNapoli and Capel, 2007 or Sekido and Lovell-Badge, 2008).

1. Two extracellular signaling molecules are expressed in BOTH male and female gonads at bipotential or indifferent stages: Fgf9 and Wnt4. Subsequently Fgf9 is upregulated in males, Wnt4 in females. Genetic manipulation in mice indicates that Fgf9 normally maintains Sox9 and represses Wnt4. In turn, Sox9 boosts Fgf9 expression. Fgf9 null mice also show male-to-female sex reversal. In contrast, Wnt4 appears to repress Sox9 and Fgf9 expression. 2. Wnt4 null XX mice show some aspects of testicular differentiation including formation of Leydig-like cells, male-like vasculature and transient activation of Sox9 and Fgf9. Wnt signaling leads to a stabilization of b-catenin, a factor that may be considered as an ovarian determinant. 3. These findings suggest that the plasticity of the bipotential gonad is controlled by mutually antagonistic signals in the gonadal field. SRY tips the balance towards testis development by boosting Sox9 expression.

-catenin -catenin

VIII. Secondary sex determination The role of hormone signaling has been referred to earlier in this lecture and discussed in other lectures. The chart below lists major human disorders resulting from abnormalities in hormone signaling. These have been referred to as cases of pseudohermaphraditism or conditions where the phenotypes of the gonads and ducts or external genitalia do not match. The term, disorder of sexual development or DSD is now preferred to refer to all congenital conditions where the development of chromosomal, anatomical, or gonadal sex is atypical. Disorder Congenital adrenal hyperplasia (excess exposure to androgens) Androgen insensitivity (abnormal receptors) 5-alpha reductase deficiency Persistent mullerian duct syndrome (loss of AMH or receptor function) Genotype XX XY Gonads ovaries testes Ducts typically female normal External Genitalia masculinized normal Other

abnormal bone growth termed testicular feminization termed testicular feminization testis descent may be blocked by female ducts





mullerian duct degenerates but wolffian duct not masculinized normal





uterus and oviducts as well as masculinized wolffian duct


IX. Summary Schematic


After Gilbert, 2006

LECTURE #8 Fertilization, Development and Human Pluripotent Stem Cells February 13, 2009, 9:50 AM 10:50 AM Gerald Schatten Ph.D.

Learning Objectives This lecture will help the students understand: 1. The numerous steps required for successful completion of fertilization including; a. Gamete transport b. Sperm capacitation c. Gamete interaction d. Oocyte activation e. Genomic union at first mitosis 2. The timing of the preimplantation development and important landmarks. 3. The basis of infertility and some of the available treatments. 4. The promise of human pluripotent stem cells.

Fertilization and Preimplantation Development At coition, approximately 300 x 106 sperm are deposited into the vagina and onto the cervix. Of these only a few hundred will make it to the site of fertilization in the ampulla of the oviduct. The vagina is a hostile environment for sperm, with an acidic pH and host defenses against invading microorganisms. The presence of sperm in the vagina results in an influx of leukocytes, hence it is imperative for fertility that sperm make a rapid progression through the reproductive tract. Sperm travel through the cervix into the uterus and into the oviducts both by sperm swimming and via a poorly understood active transport process of the female reproductive tract. Sperm are found in the oviducts within 30 minutes of deposition. This time is too short to be a result solely of sperm swimming and implies an active transport mechanism most likely due to the peristaltic contractions observed in the uterus. The cervix serves as the first barrier to sperm passage and only during the late follicular phase is the cervical mucous thin enough to allow sperm passage. At any other time during the menstrual cycle the sperm is kept out of the uterus by the cervix. Once past the cervix sperm must travel through the uterus, into and through the isthmus of the oviduct and to the ampulla (upper part of the oviduct, please see Figure 1 for timing of events throughout the female reproductive tract) where fertilization occurs.

Sperm isolated from the epididymis are differentiated, fully formed gametes but are incapable of fertilizing the oocyte. If isolated sperm are incubated in a reproductive tract for several hours they acquire the capacity to fertilize. This process is known as capacitation. During capacitation cholesterol is removed from the sperm membrane resulting in increased membrane fluidity. Biochemical events associated with capacitation include a gradual increase in the level of intracellular calcium, due, in part, to the stripping of glycoproteins from the cell membrane and unmasking calmodulin binding proteins. The increased calcium, in turn, enhances the intracellular generation of cyclic AMP (cAMP). In response to increased levels of intracellular calcium and cAMP, the spermatozoa develop a hyperactivated phenotype and become primed for the acrosome reaction. During folliculogenesis a glycoprotein layer called the zona pellucida is deposited around the oocyte. The zona serves a dual role; one of protection as the oocyte travels through the female reproductive tract, and one of specificity, as a cellspecific recognition site for spermatozoa. The zona pellucida is comprised of three glycoproteins, designated ZP 1- 3. ZP3 is the protein that provides recognition and specificity for interactions with sperm. Upon binding of the sperm to ZP3, the acrosome reaction is initiated. The acrosome reaction consists of focal fusions of the outer acrosomal membrane with the plasma membrane of the sperm, resulting in release of the contents of the acrosomal vesicle. A consequence of the acrosome reaction is that a protease on the spermatozoa surface, called acrosin, is exposed. Acrosin acts on the zona pellucida matrix, dissolving it in advance of the penetrating sperm. Additionally, the acrosome-reacted sperm can now bind to ZP2 in what is known as the second phase of sperm-zona interaction. The acrosome reaction also leaves the spermatozoon with the capacity to recognize and fuse with the plasma membrane of the oocyte. Shortly after the two membranes fuse, there are a series of periodic membrane hyperpolarization events and calcium fluxes. The calcium changes induce the secretion of the cortical granules, located on the undersurface of the plasma membrane. The release of cortical granules causes zona hardening; ZP2 is cleaved, the zona becomes less penetrable and loses the capacity to bind spermatozoa. A second block to polyspermy (fertilization by more than one sperm) is the resistance of the plasma membrane of the oocyte to fuse with additional sperm probably due to the change in polarization of the oocyte.

Figure 1: Timing of events in the female reproductive tract. Reprinted from Human Physiology 2nd edition, Stuart Ira Fox Ed. William C. Brown Publishers copyright 1984. Within the oocyte cytoplasm the sperm nucleus decondenses, and sperm protamines are exchanged with histones. The fertilized egg is now referred to as a zygote. The male and female DNA must find each other in the relatively large space of the zygotic cytoplasm. This is accomplished through formation of a large microtubule based structure, the sperm aster. Transport along the microtubules brings the female pronucleus to the male pronucleus. In mammals, the genetic material from the male and female does not mix until the first mitotic division. The first mitotic division in mammals takes about 24 hours; thereafter, subsequent divisions occur approximately every 20-24 hours. The first several mitotic divisions occur with no increase in mass, resulting in smaller cells following each division. The cells of the preimplantation embryo are called blastomeres. At this stage, one or more cells can be removed (i.e., for preimplantation genetic diagnosis) and the remainder will form an intact, normal embryo/fetus. At the early cleavage stages embryos which split in half form identical twins. These cells are able to form all parts of the developing organism and the extraembryonic contributions to the placenta and thus are termed totipotent. At the stage when the human embryo has 32 cells it will have undergone compaction. This process is a flattening of the cells against each other and represents the first differentiation or limiting of developmental competence of the developing embryo. The outer cells become a polarized epithelium. These cells will become the trophectoderm and for the extra embryonic structures of the developing embryo. This epithelium is also necessary for formation of the blastocoelic cavity as ions are pumped across the

epithelium and water follows. The inner cells are not polarized and remain pluripotent, destined to form all tissues of the embryo proper. These cells are the precursors of embryonic stem cells (see below). The blastocyst is the next stage of development and is characterized by a fluid-filled cavity surrounded by trophectodermal cells. The inner cell mass, the precursor for embryo development is found attached to the inside of the blastocyst Implantation is the adherence and attachment of the blastocyst to the endometrium following hatching from the zona pellucida. The glycoproteins of the zona allow the cleaving embryo to transit through the oviducts without sticking and implanting ectopically. However, once the blastocyst arrives in the uterus, it must escape from the zona. Once fully expanded, the blastocyst releases proteases, which digests a small hole in the zona, through which the blastocyst can squeeze out. In humans, implantation occurs during the second week, approximately 7 - 10 days following fertilization. The ovarian and uterine reproductive cycles must be in synchrony for implantation to occur. Normal implantation takes place in the endometrium of the body of the uterus, most frequently on the upper part of the posterior wall, near the midline. Prior to implantation, the ovarian hormones induce a transformation of the endometrium, which is further augmented by the trophoblast. This transformation, termed the decidual reaction, results in greater endometrial vascularization, differentiation of stromal fibroblasts to decidual cells, modification of the extracellular matrix, alterations in glandular secretory functions and cellular proliferation. If there is no implanting embryo, then the decidualized endometrium will be shed (menstruation). Implantation consists of 3 phases: attachment, invasion/degradation and proliferation. Attachment requires the down-regulation of molecules that prevent attachment and the up-regulation of molecules that promote attachment. In mammals, 2 carbohydrates, LNF-1 and heparin sulfate, support the initial attachment. In order for the blastocyst to implant it must degrade the endometrial epithelial lining. It does so by release of degradative enzymes, including collagenase, stomelysin and plasminogen activator. Once the blastocyst broaches the epithelial border, the trophectoderm will proliferate and invade the stroma. Infertility Successful conception requires all of the described steps to be completed flawlessly and timed appropriately. Infertility is defined as failure to conceive after 1 year of trying. It is estimated in this country that 15-17% of couples are clinically infertile. The consequences of infertility, while not life threatening, are demoralizing and treatment for infertility represents a growing segment of the medical industry. Most male and female infertilities can be traced to failure to properly form gametes (azoospermia, anovulation) or failure of gamete transport. A smaller number of infertilities can be traced to failures all along the fertilization pathway, for example there are sperm incapable of undergoing the acrosome

reaction and thus cannot penetrate the zona pellucida. Oocytes have been identified which cannot be activated and sperm have been found which fail to form a proper sperm aster and thus cannot unite the genomes in the zygotic cytoplasm. Failure to have a proper ovulatory cycle will be discussed in the small groups and can be regulated in some cases with hormonal therapy. Gamete transport issues can be solved with surgery to remove adhesions or blockages or by first isolating the gametes from each parent and performing in vitro fertilization (IVF). During IVF gametes are combined in a Petri dish and embryo cleavage is carried out in vitro. When the embryos have developed to the blastocyst stage they are scored for quality and transferred to the uterus of the genetic mother or a surrogate. In some cases of male infertility there are too few sperm produced to use for in vitro fertilization. Recently a technique known as intracytoplasmic sperm injection (ICSI) has been developed in which the sperm is placed directly into the oocyte using a microneedle. This technique, while successful remains controversial, as some reports have indicated twice the number of birth defects following ICSI when compared to IVF.

Human Embryonic Stem Cells In 1998 two independent research groups successfully derived human embryonic stem cells. These cells were notable for their pluripotent nature and the ability to remain undifferentiated in culture indefinitely. Totipotent refers to cells capable of forming all cells of the fetus and extracellular membranes. The only commonly accepted totipotent cells are those of the precompaction embryo. Pluripotent cells are capable of forming many lineages but not all. hESC are generally considered to be pluripotent but there is evidence that these cells can form some of cellular types associated with the extraembryonic membranes. The generation of these cell lines sparked both immediate and emotionally charged public debate, as well as fantastical predictions about the many diverse ways in which these cells could be used in medicine. The use of Federal research monies to study these cells was originally banned as part of a long running policy banning Federal funding of research involving human embryos. This changed on August 9, 2001 as Former President Bush allowed Federal funds to be used to study a number of existing lines but not to be used for the derivation of new cell lines. President Obama is likely to modify this policy significantly

There are currently several types of stem cells and the arbitrary distinction between and among them is now beginning to be eliminated as biologists learn more about their fundamental natures. The following are some of the common terms, though most investigators are particularly intrigued by the entire class of Pluripotent Stem Cells (PSCs). PSCs include the following: 1.) Embryonic stem (ES) cells derived from the inner cell mass of blastocyst stage embryos; 2.) Embryonic germ (EG) cells, which are the precursors to the gametes (isolated from the primordial germ cells) obtained from aborted first trimester fetuses; 3.) The first pluripotent stem cells isolated were derived from germ cell tumors (teratomas) and are named embryonic carcinoma (EC) cells. These cells currently attract the least attention among stem cells but are being used in clinical trials for the treatment of stroke; and 4.) induced Pluripotent Stem Cells (iPS). iPS was developed in mice in 2007 and translated to humans in 2008. Through the use of four or fewer transcription factors it is possible to reprogram somatic cells back into their embryonic-like pluripotent state, similar to the events thought to occur during nuclear transfer or therapeutic cloning which has succeeded in mice and monkeys, but not yet humans. iPS was SCIENCEs 2008 Breakthrough of the Year.

Four experimental routes for nuclear reprogramming. Blue components represent the normal process of cell differentiation during development from a fertilized egg to adult cells or tissues. Red arrows represent nuclear reprogramming (A) by nuclear transfer to eggs, (B) by induced pluripotency iPS, (C) by lineage switching back to a branch point and out again in a different direction, and (D) by direct conversion. The lower part of the figure shows reprogramming by the generation of ES cells; these can be aggregated into an embryoid body (EB), made to differentiate in culture (diff), or transplanted to a blastocyst. In each case, various types of adult cells can be formed. [From: Nuclear Reprogramming in Cells. J. B. Gurdon and D. A. Melton Science 19 December 2008: Vol. 322. no. 5909, pp. 1811 1815]. Differentiation refers to that process whereby a cell acquires distinct and predictable characteristics. Human ES, EG, EC and iPSCs cells have demonstrated the capacity to differentiate into all of the tissues of the adult organism. The key to the use of these cells in therapy is to ensure that they do not form cancers when transplanted and also to differentiate them into the stable cellular lineages, while preventing differentiation towards undesirable cell lineages. The first diseases likely to benefit from stem cell technologies are those resulting from a single specific cellular defect i.e., Parkinsons disease (dopaminergic neurons), or Type 1 diabetes (beta-islet cells). Directed differentiation towards dopaminergic neurons has been accomplished and preclinical trials are underway in animals. Only limited progress has been made towards the differentiation of beta-islet cells but large research resources are aimed at solving this problem. One of the hurdles to the directed differentiation harkens back towards embryology when specific 3-dimensional architecture is needed to form organ structure and tissue gradients are necessary to establish differentiation.

LECTURE # 9 The Placenta Friday, February 13, 2009 11:00 am- 12:00 pm Georgia K. Duker, Ph.D. Learning Objectives: The general learning objectives of this lecture are to: 1. Differentiate among the extraembryonic membranes and how they contribute to placental formation. 2. Understand the development, anatomy and histology of the normal placenta 3. Know the cells that contribute to the placenta: fetal (cytotrophoblasts & syncytiotrophoblasts) and maternal (decidual cells & spiral arteries) 4. Discuss the process of implantation, including the chemical signals and receptors. 5. Illustrate the difference between a primary, secondary, tertiary and term chorionic villus. 6. Discuss the remodeling of maternal spiral arteries, and complications of preeclampsia. 7. List the physiological functions of the placenta. 8. List the hormones secreted by the placenta.

Introduction: The placenta is a unique organ. A complex structure, in a mere 40 weeks, the placenta grows to weigh just under 500 grams, and then the entire organ is sloughed off as afterbirth. It is a intermingling of fetal and maternal tissues in a very precise arrangement. The placenta is as invasive as a tumor. Yet, for the fetus it serves as lung, intestine, endocrine gland, kidney and immune shield.

Transport Functions of the Placenta Given its diverse functions, a variety of transmembrane transport capabilities are necessary for the fetal placental cells (syncytiotrophoblasts).

gases IgG ions glucose amino acids LDL pathogens

Passive diffusion Receptor-mediated endocytosis Active transport ATP-pumps Facilitated diffusion Facilitated diffusion Receptor-mediated endocytosis alcohol drugs Rubella, cytomegalovirus, herpes simplex, syphilis, HIV

Formation of the Fetal Membranes The fetal membranes include the chorion, the amnion, the yolk sac and the allantois. With the exception of portions of the yolk sac and the allantois, the fetal membranes do not form part of the fetus, but are extra-embryonic structures. The chorion is a complete ball. At 5 weeks gestation, chorionic villi extend out in all directions. As the embryo rapidly expands (8-10 weeks gestation), the overlying chorion is pressed flat, and villi persist only at the implantation site. In these same figures, note the amnion that develops from the inner cell mass to directly surround the developing embryo. Since the embryo has been completely embedded into the maternal endometrium, there is also a layer of decidual cells that surrounds the chorion. Therefore, in the early weeks of gestation, one can distinguish the following layers: the embryo, a distinct amniotic cavity, amnion, chorionic cavity, chorion, decidual layer and uterine cavity.

By 15 weeks gestation, the rapidly growing embryo and amnion have expanded, obliterating the chorionic cavity and fusing the amnionic membrane onto the chorionic plate. During labor, this amniochorionic membrane is what will rupture to release the aminotic fluid. By 22 weeks, the overlying decidual cells have been pushed across the uterine lumen and are now fused with decidual cells on the opposite wall, thus obliterating the uterine cavity.

Placental Development: The conceptus After fertilization in the mid-Fallopian tube, the conceptus is propelled toward the uterus, all the while undergoing mitotic divisions: Day 2-3 (post-fertilization) the morula (~100 cells) arrives in the uterus Day 5 (post-fertilization) - a blastocyst develops with a fluid filled center, and cells differentiated into surface trophoblasts (give rise to extraembryonic membranes), and the inner cell mass (give rise to the embryo proper). While in transit, an outer glycoprotein coat, the zona pellucida, has prevented binding to the maternal tissues. Day 6 (post-fertilization) - the blastocyst releases a protease that digests the zona. The blastocyst now hatches from the zona pellucida, and attachment is now free to begin. Days 7-8 (post-fertilization) - Normal implantation takes place in the endometrium, most frequently on the upper part of the posterior wall of the body of the uterus. Day 9-10, post-fertilization, the embryo is completely embedded in the uterine wall, and has been sealed off by the healed epithelium.

Hillier, Kitchener, Neilson, Scientific Essentials of Reproductive Medicine

Implantation Implantation can only occur in a hormonally primed uterus. The current menstrual cycle has prepared the endometrium: glands fill with nutrient-rich secretions stromal cells transform into glycogen and lipid-ladened decidual cells endometrial vasculature has proliferated endometrial epithelial cells express surface adhesion molecules.

Remember, the endometrial glands only produce one cycle of secretions for immediate nutrition. During formation of the placenta, it is the maternal decidual cells that coordinate implantation. These specialized stromal cells secrete cytokines to increase trophoblast attachment, and also fibronectin and laminin to impede the depth of trophoblast invasion. A decidual reaction can occur in stromal cells of the peritoneum, ovary or Fallopian tubes, and accounts for the possibility of ectopic implantation.

Days 20-24 of a regular 28 day menstrual cycle are the optimal window for implantation. If there is no implantation, then the decidualized endometrium will be shed during menstruation. Implantation consists of three steps: attachment, invasion and proliferation A. Attachment Uterine receptivity depends on signals from both the endometrium and the trophoblasts. The endometrium secretes leukemia inhibiting factor (LIF) and displays heparan-binding epidermal growth factor-like factor (HB-EGF). The trophoblasts express EGF-receptors and heparan sulfate proteoglycans. The interactions of EGF and heparan sulfate are obvious. The requirement for LIF is more circumstantial; diminished secretion of LIF is associated with recurrent pregnancy loss. These interactions (and probably others) stimulate the trophoblast cells to differentiate into two populations: the syncytiotrophoblasts, an outer, fused layer that interfaces with the endometrium; and the cytotrophoblasts, a proliferative cell layer facing the blastocyst cavity. At the same time, these interactions between the trophoblasts and the uterine epithelium initiate changes in the endometrial decidual cells. These stromal cells activate a variety of signaling genes that result in decidual remodeling and facilitate trophoblast invasion. The decidual cells produce proteases and prostaglandins.

Norwitz et. al. NEJM 345:19, 1400-1408.

The presence of a blastocyst ready to implant is required for decidual cell induction of COX2 enzymes for prostaglandin synthesis. Prostaglandin I2 is required for implantation. COX2 production is induced only at the site of implantation. Elsewhere in the uterus, COX 2 is not induced, and COX 1 synthesis is blocked by rising progesterone and estrogen. Clinical implications of this are evident in rheumatoid arthritis. In RA, successful implantation can be a problem when women of reproductive age are (or at least were?!?) liberally treated with COX2 inhibitors. On the other hand, administration of exogenous prostaglandins (intraveneously, intraamniotically or vaginally) induces abortion at any stage of gestation. So, prostaglandins are lower in the uterus throughout pregnancy, except for the specific site and time of implantation, and, at the very end of pregnancy, partuition. B. Invasion A plethora of fetal and maternal changes facilitate invasion of the blastocyst. 1. Desmosomesapoptosis Fetal/maternal interactions decrease desmosomes in the endometrial epithelium. This eventually results in apoptosis and permits syncytiotrophoblast invasion. 2. Proteinases - Syncytiotrophoblasts secrete proteinases (matrix metalloproteinases) that digest through the epithelium and into the decidual cell stroma. The decidual cells release their stores of lipid and glycogen to nourish this pre-placental phase. These erosive enzymes also open up maternal blood vessels, which deposit blood in growing gaps within the syncytial layer. 3. Integrins - Invading syncytiotrophoblasts increase expression of vascular adhesion molecules (integrins), and decrease production of adhesion molecules of the cytotrophoblast stem cells. 4. Immune modulation - Syncytiotrophoblasts express high levels of HLA-G, a MHC I molecule with very limited polymorphisms. Therefore, syncytiotrophoblasts will not vary significantly from the HLA-G of the mother, decreasing the possibility of rejection. Syncytiotrophoblasts also secrete interleukin-10, a cytokine that inhibits lymphocyte responses. The maternal tissues also contribute to the immuneprotective environment. Decidual cells secrete localized prostaglandins (at implantation) that suppress T-cells. Maternal leukocytes secrete interleukin-2 to interfere with maternal rejection. 5. Progesterone maintenance - Syncytiotrophoblasts also secrete human chorionic gonadatropin (hCG), which acts as a signal to the corpus luteum to continue secretion of progesterone (and estrogen). Unless supplied with continuous progesterone, the uterine endometrium will be shed in a menstrual bleeding. Excess hCG spilling into the urine becomes the basis for the pregnancy test. Later, by 8-10 weeks, the synctiotrophoblasts themselves will take over synthesis of progesterone.

Subsequent production of estrogens requires the cooperative effort of the fetal adrenal gland. 6. Invasion depth Proper syncytiotrophoblast invasion depth is essential for placental function. Normally, the invading tissue extends through the endometrium and into the outer third of the myometrium. The importance is most apparent when defects of excessive or inadequate invasion occur. Excessive invasion Deficient decidual development and firmer placental attachment into or through the myometrium can result in maternal hemorrhage. Inadequate invasion Associated with preeclampsia. With limited endovascular invasion, syncytiotrophoblasts fail to switch to vascular adhesive molecules. As a consequence, the uterine arterioles remain small-bore, high-resistance vessels, and cannot meet the circulation demands of later pregnancy.

C. Proliferation Cell proliferation continues, and chorionic villi are formed in three stages: 1. Primary villi form from the initial trophoblast shell, as the cytotrophoblasts proliferate with an outer, fused, syncytiotrophoblast layer exposed to the maternal tissues. Gaps, called lacunae, form in the syncytiotrophoblast layer as it invades into maternal blood vessels. These lacunae are filled with maternal blood that now bathes the syncytiotrophoblasts.
Hillier, Kitchener, Neilson, Scientific Essentials of Reproductive Medicine, p313 d,e,f

1o villi

2o villi

2. Secondary villi form from the invasion of extraembryonic mesoderm into the primary villi. Together these three layers: syncytiotrophoblast, cytotrophoblast and extraembryonic mesoderm form the chorion.

3. Tertiary villi form when embryonic blood vessels invade the connective tissue core of the secondary villi. Tertiary villi are functioning at about 4-5 weeks postfertilization. The embryonic heart is already beating, and red blood cells are being produced in the yolk sac. Cytotrophoblast cells break through at the bottom to anchor the villi to the maternal decidua.

3o villi

As gestation proceeds, the tertiary villi continue to mature. By the third trimester of pregnancy, the blood vessels occupy a larger proportion of the villi. Few cytotrophoblast cells are seen, and the nuclei and cytoplasm of the synctiotrophoblast are cluster into synctial knots. The actual diameter of the villi also decreases with maturation, as branching increases. Therefore, the final diffusion barrier of the placenta is: the attenuated syncytiotrophoblast, its basement membrane, the basement membrane of a fetal blood vessel, and the fetal endothelial wall. Chorionic villous sampling is used to detect abnormalities in fetal chromosome number. Samples of villous tissue can be obtained after 10 weeks of gestation using ultrasound guidance. Because the trophoblast cells are rapidly dividing, karyotypes can usually be obtained within 24 hours. Placental Villous Tree Structure Placental villi are organized into lobular units called cotyledons, which are visible from the maternal side of the placenta. Cotyledons form when wedge-like septa of decidual tissue grow from the maternal side toward the chorionic plate. The structure of villi is reminiscent of tree branches; as one moves from the chorionic plate towards the terminal villi, the branches get smaller and are more numerous. This branching allows for maximal surface area for gas and nutrient exchange.

Fetal Hemoglobin Binding Curve The maternal blood in the open intervillous spaces is a mixture of incoming arterial blood and exiting venous blood. Therefore, while the O2 content of maternal arterial blood is near saturation, 100% (at 80 mm Hg partial pressure O2), the lacunar blood is about 70-80 % saturated. By the time the fetal placental layers impede diffusion, the partial pressure of O2 in the fetal umbilical vein is 25-30 mm Hg. It is therefore essential that the fetal form of hemoglobin has a higher binding affinity for O2 than adult hemoglobin. This assures that the fetal hemoglobin (Hb, 2, 2) is capable of attracting O2 away from the maternal Hb. The P50 value for fetal hemoglobin (i.e., the partial pressure of oxygen at which the protein is 50% saturated; lower values indicate greater affinity) is roughly 19 mm Hg, whereas adult hemoglobin has a value of approximately 26.8 mmHg.

Maturation of Uteroplacental Blood Flow Where branches of tertiary villi contact the maternal endometrium, cytotrophoblast cells penetrate the synctiotrophoblast to create an anchoring villi. Proteases from invading cytotrophoblasts erode and remodel branches of the uterine spiral arteries. These enlarged vessels empty into the lacunar spaces between developing villi to establish the primitive uteroplacental circulation (see diagram above). Cytotrophoblast cells and maternal vascular smooth muscle cells regulate maternal blood access. Notice that the fetal circulation is within closed vessels of the villi, while the maternal circulation pools in the open intervillous spaces, lacunae.

Hormone Production by the Placenta Syncytiotrophoblast (Fetal) 1. Proteins a. Human Chorionic Gonadatropin, hCG Secreted as early as 6-7 days after fertilization; Binds to the LH receptor to maintain the corpus luteums secretion of progesterone. Production drops after 8-10 weeks. b. Human Placental Lactogen, hPL (or Human Chorionic Somatotrophin) Breast and general growth 2. Steroids a. Progesterone Synthesized from cholesterol precursors; express LDL receptors to bind LDL from maternal blood. Begin by day 16; major producer of by 5-6 weeks. b. Estrogen - Placental progesterone travels to the fetal adrenal cortex where it is converted into androgens. These, in turn are used by the syncytiotrophoblasts to form estrogens.

Decidua (Maternal endometrial stromal cells) Under the influence of progesterone, the stromal cells of the endometrium become decidual cells. This transformation takes place to a small degree during the secretory phase of every monthly menstrual cycle. Decidual cells develop a dense secretory cytoplasm with stores of glycogen and lipid for nutrient support. Dercidual cells also releasing fibronectin, laminin and cytokines that serve to increase trophoblast adherence (therefore limiting invasion) and to promote blood vessel proliferation. Note that decidualization can also occur in stromal cells of the ovary, fallopian tubes, or even the peritoneum, thus facilitating ectopic pregnancies. 1. Prolactin (in addition to pituitary prolactin) 10-30 weeks, stimulate breast. 2. Relaxin - at partuition - soften cervix, pelvic ligaments & pubic symphysis 3. Prostaglandins - at implantation site = immune modulation - at partuition - form gap junctions in myometrium = coordinated contractions. 4. Cortisol, CRF and GnRH 5. Cytokines including VEGF and interleukin-15

Monday February 16, 2009 8:30 am 10:00 am Resolution of PBL #1 Initial Presentation of PBL #2
Proceed directly to small group rooms

Workshop #1: Androgens Monday, February 16, 2009 10:15 am 11:00 am Learning Objectives: 1. To gain an appreciation of the differing roles of androgens throughout development of the reproductive system in the male. 2. To gain an appreciation of the multiple sites of possible perturbation of androgen signaling and of the variable impact such perturbations can have on sexual development. 3. To review the use of the OMIM database in the evaluation and diagnosis of a patient. 4. To consider the role of phenotype and sex of rearing on gender identity.

The following material should be reviewed by students in preparing for this workshop. Cases to be discussed are attached. Dr. Lance-Jones' lecture on Sexual Differentiation Dr. Orwig's lecture on Reproductive Physiology of the Male In addition, please review your notes from Endocrinology relevant to todays topic. Come to class prepared to discuss the testosterone synthesis pathway, androgen binding and signaling. PROCEED DIRECTLY TO THE SMALL GROUP ROOMS.

Case 1 A patient is referred to your endocrine clinic because she is 17 years old and has not as yet shown breast development, nor has she had a menstrual period. Physical exam revealed female external genitalia with triangle shaped pubic hair.

Work up: Her karyotype reveals 46 XY; pelvic ultrasound shows no internal genitalia and bilateral masses in the inguinal region. The vagina ends in a blind pouch.

Plasma hormone levels are reported below: Basal Testosterone Testosterone precursors DHEA 17 hydroxyprogesterone androstenedione, etc LH FSH Inhibin markedly elevated normal normal no increase in Testosterone exaggerated increase in LH normal FSH response normal result low (0.7 nmol/L)

all low

hCG stimulation test: GnRH stimulation test: ACTH stimulation test: Question 1:

What conclusion arises from the observed absence of internal genitalia?

Question 2:

What is the likely explanation of the female phenotype with a 46XY genotype?

Question 3: Based on the hormone data, describe the likely appearance of a biopsy of the inguinal mass.

Question 4: Discuss the information obtained in the hormone data and stimulation tests to identify a likely cause of the defect in this patient. Suggest at least two molecular defects which could present with this phenotype and explain how one could differentiate between them.

Case 2 This patient is a 27 year old woman who presented with primary amenorrhea. Physical exam revealed normal female external genitalia, a right inguinal hernia and a 6 cm deep, blind-ended vagina. Karyotype of this patient was 46XY; pelvic ultrasound showed no evidence of internal Mullerian structures. Breast development had occurred at 14 years. Pubic hair was course and dark and grew in an indeterminant pattern. Hormone studies of this patient were as follows: Plasma LH: elevated FSH upper end of normal range Testosterone: normal range for mature male hCG stimulation test: normal (for male) increase in Testosterone Other endocrine tests: normal

Question 1: What is a likely diagnosis based on phenotype and hormone data?

Question 2: Does this person have gonads? If so, predict the histological appearance of the gonad. Predict the appearance of any internal genitalia. Explain your conclusions.

Question 3: What finding is inconsistent with a diagnosis of complete testicular feminization?

Question 4: How would you proceed in this case? Give several examples of tests to be used to further understand this case.

Question 5: Discuss treatment issues regarding this patient.

Case 3 A 13 year old girl is brought to your office in some distress over what appears to be progressive virilization. Her muscularity is increasing, her voice is cracking, and she has felt a mass in one of her labia. Physical exam reveals a healthy 13 year old girl with female external genitalia. There is a palpable mass in her left labia, and a slight increase in labial pigmentation. She has no evidence of breast development and has not started her periods. Pelvic ultrasound reveals no ovaries and no uterus. However, there is one abdominal testis, and what appears to be normal vas deferens, seminal vesicles and epididymis. The labial mass is testicular tissue. A karyotype reveals 46XY Endocrine work up: LH: elevated FSH: normal Testosterone: elevated Adrenal status: normal

Question 1: Using the hormone data and physical findings, explain the likely defect in this patient.

Question 2: What additional data would you like to have on this patient?

Question 3: What is the prognosis for this patient? What would you recommend as treatment, if any?

Workshop #2 Monday, February 16, 2009, 11:00 am 12:00 pm The following table describes the work-ups of seven phenotypically female patients who presented with infertility. In the spaces provided, predict what you think hypothalamic GnRH levels will be, identify the problem for each patient and suggest a treatment to restore fertility. Patients 1 2 3 4 5 6 7 Physical exam/ Immature nl adult nl adult nl adult app/ nl adult/ no nl adult/ no m. nl adult/ regular Symptoms appearance/ appearance/ appearance/ 10 amenorrhea/ recent menstrual cycles recently/ menstrual cycles 0 0 0 1 amenorrhea 2 amenorrhea. 2 amenorrhea. no pubic hair cycles lactating FSH/LH levels low low both high both high low low normal/ normal normal/ normal

Estradiol/ Progesterone Predicted GnRH level Response to GnRH injection

low/ undetectable

low/ undetectable

low/ undetectable

low/ undetectable

very high/ undetectable

low/ undetectable

positive (increased LH)

negative (no increase in LH)

positive (increased LH)

positive (increased LH)

positive, but amplitude of LH response decreased

positive (increased LH)

positive (increased LH)


a. Under physical exam, nl adult means the patient looked like a mature adult female. 10 amenorrhea means the patient never had a menstrual cycle; 20 amenorrhea means that at some time in her life, menstrual cycles were normal, or had at least occurred. b. Under response to GnRH injection, patients receive an i.v. injection of a standard dose of GnRH, and then blood samples are collected to characterize the induced secretion of LH and FSH. All patients are < 30 years of age.

CONTRACEPTION Jennifer Hayes, M.D. Lecture 10 - February 17, 2009 8:30 am

Learning Objectives: 1. To understand the mechanism of action of each major contraceptive method. 2. To learn the difference between theoretic use and (actual) effectiveness of a contraceptive method. 3. To understand the non-contraceptive advantages of each method. 4. To discuss issues of compliance and convenience for each method. 5. To learn how to talk to patients about contraception. REFERENCES: Appropriate sections in Comprehensive Gynecology, Herbst AL (editor), The Menstrual Cycle, Ferin M (editor), Understanding Human Sexuality, Hyde JS (editor), A Clinical Guide for Contraception, Speroff N and Darney P (editors). Websites: Association of Reproductive Health Professionals (http://arhp.org) Educational materials available on family planning, contraceptives and other reproductive health issues, including STDs, HIV, urogenital disorders, menopause, sexual health and infertility. CME-accredited programs available to professionals. Alan Guttmacher Institute (http://www.agi-usa.org) Research and policy organization affiliated with Planned Parenthood. Extensive resources on multiple topics in sexual health, such as statistics and policy papers related to contraception, abortion, STDs, youth sexuality practices, and pregnancy.


Why does the medical profession care about contraception?

Each day approximately 100 million sex acts occur worldwide. Of the 175,000,000 pregnancies that occur annually, approximately 75,000,000 (~43%) are unintended. Depending on the study population, the unintended pregnancy rate is as high as 64%. Over 50,000,000 abortions occur worldwide each year. In the United States, approximately half of pregnancies are unintended; half of these will result in abortion as an outcome. In half of

the 1.3 million abortions occurring in the U.S. each year, a contraceptive method being used failed to prevent pregnancy. The importance of good family planning is underscored by the fact that unintended pregnancies are associated with higher risks for both mother and infant. In the United States the annual maternal mortality rate is 7.5 maternal deaths per 100,000 live births, which far exceeds the goal of 3.3 deaths/births set in 1987 for Healthy People 2000. At least 20 other countries have lower maternal death rates than does the U.S. (Source: National Institutes of Health, 2000
and National Center for Health Statistics, CDC)

Essential background information needed for patient care needs relative to contraception include principles of sexual history taking, patient education, counseling, and informed consent. It is also imperative that the physician has an awareness of the patients social, religious, and cultural background, as well as a thorough understanding of the basic reproductive anatomy and physiology, i.e. the contents of this course. II. Definitions and terms relevant to contraceptives. A. Mechanism of Action. Mechanism of action refers to the specific way that a contraceptive method prevents pregnancy. Importantly, some methods have multiple actions to prevent pregnancy. Mechanism is also an important factor in the discussion of prevention of sexually transmitted diseases. Students should understand how each contraceptive functions and to develop the ability to tailor their approach to each patients needs. B. Effectiveness. Effectiveness addresses the degree of reliability of each contraceptive method to prevent pregnancy. Effectiveness is often described in terms of perfect use and typical use over one year. These rates are usually expressed as percentages. For example, 90% efficacy implies that 90 of 100 women using that method over one year would not get pregnant and 10 would get pregnant. The three major components of effectiveness include the type of contraception (method of action), the performance of the user and the degree to which the clinician has informed and educated the patient. C. Cost. Health care providers need to be aware of cost and how that may influence patient decisions regarding contraceptive choices. Of note, many insurance plans will not cover the cost of prescription contraceptives. D. Non-contraceptive advantages. An essential component of any discussion of contraception must include the potential health benefits of a particular method. Additionally, it is important to have a simultaneous discourse on prevention of sexually transmitted diseases (STDs). E. Disadvantages and Cautions. Each patient is unique both biologically and emotionally. A discussion of potential negative effects should be

presented for each contraceptive for any particular patient. In addition, possible negative effects should be compared with other contraceptive options. Remember that pregnancy is a negative effect. III. TYPES OF CONTRACEPTION A. Natural Family Planning: NFP utilizes signs, symptoms and menstrual cycle data to determine times of low fertility. NFP requires a good understanding of the menstrual cycle, rigorous monitoring of fertile periods, and strict adherence to abstinence during potentially fertile times. The three components of NFP are as follows. 1. The calendar calculation. A record of menstrual history is maintained for 6 cycles. The shortest and longest cycle lengths are then used to estimate the timing of the fertile period. The onset of fertility is calculated as the shortest cycle length minus 20 days. The last day of the fertile period is calculated by subtracting 11 from the longest cycle. Sperm viability is generally about 2-3 days but can be as long as 6-7 days. The ovum can only be fertilized within the ampullary portion of the fallopian tube during the first 24 hours after ovulation. 2. Cervical mucus changes. The woman needs to check her cervical mucus daily for amount and consistency in order to identify when ovulation has likely occurred. Typically, the woman must use her fingers to feel for her mucus at the cervical os. Following menses, estrogen levels are low and a minimal amount of cervical mucus is produced. As the dominant follicle develops and estrogen levels increase, cervical mucus production increases. The mucus is initially thick and rubbery and gradually becomes more thin, stretchy and transparent as estrogen levels peek and ovulation approaches. After ovulation, a rapid increase in progesterone levels causes an abrupt drying of the mucus, which again appears as thick and rubbery. 3. Basal body temperature. This method is based on the assumption that early morning body temperature (prior to getting out of bed) will increase noticeably (0.40 0.80 F) with ovulation. The elevation in BBT should persist for 3 days, after which the woman will enter a postovulatory infertility period. Obviously, this method is not for predicting the beginning of the fertile period. Use of BBT can get tricky as a woman enters perimenopause. Proper performance of NFP requires significant instruction from a health care professional and offers no protection from STDs. Ideally, the woman needs to use all three methods concurrently since menstrual cycle irregularities can occur with stress and as a woman gets older, emphasizing the need to confirm when ovulation has occurred with changes in cervical mucus and BBT.

B. Withdrawal: The male removes his penis from the vagina prior to ejaculation, which prevents the deposition of sperm into the female reporoductive tract. Because ejaculation of semen usually begins before the male senses climax and ejaculation, the method has a high failure rate. C. Lactational Amenorrhea Method: LAM is a highly effective, natural method of contraception thought to work by suppressing ovulation. Requirements for use include a mother that is nursing at least 80% both day and night, with no regular nutritional supplements to the infant, and no return of menstruation. LAM is highly effective for the first 6 weeks after delivery but is not recommended as an effective method of contraception beyond 6 months after delivery. D. Barrier methods 1. Male condom: Currently the only reversible contraceptive for men. Condoms are readily available and relatively inexpensive. They function by preventing direct contact of sperm with the ovum by preventing release of sperm into the vaginal canal. Most condoms are made of latex. Polyurethane (plastic-based) condoms should be used if EITHER partner has a latex allergy. Although latex condoms are currently the gold standard for protection against STD transmission, they are not 100% effective, especially for the viral STDs such as hepatitis B virus, herpes simplex virus and HIV. Data is lacking regarding the efficacy of polyurethane condoms for STD prevention. 2. Female methods: female controlled barrier methods are intended for use within the vagina, potentially without the knowledge of the male partner. a. Female condom: The Female Condom (TFC) is a polyurethane sheath with a flexible, polyurethane ring at each end, pre-lubricated on the inside with a silicone-based lubricant. Unlike the male polyurethane condom, the female condom does provide both pregnancy prevention and STD prevention. On the positive side, a woman can usually control the use of the condom without having to enter a debating contest. On the negative side, it can be difficult to figure out how to place the condom correctly. There is also the possibility that the polyurethane ring will rest on the clitoris; some women find this stimulating, while others feel this is irritating or even painful. b. Diaphragm: A properly inserted diaphragm will cover the cervix and prevent sperm from entering the uterus. Structurally, the diaphragm is a dome-shaped latex cup with a flexible rim (there are a variety of rim constructions that affect flexion of the diaphragm for insertion and removal). The diaphragm is used concurrently with

spermicide, which is applied to the concave surface. The posterior rim of the diaphragm rests in the posterior fornix, and the anterior rim is placed behind the public bone. Diaphragms are sized by a clinician to fit an individuals vagina. c. Cervical cap: There are two cervical caps available in the United States. Spermicide should be added to the cap before insertion. The FemCap (www.femcap.com) is made of silicone and is shaped like a sailors cap, with the brim pushing out into the fornices, holding a dome over the cervix itself. A soft strap across the top of the dome The cap comes in three sizes based on gravidity and parity. The small size is for nulligravid women, the medium size is for gravid women who are nulliparous, and the large is for parous women. Leas Shield (www.leasshield.com) is made of silicone and has a cup-shaped bowl that covers the cervix without resting on it. A ring protrudes up from the bowl to rest behind the symphysis pubis and a one-way valve allows air and fluid to flow out of the bowl (creating a suction fit). One size fits all female patients. d. Spermicides: Spermicides consist of a chemical detergent, typically nonxynol-9, and a carrier. Nonoxynol-9 is a surfactant that works to destroy the sperm cell membranes. Spermicides can be administered as a foam, gel, cream, film or suppository. The best protection is provided when they are used in conjunction with another barrier method (i.e., condom, diaphragms, or cap) but they can be used alone. Significant protection against gonorrhea, chlamydia, hepatitis B and HPV was suggested in laboratory testing but did not occur in human clinical trials. The relationship of spermicide use with transmission of HIV is complex due to the cidal effect on epithelial cells; some recent studies suggest that use of nonoxynol-9 products by sex workers may increase the transmission of HIV. Spermicides were released on the market decades ago without the usual FDA testing that would occur today. Studies are currently underway to test microbicides (www.microbicide.com), which are spermicides that also prevent sexually transmitted diseases. Unfortunately, the first few products to get to the HIV testing stage have failed to be successful. e. Contraceptive sponge: Although removed from the market in the USA in 1995 for reasons unrelated to product efficacy, the Today sponge (www.todaysponge.com) is available again as of 2005. This sponge consists of a polyurethane foam carrier with 1000 mg of nonoxynol-9. It acts as a mechanical barrier that holds the nonoxynol-9 over the cervix. Because it comes in a single size, women with large cervixes (mainly women who are parous) have higher failure rates. Sponges do not provide very good protection

against transmission of STDs, especially HIV. In fact, sponge use may result in an increased risk of HIV transmission by eroding the vaginal epithelium. D. Hormonal Methods of Contraception 1. Progestin-only (mini) pills: These are what they sound like, free of estrogen. The mini-pill contains 3 to 10 times less progestin (depending on formulation) than what is contained in COCs. The minipill functions primarily to thicken cervical mucus (see notes on fertilization). Since there is no estrogen in these pills, there are no estrogenic side effects. As with COCs there is less cramping and generally women will experience easier periods. The major side effect is intermenstrual bleeding or spotting. These menstrual cycle changes are sometimes disconcerting to women and are a frequent reason for discontinuing these pills. Mini-pills require strict regularity in pill-taking and attention to timing. When failures occur, the pregnancy is more likely to be ectopic (tubal) compared to rates of ectopic pregnancy in the general population. 2. Combined Hormonal Contraceptives contain an estrogen and a progestin. ESTROGEN: All currently marketed products contain the synthetic estrogen mestranol or ethinyl estradiol. Mestranol is only found in some oral contraceptive formulations containing 50 mcg of estrogen. EE is the estrogen that is used in all products that will be routinely used by clinicians in this facility. PROGESTIN: A progestin is a synthetic version of progesterone. There are 3 different types of progestins used in combined hormonal contraceptives: o Estranes: these are norethindrone, norethindrone acetate, and ethynodiol diacetate. These are only found in oral contraceptives. o Gonanes: these are norgestrel, levonorgestrel, norgestimate, norelgestromin, desogestrel and etonogestrel. Norgestrel, levonorgestrel, norgestimate and desogestrel are found in oral contraceptives. Norelgestromin is the active metabolite of norgestimate and is present in the contraceptive patch. Etonogestrel is the active metabolite of desogestrel and is found in the contraceptive ring. o Spirolactones: there is only one progestin in this family, drosperinone.

The mechanism of action of all Combined Hormonal Contraceptives is the same; inhibit ovulation by suppression of FSH (suppress

follicular development) and LH (suppress ovulation). Whereas progestin-only pills have just enough progestin to thicken the cervical mucus, the addition of estrogen in Combined Hormonal Contraceptives works to thin the cervical mucus. So as to maintain a balanced effect that leads to thickening of the cervical mucus, Combined Hormonal Contraceptives contain more progestin than is found in progestin-only pills. This higher amount of progestin is not only enough to create a thick cervical mucus in the presence of estrogen, but is also enough to inhibit ovulation and ovarian steroidogenesis. Combined Hormonal Contraceptives are marketed by the FDA to be used primarily in a cyclic fashion of 28 day cycles. Any CHC that contains a steady amount of hormone can be used in an extended regimen which continues the hormone for a longer period of time. Extended cycling will decrease the number of scheduled menses that a woman will experience although she may have more breakthrough bleeding, especially in the first few months of use. Only 2 products are approved by the FDA for extended cycling (see COCs). Combined Hormonal Contraceptives are highly effective when used as directed, with failure rates of 2% or less in one year. However, because all of these methods require user compliance, real life (or typical) failure rates are much higher than in studies. Typical use failure rates for COCs average around 7-8%. This rate will be slightly lower in a highly experienced and motivated COC user. However, the rate will be much worse in populations at higher risk for poor compliance. For example, inner city adolescents have a one year failure rate of approximately 20%. Typical use failure rates have not yet been determined for the contraceptive patch or vaginal ring. Combined Hormonal Contraceptives are highly effective, easy to use, and relatively inexpensive. Benefits include a decrease in the amount of blood lost during menstruation, regulation of irregular periods, shorter periods, decreased menstrual cramps and a reduced incidence of functional cysts. There is also a protective effect against ovarian and endometrial cancers (proven with COCs and likely to be similar for other Combined Hormonal Contraceptive methods). When desired, Combined Hormonal Contraceptives can be used to control the timing of menses for personal reasons (i.e., weekends and vacations). Of course, there are some negatives. Some women will experience headaches, nausea, mastalgia, mood changes or intermenstrual bleeding when they first start using the pill. The use of Combined Hormonal Contraceptives is NOT recommended for women who are

smokers over 35 years of age, hypertensive, or have a history of cardiovascular disease. In these circumstances other forms of contraception may be more appropriate. Despite good epidemiologic evidence to the contrary, the question of whether Combined Hormonal Contraceptives are associated with breast cancer will forever be of concern to women. It appears that exogenous estrogens do not cause new breast cancers; however, they may help to promote existing tumors. This may not be all bad, as these lesions are detected and thus treated earlier and these women have better survival. A. Combined Oral Contraceptives (COCs) Commonly referred to as the pill, COCs are oral pills that are to be swallowed once daily. Ideally the pill should be taken at the same time every day. One product (Femcon Fe) is available in a chewable product. Some formulations are monophasic (same amount of progestin and estrogen in each active tablet) and some are multiphasic (varying amounts of progestin and/or estrogen in each active tablet) products. Most products are marketed for one 28 day cycle of use. Most of these contain 21 active hormone pills and 7 placebo pills. Menses typically occurs on the second or third day of the pill-free interval due to hormone withdrawal. Return of fertility is immediate if a new package of pills is not started. Variations the standard 21/7 regimen include the following: Inclusion of iron pills instead of placebo pills (found in some LoEstrin products) Uses of hormones during the last 7 daysMircette contains 2 days of placebo followed by 5 days of EE 10 mcg (which is half the amount of EE used in the first 21 days) Cycling with 24 days of hormone and 4 days of placebo (ie, LoEstrin 24 and Yaz) Extended regimen with 84 active pills and 7 placebo pills (Seasonale). This product contains the same hormone in each pill as Nordette, Levlen and their therapeutic equivalents. Extended regimen with 84 active pills and 7 low dose estrogen pills instead of placebo pills (Seasonique). The 84 active pills contain the same amount of hormone as is found with Seasonale. The 7 low dose estrogen pills contain EE 10 mcg (like with Mircette). Many COCs are available in FDA-approved therapeutic equivalents. These are commonly called generic pills. Generic COCs are commonly given names, like brand pills, which can be confusing.

B. The Nuvaring (www.nuvaring.com) delivers ethinyl estradiol and etonogestrel, which is the active metabolite of the progestin desogestrel. Basically, this is a birth control pill in a ring. The ring is made of ethylvinyl acetate (a soft plastic) and is 5.5 cm in diameter, which is smaller than most diaphragms. The ring is inserted in the vaginathere is no right or wrong way to insert it and it rarely falls out (2.5% of women will experience one event over a one-year period where it falls out). The ring is designed to be left in for 3 weeks and then removed for one week. The ring is not typically removed for sexual intercourse. There is enough hormone in the ring to be effective for 4-5 weeks. Thus, a woman could choose to use the ring for 4 weeks and then take a week off (a 5 week cycle or 10 periods a year instead of 13) or even use the ring continuously, changing it every 4 weeks. Unlike with the patch or pill, women who want to use the ring continuously dont have to buy any extra contraceptives. Each ring releases about half the levels of hormone as an oral contraceptive. T C. Contraceptive Patch The OrthoEvra transdermal system (www.orthoevra.com) delivers ethinyl estradiol and norelgestromin, which is the active metabolite of the progestin norgestimate. Basically, this is a birth control pill in a patch. The pill that it is most similar to is OrthoCyclen. The patch is applied to the abdomen, buttock, upper outer arm, or upper torso (excluding the breast). The patch is applied weekly for three weeks. It is applied on the same day of the week each week the patch change day. It is preferable for the woman to place the new patch on a fresh area of skin to avoid skin reaction (although efficacy will not be affected if it is put in the same spot). There is a one-week patchfree period. Withdrawal bleeding usually occurs during the patchfree week. Ortho Evra may also be used continuously but estrogen related side effects and discontinuation for these side effects in continuous patch users is greater than in women using the patch cyclically. Following application of the Ortho Evra, the hormones reach reference levels within 48 hours. The patch can maintain serum concentrations of NGMN and EE in the target range through nine full days for the second and third patches being used in a given cycle. Hormone absorption was also tested under various environmental conditions such as a health club (sauna, whirlpool, treadmill) and in a cold water bath. With Ortho Evra use, 60% more estrogen is absorbed over a 21 day cycle than occurs with average COCs. There is no clinical evidence that this leads to more serious adverse events such as MI, stroke or pulmonary embolus. Studies are contradictory as to whether there might be a slight increase in the number of women who develop VTE, although this rate would still not exceed the rate of VTE in

pregnancy. Approximateley 3-4% of patches will partially or completely fall offthis translates to an average of 1-2 patches per year for each user. Overall, compliance is significantly better with the patch than the pill, but this does not translate into significantly higher efficacy. The compliance benefits are most notable for teens and young adults. 3. Implants: a. Norplant System: consists of six polyurethane rods containing a total of 35mg of levonorgestrel, a synthetic progestin. The hormone is released at a low, steady rate of 85 g daily initially, decreasing to 50 g at nine months, 35 g at 18 months, and 30 g a day thereafter. Contraceptive levels of levonorgestrel are maintained for at least 7 years although the method is only approved for use in the United States and other countries for 5 years. Norplant System is no longer distributed for new insertions in the United States. Norplant System works primarily by thickening cervical mucus. The capsules are inserted by a clinician under the skin (between the dermis and subcutaneous tissue), usually in the non-dominant arm. A major advantage is that use of this highly effective method requires no thought or effort for up to 5 years. Also, because this is a progestin-only method, there are no estrogenic side effects. On the down side, there are almost always menstrual cycle disturbances resulting in unpredictable bleeding episodes. However, with continued use, regular bleeding patterns become more common, although they are not guaranteed. Other side effects can include weight gain, mood changes, acne, mastalgia, and, rarely, hair loss. As with oral contraceptives, there is an interaction with anticonvulsants (except valproic acid), which results in increased failure rates. While about 15% of women who become pregnant while using Norplant System will have an ectopic pregnancy. Still, because pregnancy rates overall are so low, the overall risk of ectopic pregnancy is reduced compared to the general population. Finally, there is occasional difficulty removing the capsules when fertility is desired again, if insertion or removal is performed by an inexperienced provider. After removal, return of fertility is immediate. b. Norplant II (Jadelle in Europe): approved by the FDA but likely never to be marketed in the United States. The rods are made of a silicone elastomer containing a solid core of levonorgestrel. Each of the two capsules contains 75 mg of LNG which diffuses through the thin silicone elastomer in continuous low doses. The initial release rate is approximately 98 g/day which declines to 44g/day by 9 months, 39 g/day by 12 months, and 29g/day by 36 months.

Contraceptive protection is afforded within 24 hours of insertion and continues until removed or up to 3 years. After removal, the woman returns to her pretreatment level of fertility within 24-48 hours. Side effects are similar to Norplant System. Contraceptive levels of levonorgestrel are maintained for at least 5 years although the method is only approved for use in the United States and other countries for 3 years. c. Implanon: This single rod implant contains etonogestrel (the active metabolite of desogestrel). The method is effective for 3 years. In large trials, no pregnancies have been reported. Randomized trials comparing Implanon to Norplant show easier insertion and removal, as expected. Insertion of Implanon was completed in under three minutes in 90% of women (median one minute), and removed in under five minutes in 83% of women (median two minutes). Norplant, by comparison was inserted in under three minutes in 37% of women (median four minutes) and removed in under five minutes in 36% (median eight minutes). Unlike Norplant, bleeding is irregularly irregular (with Norplant, bleeding tends to become more regular over time). Although ovulation is centrally suppressed, estrogen production by the ovary is maintained. Very rarely, ovulation can occur in the third year of use but efficacy is maintained because of the thickened cervical mucus. 4. Injections: a. Depo-Provera: contains depo-medroxyprogesterone acetate (DMPA), a synthetic derivative of 17-hydroxyprogesterone. A single IM injection of 150 mg DMPA will reliably inhibit ovulation for three months by suppressing FSH and LH levels and eliminating the LH surge. Other contraceptive actions include development of a shallow, atrophic endometrium and thick cervical mucus. Again, there is no estrogen involved. It is a highly effective, long-term contraceptive that is reversible. The only drug that decreases the effectiveness of DMPA is aminoglutethimine, which is used to suppress adrenal function in selected cases of Cushings syndrome. As with implants, menstrual cycle disturbances are common. Weight gain, depression, breast tenderness, and menstrual irregularities may continue until the DMPA is completely cleared from the womans body about six to eight months after the last injection, (but contraception is maintained only for 3 months). About 15% of women who become pregnant while using DMPA will have an ectopic pregnancy. Still, because pregnancy rates overall are so low, the overall risk of ectopic pregnancy is reduced compared to the general population. A subcutaneous formulation (which could be self-injected) is also available (104 mg) which has

the same efficacy and side effects as DepoProvera. Recent concern over the low levels of estrogen that occur in users of DepoProvera has led to warnings regarding potential effects on bone mineral density with long term use. These warnings, though are not based on any data that demonstrates that reproductive age women actually experience more fractures. The limited data available suggests that any changes seen in bone mineral density do not correlate with increased fracture risk. b. Lunelle: no longer available although similar combined products (Perlutal) are available in various countries throughout the world. Lunelle contains 25 mg medroxyprogesterone acetate (MPA) and 5 mg estradiol cypionate (E2C) in a monthly injection. Perlutal contains 10 mg estradiol enanthate combined with 150 mg dihydroxyprogesterone acetophenide. Compared to DMPA, the method is more inconvenient because it requires a monthly injection but has the benefit of predictable, monthly menses. No pregnancies have been reported in U.S. clinical trials (compared to a Pearl Index of 0.34 with COC). Clinical trials demonstrate side effects similar in nature to those experienced with COCs. U.S. trials were not randomized and suggest that some side effects, like weight gain, mastalgia, mood changes and acne were more common with Lunelle. Importantly, despite requiring a monthly visit to the clinicians office, women were very happy with the method; 84% of women felt their experience with Lunelle was very or somewhat favorable and 90% would recommend it to a friend. F. Intrauterine Devices: IUDs currently available in the United States include copper containing devices and hormone delivery systems. IUDs are very safe and effective methods for women in long-term mutually monogamous relationships. Risk of intrauterine infection with IUD use is mainly related to monogamy/relationship status, although there is a slightly increased incidence in the first 3 weeks after insertion. However, this risk is so low in appropriate candidates that the use of antibiotic prophylaxis at the time of insertion does not effect infection rates. Overall, the IUD is a relative financial bargain that requires little thought once put into place by a clinician. Women who have contraindications for hormonal contraceptives can safely use IUDs. 1. Copper-containing IUD: The Paragard 380A is a T-shaped device with 380 mm2 of copper copper wire is wrapped around the stem and two bands of copper are present on each arm. Whereas older types of copper-containing IUDs contained less copper and had a shorter duration of action (4 years), this IUD, with its extra copper, provides contraceptive efficacy for at least 10

years. The bands of copper on the arms of the T provide the extra copper, hence the importance of the A (for arms) in the name of the device. The plastic T is composed of polyethylene to which barium sulfate is added (to create X-ray visibility) with a string attached to the bottom of the vertical stem. The copper IUD acts mainly to create a reaction within the uterine cavity that kills or deactivates sperm. The copper IUD may also effect the ability of a fertilized egg to move down the fallopian tube, impair implantation process, effect the uterine lining, or have other mechanisms not yet understood. Efficacy of this device is 98% over 10 years. This very low cumulative failure rate is identical to female sterilization for women <34 years of age. Major side effects include increased dysmenorrhea and menstrual flow, although these usually respond well to NSAID treatment, resolve within a few months of use, or are easily tolerated. In 2% to 10% of users, the device will be expulsed within the first year. If pregnancy should occur while an IUD is in place, it should be removed, as severe pelvic infections are more likely if the IUD is left in place. Approximately 50% of pregnancies conceived while an IUD was present will abort spontaneously, while about 6% of pregnant women with an copper IUD will have an ectopic pregnancy. Still, because pregnancy rates overall are so low, the overall risk of ectopic pregnancy is reduced compared to the general population. 2. Levonorgestrel IUD (Mirena): This hormonal intrauterine contraceptive system has been marketed in Europe for more than 10 years, and is currently available in 54 countries, including the United States (as of 2001). This system consists of a T-shaped plastic frame impregnated with barium sulfate (to create X-ray visibility) with a steroid reservoir around the vertical stem containing 52 mg of levonorgestrel. The reservoir is covered by a polydimethylsiloxane membrane which regulates the release of levonorgestrel. Two dark monofilament polyethylene threads are attached to the lower end of the vertical arm. Levonorgestrel is released into the uterine cavity at a rate of 20 g per 24 hours for the first year. The release rate decreases gradually to 14 g per 24 hours after 5 years, a level above that needed for maximum efficacy. This level is 4-13% that achieved using an oral contraceptive with 150 mg of levonorgestrel. This low dose in the local uterine environment is minimally absorbed resulting in associated systemic side effects (vaginal dryness, flushing, headaches, hirsutism, nausea, acne, and mood changes) in <3% of users; these complaints decrease with increasing duration of use and older age of the women.

The LNG IUS is a highly effective contraceptive method, with failure (pregnancy) rates comparable to tubal sterilization. In contrast, the LNG IUS is reversible and does not require surgery (with its inherent risks) to initiate. The first year life-table pregnancy rate is 0.14/100 women with a cumulative 5-year rate of 0.71/100 women. As would be expected with such a low rate of pregnancy, the ectopic pregnancy rate is 0.07/100 woman-years, a rate lower than the general population (1.9/100 women). The mode of action of the LNG IUS is mostly related to local action within the cervix and uterine cavity. The cervical mucus becomes thickened and the endometrium suppressed (by hormonal and local inflammatory mediator effects). inhibiting the passage of sperm from the vagina into the upper reproductive tract. Additionally, the low concentrations of levonorgestrel absorbed into the circulation affect the hypothalamic-pituitary-ovarian axis resulting in ovarian inhibition in 2555% of users. After one-year of use, 85% of cycles are ovulatory. Studies also demonstrate that normal development of ova and interaction between sperm and ova (by Glycodelin A) are inhibited, implying that the LNG IUS also inhibits fertilization. Lastly, implantation of a fertilized ova is inhibited based on electron microscopy studies of the basal lamina of the endometrium and the lack of early chorionic gonadotropin activity in LNG IUS users. This highly effective form of contraception is also well-tolerated, with a one-year continuation rate of approximately 80%. The most common side effect is a change in the menstrual pattern, which can include spotting, irregularity, oligomenorrhea, or amenorrhea. Irregular bleeding and spotting are most common during the first few months following insertion after which the amount of bleeding diminishes. Overall, menstrual blood loss is reduced and hemoglobin levels increase. This effect has significant therapeutic relevance for women with menorrhagia. G. Sterilization 1. Male: a vasectomy causes mechanical blockage of the vas deferens that interrupts the path for sperm to move from the testicle and epidydimis to the penis for ejaculation. It is highly efficacious, permanent, safe and has no adverse drug interactions. Although reports have surfaced potentially linking vasectomy to prostate cancer and cardiovascular disease, large-scale studies have easily refuted these claims. Of course, people do change their minds, and vasectomies are somewhat tricky to reverse, with no guarantee of restored fertility. A major problem is the lack of data on efficacy beyond 1-2 years after the procedure.

2. Female: commonly referred to as a tubal ligation, there are actually multiple methods that can be used to mechanically block the passage of the ovum and sperm. These methods include tubal occlusion, or hysteroscopic sterilization. Tubal occlusion is most commonly performed immediately after a delivery or at the time of cesarean section (post-partum tubal ligation [PPTL]) or laparoscopically (laparoscopic tubal ligation [LTL]). Failure rates for tubal occlusion methods, unlike with vasectomy, have been well-studied and are age dependent, with higher rates in younger women. Failures occur equally across time in 10-year follow-up studies and approximately 33% of all failures result in ectopic pregnancy. Younger women are more likely to express regret later in life and consider reversal. However, reversal is costly, difficult, and requires another surgery. Hysteroscopic sterilization (Essure) provides a less invasive method of sterilization that is more effective than tubal occlusion and can be performed in an office setting. Coils are placed in the tubal ostia that hold a polyethylene fiber in the proximal portion of the fallopian tube. Over a 3 month period, fibroblast in-growth occurs to block off the tube. Correct placement can be obtained in 90-98% of women. A hysterosalpingogram is performed after 3 months to prove tubal blockage. No pregnancies have been reported in any women for whom both coils were successfully placed and the HSG demonstrated occlusion. Additional hysteroscopic methods and use of intrauterine medications that lead to scarring of the tube are under development. Adiana, a method whereby stents are placed in the tube the lead to scarring in a similar fashion to Essure, just received preliminary FDA approval. Attempts to use quinacrine sulfate as an intrauterine instillate to cause tubal occlusion have proven to be less effective than hoped with evidence of potential carcinogenicity. H. Emergency Contraception (EC): a method of contraception that prevents pregnancy after sexual intercourse has occurred. Inappropriately called the morning after pill because methods are available that can be effective as long as 5 days after unprotected intercourse and not all unprotected intercourse occurs at night! The risk of pregnancy with one act of intercourse ranges from approximately 0-30%, depending on the day of the cycle. EC cannot eliminate this risk when used after intercourse, but will decrease the chance that pregnancy (implantation) will occur. Mechanism of action will be dependent on method and timing of use relative to ovulation. Hormonal methods will work to delay ovulation if used prior to ovulation. If ovulation has already occurred, then the exact mechanism of action is not understood but can include impairment of morula or blastocyst transport, effecting the blastocyst to inhibit implantation, effecting the decidual lining to make it inhospitable (out-ofphase) for implantation, or some other process. The sooner the method is

used, the more effective it is to prevent pregnancy. Studies have also demonstrated that keeping EC at home in case of need does not increase rates of unprotected intercourse but does result in fewer pregnancies. The greatest barrier to EC use is lack of knowledge, usually because of lack of patient education by the health care provider. a. Yuzpe regimen: the original method of modern EC, this regimen consists of 2 doses of 500 g of levonorgestrel plus 100 g of ethinyl estradiol, taken 12 hours apart. The overall risk of pregnancy after use of the Yuzpe regimen is about 3%, and the expected pregnancy rate is decreased by about 60% to 70%. A dedicated product called Preven is currently marketed, although this method can also be administered by breaking up packs of certain combined oral contraceptives containing the same hormones. The method has proven efficacy when the first dose is administered within 72 hours of unprotected intercourse. The most common side effects are nausea and vomiting, reported in 50% and 20% of users, repectively. The incidence and severity of these side effects can be reduced by using Meclizine 30 minutes prior to the first dose of EC; however, about 30% of women will feel lethargic secondary to the Meclizine. Some women also experience breast tenderness, abdominal pain, headaches, or dizziness. Almost all (98%) of women will bleed within 3 weeks of EC use. b. Levonorgestrel-only regimen: this newer method of EC was proven in a multinational, prospective, double-blind randomized trial to be significantly more effective and have fewer side effects compared to the Yuzpe regimen. This method, called Plan B, consists of two doses of levonorgestrel 750 g taken 12 hours apart within 120 hours (5 days) of unprotected intercourse. The overall risk of pregnancy is about 1%, and the expected pregnancy rate is decreased by about 85%. In fact, this regimen is more effective when used between 25 and 48 hours after intercourse compared to using the Yuzpe regimen within the first 24 hours. The same types of side effects can occur as with the Yuzpe regimen; however, the rates of nausea and vomiting are only 25% and 6%, respectively. Although approved for use in 2 doses, the two pills can be taken simultaneously with the same efficacy and side effect rates as when the two pills are taken 12 hours apart. Plan B was (finally!) approved by the FDA for behind the counter sales, which was already available in most European countries (in France, for example, EC is available in schools). c. Antiprogestins (mifepristone; RU486): antiprogestins are also very effective methods of EC. Studies using mifepristone (MifeprexTM) in doses as low as 10 mg demonstrate that this method can decrease the chance of pregnancy for up to 5 days after intercourse. Efficacy are

side effects are comparable to a levonorgestrel-only regimen. The only difference is that women who use an antiprogestin are more likely to have a delay in the onset of the expected menses. The major drawback of use in the U.S. is cost and access. Mifepristone will be of benefit for women who seek treatment more than 72 hours after unprotected intercourse. However, the lowest dose available in the U.S. is 200 mg and treatment will only be available from physicians and hospitals that have mifepristone on hand for abortion. d. Copper-containing IUD: for women who are appropriate candidates, copper-containing IUDs (Paragard) are the most effective form of EC available. The IUD can be inserted within 5 days of unprotected intercourse and is a reasonable option in women who also desire longterm contraception. Side effects are limited to those associated with IUD use. The insertion will trigger an inflammatory response, which makes the endometrium unsuitable for implantation and interferes with fertilization and transport. IV. ABSTINENCE

Abstinence is the avoidance of sexual contact and is actually a lifestyle choice, not a method of contraception. By definition, contraception is used to avoid pregnancy that can result from sexual intercourse. Abstinent individuals are not sexually active and, by definition, do not need contraception. It is 100% effective but the woman and her partner should be prepared to use contraception if they should decide to become sexually active. Some studies have suggested that abstinence can increase self esteem and self image when it is a selfselected choice; however, it is unclear whether these result from choosing abstinent or if women who choose abstinence are more likely to have high selfesteem and self-image.

See tables and charts on the following pages.

Method Pill Tubal sterilization Male condom Vasectomy 3-month injectable Withdrawal IUD Periodic abstinence (calendar) Implant, 1-month injectable, patch Periodic abstinence(natural family planning) Diaphragm Other* TOTAL No. of users (in 000s) 11,661 10,282 6,841 3,517 2,024 1,513 774 450 461 133 99 354 38,109 % of users 30.6 27.0 18.0 9.2 5.3 4.0 2.0 1.2 1.2 .4 .3 .9 100.0

* Includes the sponge, cervical cap, female condom and other methods.

From http://www.guttmacher.org/pubs/fb_contr_use.html

Table 2. Percentage of women experiencing a contraceptive failure during the first year of typical use and the first year of perfect use and the percentage continuing use at the end of the first year, in the United States % of Women Experiencing an Accidental Pregnancy within the First Year Of Use ____________________________________ Typical Use Perfect Use 85 26 25 9 3 2 1 19 40 20 40 20 20 21 14 5 4 26 9 20 9 6 5 3 0.5 0.1 2.0 0.8 0.1 3.0 0.05 0.5 0.15 1.5 0.6 0.1 0.3 0.05 0.5 0.10 81 78 81 70 88 100 100 42 56 42 56 56 56 61 71 85 6 40 63 % of Women Continuing Use At One Year

Method Chance Spermicides Periodic Abstinence Calendar Ovulation Method Sympto-Thermal Post-Ovulation Withdrawal Cap Parous Women Nulliparous Women Sponge Parous Women Nulliparous Women Diaphragm Condom Female(Reality) Male Pill Progestin Only Combined IUD Progesterone T Copper T 380A LNg20 Depo-Provera Implants Female Sterilization Male Sterilization

Yearly Cost for Contraceptive Methods and Associated Services

Source: Trussell, et al., Am J Public Health 1995;85:494-503 $2,500.00



* Figures based on 72 acts of intercourse per year. ** Figures include the drug/device, insertion, and removal.



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Pregnancy maintenance and placental function February 17, 9:15 AM Yoel Sadovsky, MD Full address Yoel Sadovsky, MD Scientific Director, Magee Womens Research Institute Elsie Hilliard Hillman Professor of Womens Health Research Dept of OBGYN and Reproductive Sciences University of Pittsburgh 204 Craft Avenue Pittsburgh, PA 15213 Phone (412) 641-2675 Fax (412) 641-3899 Email ysadovsky@mwri.magee.edu

Learning objectives Students attending this lecture will: Recognize mechanisms that are essential for maintenance of pregnancy Describe the consequences of inadequate pregnancy support Understand the critical role of the placenta in supporting the developing fetus.

Outline A. Hormones that govern the maintenance of pregnancy Cross talk between the implanted embryo, ovarian corpus luteum, the maternal uterine endometrium and maternal systemic hormones is essential for the maintenance of pregnancy. Several hormones, including steroids, protein and glycoproteins play a key role in that process. We will review the production and function of these hormones in the context of early pregnancy development. 1. Protein hormones a. Human chorionic gonadotropin (hCG) b. Human placental lactogen (hPL) c. Prolactin d. Alpha fetoprotein (AFP) e. Relaxin f. IGF and other growth factors 2. Steroid hormones a. Progesterone b. Estrogen c. Corticosteroids

Yoel Sadovsky, MD: Pregnancy maintenance and placental function February 17, 9:15 AM Page 2

B. Placental function The placenta plays an active role in supporting the pregnancy and maintaining fetal growth. Once established, the placenta provides the fetus with oxygen and nutrients, removes CO2 and waste products, and synthesizes diverse proteins, growth factors and hormones that are essential to the maintenance of pregnancy. We will review the following: 1. Placenta growth 2. Placental blood flow 3. Placental transport functions: a. Oxygenation and transport of other gases b. Amino acids c. Glucose d. Fat e. Other essential nutrients 4. Other functions C. Early pregnancy loss Between 50-75% of all pregnancies end in spontaneous abortion, mainly prior to implantation. More than 80% of the losses occur before 10 weeks of pregnancy. Approximately 5% of couples will have 2 consecutive miscarriages and about 1% will have 3 or more consecutive miscarriages (termed recurrent pregnancy loss). The risk of another miscarriage does not change after a single miscarriage. After two losses, the chance of another loss is approximately 25%; after 3 losses, the rate is 30-45%. The main causes of pregnancy loss are: 1. Structural Abnormalities a. Chromosomal and other genetic abnormalities b. Non-genetic structural anomalies. 2. Uterine abnormalities 3. Infection and inflammation 4. Abnormalities of thrombosis and auto-immune disorders 5. Hormonal disorders and severe medical problems 6. Drugs and toxins 7. Trauma D. Causes of late fetal injury (fetal death and growth restriction) 1. Placental dysfunction a. Vascular disease and hypoperfusion b. Villous disease 1) Hypoxia and infarct 2) Fetal thrombotic vasculopathy 3) Perivillous fibrin 4) Villitis c. Macroscopic lesions 1) Abruption 2) Placenta previa d. Confined placental mosaicism

Yoel Sadovsky, MD: Pregnancy maintenance and placental function February 17, 9:15 AM Page 3

2. Genetic factors 3. Congenital anomalies (e.g., cardiac anomalies) 4. Infections a. CMV b. Rubella c. Varicella d. Toxoplasmosis 5. Multifetal gestation 6. Maternal diseases a. Hypo-nutrition b. Collagen vascular disease c. Diabetes with vasculopathy d. Hypertension e. Antiphospholipid antibody syndrome f. Hypoxia 1) Systemic disease 2) Cardio-pulmonary disease 3) High altitude g. Maternal endocrine disorders h. Environmental toxins 1) Smoking 2) Alcohol 3) Cocaine 4) Teratogens (e.g., anticonvulsants, chemotherapeutic agents) E. Consequences of late fetal injury 1. Antenatal morbidity a. Intrauterine fetal demise b. Preterm delivery and its complications c. Intra-partum morbidity d. Greater need for cesarean section 2. Neonatal morbidity a. Prematurity b. Hypoxia, acidosis and their sequelae c. Hypoglycemia d. Polycythemia e. Hyperbilirubinemia f. Hypothermia 3. Childhood a. Developmental delay b. Neurobehavioral dysfunction c. Learning disability 4. Long-term outcome (Barker hypothesis) a. Insulin resistance and type-2 DM b. Hyperlipidemia c. Hypertension d. Coronary artery disease

Yoel Sadovsky, MD: Pregnancy maintenance and placental function February 17, 9:15 AM Page 4

F. Suggested reading 1. Speroff L and Fritz MA. Endocrinology of Pregnancy (chapter 4), in Clinical Gynecologic Endocrinology and Infertility, 7th ed. Philadelphia: Lippincott, Williams and Wilkins. 2. Cunningham G, Leveno KL, Bloom SL, Hauth JC, Gilstrap III LC, Wenstrom KD Implantation, Embryogenesis, and Placental Development (chapters 3), and Fetal Growth and Development (chapter 4) in Williams Obstetrics, 22nd ed . New York: McGraw-Hill Publishers, Inc. 3. Barker DJP. The origins of the developmental origins theory. Journal of Internal Medicine 2007;261:412417 (attached).

Embryonic and Fetal Development Robert W. Powers, Ph.D. Lecture 12 - February 17, 2009 11:10 am Learning Objectives: The learning objectives of this lecture are to introduce early human embryology including a broad overview of developmental timing and critical periods of development. Specific areas of focus will include: 1. The early embryo structures 2. The early conceptus 3. Gastrulation, the development of the three germ layers and the tissues that eventually develop from them. 4. Developmental abnormalities Overview of early human embryology. The history of man for the nine months preceding his birth would probably be far more interesting, and contain events of greater moment, than all the three score and ten years that follow it. Samuel T. Coleridge (1772-1834) Week 1. 1. Fertilization: one day post-ovulation. Fertilization begins when a sperm penetrates an oocyte (an egg) and it ends with the creation of the zygote. Fertilization takes about 24 hours. Within 11 hours following fertilization, the oocyte has extruded a polar body with its excess chromosomes. The fusion of the oocyte and sperm nuclei marks the creation of the zygote and the end of fertilization. 2. Cleavage, First Cell Division, Blastomeres: 1.5 - 3 days post-ovulation. The zygote undergoes cell division approximately every twenty hours. The zygote becomes a morula (mulberry shaped) at the Hatching blastocyst. sixteen cell stage, leaves the fallopian tube and enters the uterine cavity three to four days after fertilization. 3. Early Blastocyst: around four days postovulation. Cell division continues, and a cavity known as a blastocele forms in the center of the morula. Cells flatten and compact on the inside of the cavity while the zona pellucida remains the same size. With the appearance of the cavity in the center, the entire structure is now called a blastocyst. The presence of the blastocyst indicates that two cell types are forming: the embryoblast (inner cell mass on the inside of the blastocele), and the trophoblast (the

cells on the outside of the blastocele). 4. Implantation: around five days post-ovulation The blastocyst "hatches" from the zona pellucida as the blastocyst enters the uterus. The trophoblast cells secrete enzymes which erode the epithelial uterine lining and create an implantation site for the blastocyst. The ovary is induced to continue producing progesterone while human chorionic gonadotropin (hCG) is released by the trophoblast cells of the implanting blastocyst. The ideal implantation site is the back wall of the body of the uterus towards the mother's spine. Week 2. The week of twos 5. Implantation Complete: seven to twelve days post-ovulation. The trophoblast cells engulf and destroy cells of the uterine lining creating blood pools (lacunae), both stimulating new capillaries to grow and foretelling the growth of the placenta. The inner cell mass divides, rapidly forming a two-layered disc. The top layer of cells, the epiblast, will become the embryo as well as line the to be formed amniotic cavity, while the lower cells, the hypoblast, will become the yolk sac. 6. Many events occur in twos during the second week. Although there are exceptions, the mneumonic week of twos is handy for remembering many of the events of the second week. Embryoblast splits into two germ layers: epiblast and hypoblast. Trophoblast gives rise to cytotrophoblast and syncytiotrophoblast. Blastocyst cavity is remodeled twice: primary yolk sac and then the definitive yolk sac. Two novel cavities appear: the amniotic cavity and the chorionic cavity. Extraembryonic mesoderm splits into two layers that line the chorionic cavity. Embryo at the end of week 2.

Week 3. 7. Gastrulation: "It is not birth, marriage, or death, but gastrulation, which is truly the most important time in your life." Lewis Wolpert (1986) At the beginning of gastrulation, a narrow line of cells appears on the surface of the epiblast. This primitive streak is the future axis of the embryo and it marks the beginning of gastrulation, a process that eventually gives rise to all three layers of the embryo: ectoderm, mesoderm and endoderm. The primitive streak contains a depression called the primitive pit that is surrounded by a small elevation of epiblast called the primitive node and finally the primitive groove which is the channel through which epiblast cells migrate down into the potential space between the epiblast and the hypoblast. The first wave of migrating epiblast cells invade the hypoblast, displacing the original hypoblast cells and replacing them with a layer of definitive endoderm. The second wave of migrating epiblast cells migrate laterally and cranially between the endoderm and the epiblast and coalesce to form the third germ layer, the intraembryonic mesoderm. After gastrulation is complete the epiblast is called the definitive ectoderm. Gastrulation does more that convert the bilaminar germ disc into a trilaminar germ disc. It also establishes the craniocaudal axis and bilateral symmetry of the embryo. During gastrulation two faint depressions appear in the ectoderm, one at the cranial end and the other at the caudal end behind the primitive streak. The ectoderm in these two depressions fuses tightly with the underlying endoderm, excluding the mesoderm and form two bilaminar membranes. The cranial membrane is the buccopharyngeal membrane which will eventually break down during the fourth week of development to form the opening to the oral cavity. The caudal membrane is the cloacal membrane which will eventually break down during the seventh week to form the openings of the anus, urinary and genital tracts. Overview of gastrulation.

Gastrulation also brings subpopulations of cells into close proximity so they can interact to produce tissue precursors that eventually give rise to organ systems of the body. The primary germ layers (endoderm, mesoderm, and ectoderm) are formed and organized in their proper locations during gastrulation. In general, the endoderm, the most internal germ layer, forms the lining of the gut and other internal organs. ectoderm, the most exterior germ layer, forms skin, brain, the nervous system, and other external tissues. mesoderm, the middle germ layer, forms muscle, the skeletal system, and the circulatory system. Tissues formed by the definitive ectoderm: Skin Nails Hair Lens of eye Lining of the internal and external ear, nose, sinuses, mouth, anus Tooth enamel Pituitary gland Mammary glands All parts of the nervous system Tissues formed by the mesoderm: Muscles Bones Lymphatic tissue Spleen Blood cells and vasculature Heart Kidneys Reproductive and excretory systems Tissues formed by the definitive endoderm: The lining of lungs Liver Tongue Thyroid Pancreas Tonsils Bladder Digestive tract. 8. Induction of the neural plate neurulation. The two most prominent precursor structures that arise during the third week are the somites and the neural plate. On the 19th day the neural plate appears. It represents the first step in the genesis of the nervous system. The neural plate is identifiable as the medio-sagittal thickening of the ectoderm on either side of the midline, cranial to the primitive node. At the cranial end, the neural plate is wider and consists of the region where the brain will arise. At the caudal end it is narrower and gives rise to the spinal cord.

Approximately 50% of the ectoderm becomes the neural plate and the remainder forms the epidermis. During the fourth week the neural plate will indent along the midline and fold into a tube, the neural tube, which is the precursor of the central nervous system (brain and spinal cord). The neural folds approach each other after the 23rd day and merge to form the neural tube. The closure of the neural tube begins in the cervical area (in the middle of the embryo) and extends from there in both the cranial and caudal directions. The anterior neuropore (cranial) closes itself on between 23 and 24 days. The posterior neuropore (caudal) closes between 25 and 28 days. The top of the anterior neuropore corresponds to the terminal lamina of the adult brain and the posterior neuropore to the terminal filum at the end of the spinal cord. If the posterior neuropore does not close, a spina bifida occurs. If, on the other hand, the closure of the anterior neuropore fails to take place, an anencephaly results. While the neural tube is in the process of closing, cells on the lateral side of the neural plate detach themselves and form the neural crest. The neural crest cells form, so to speak, a 4th embryonic germinal layer. This contains a partial segmentation that contributes to the formation of: the peripheral nervous system (neurons and glia cells of the sympathetic, parasympathetic and sensory nervous systems) epidermal pigment cells (melanocytes) calcitonin cells of the thyroid gland cells of the adrenal medulla facial cartilage dentin of teeth During neurulation, somites form in pairs flanking the neural tube. The somites form in crainocaudal succession from whorls of paraxial mesoderm; and eventually give rise to the vertebral column, ribs, muscles, and skin. Week 4. Embryonic folding. 9. Embryonic folding. During the brief span of the fourth week, the embryo undergoes a complex process of embryonic folding that converts it from a flat germ disc into a threedimensional structure that is recognizable as a vertebrate. The main force responsible for embryonic folding is the differential growth of different portions of

the embryo. The embryonic disc grows vigorously, particularly in length, whereas the growth of the yolk sac stagnates. Since the outer rim of the embryonic endoderm is attached to the yolk sac, the expanding disc is forced to bulge into a convex shape. As a result of folding, the cephalic, lateral and caudal edges of the germ disc are brought together along the ventral midline. The ectodermal, mesodermal and endodermal layers of the embryonic disc each fuse to the corresponding layer on the opposite side creating a fishlike three-dimensional body form. The process of midline fusion transforms the flat endoderm into a gut tube which has blind ends until the complete breakdown of the buccopharyngeal and cloacal membranes. The midgut remains open to the yolk sac, and the neck to the yolk sac is reduced to a slender vitelline duct. Lastly, as a result of folding, the amnion, which initially arises from the dorsal margin of the germ disc ectoderm, is carried ventrally to enclose the entire embryo. Between the fourth and eighth weeks, an increase in the production of amniotic fluid causes the amnion to swell until it completely takes over the chorionic space. When the amnion contacts the chorion, the layers of the extraembyonic mesoderm covering the two membranes fuse loosely and the chorionic cavity disappears. The expanded amnion creates a roomy weightless chamber in which the fetus can grow and develop. After embryonic folding is complete, the amnion takes origin from the umbilical ring surrounding the roots of the vitelline duct and connecting stalk. The progressive expansion of the amnion creates a tube of amniotic membrane that encloses the connecting stalk and vitelline duct, and this composite structure is now called the umbilical cord. The main function of the umbilical cord is to circulate blood between the embryo and the placenta. 10. Critical periods of development and embryopathies. The term embryopathies refers to congenital abnormalities that appear during the embryonic period. Before the 14th day an insult (injury) to the embryo will typically not cause an abnormality because the embryo either repairs the damage or dies (spontaneous miscarriage). However, during the embryonic period (weeks 3 to 8), during which numerous mitotic

divisions and organogenesis occur, the embryo is particularly vulnerable and most congenital abnormalities occur at this time. On the other hand, fetopathies are abnormalities that occur after the 8th week and up to delivery. Sensitivity to injurious substances is reduced in the last two trimesters of pregnancy. Most of the organs already exist by this time and are thus less susceptible. One exception is the cerebral cortex which, between the 8th and 15th weeks, is especially sensitive to injurious substances. The classification of congenital abnormalities can be defined as all those that appear as a result of an interruption or deviation of normal development and lead to changes in form and/or structure. In this definition, the cause is ignored. Some classifications of abnormalities include: Primary abnormalities: Defects (genetic anomaly) in the structure of an organ or part of an organ that can be traced back to an anomaly in its development (spina bifida, cleft lip, congenital heart defect). Secondary abnormalities ("disruption"): Interruption of normal development of an organ that can be traced back to external influences, either teratogenic agents (infection, chemical substance, ionizing radiation) or trauma. Dysplasia: Abnormal organization of the cells in a tissue (e.g., osteogenesis imperfecta). Numerous dysplasias are genetically caused (e.g., achondroplasia). Agenesia: The absence of an organ due to a development that failed to happen during the embryonic period. Syndrome: A syndrome comprises a group of anomalies that can be traced to a common origin (Down syndrome occurs due to a trisomia of chromosome 21 and leads to a number of characteristic anomalies). Primary abnormalities can be divided into three subgroups: Genetic aberrations: Genetic aberrations (either monogenetic or polygenetic mutations) account for roughly 7.5% of congenital abnormalities. Chromosomal aberrations (structure or quantity): Account for roughly 0.5 % of congenital abnormalities. Multifactorial anomalies: Anomalies that can be traced back to several genes and can be influenced by environmental factors (medications, chemical products). This includes all abnormalities of the neural tube, harelips and cleft palates, as well as cardiac-circulation-disorders, dysplasia of the hips, and cryptorchism. Some examples of primary abnormalities: Caudal dysplasia: This is a general term that comprises a group of syndromes that range from small anomalies in the area of the lower vertebrae to complete fusion of the lower extremities (sirenomelia). VATER, comprises vertebral and vascular abnormalities, anal atresia, tracheooesophageal fistela, esophageal atresia, renal anomalies and radial dysplasia. The heterogeneity of these abnormalities precludes a common mechanism of origin. However, these malformations are likely connected with erroneous growth and disturbed migration of the mesoderm during the third week (gastrulation).

Spina bifida: The largest portion of spinal cord abnormalities can be traced back to an unsuccessful closure of the neural tube, caudal to the 4th somite pair during the fourth week. This defect is termed spinal dysraphia and it influences the development of the central nervous system and the vertebral arches that lie above it. This is evidenced by a more or less strongly pronounced opening of the vertebral canal (spina bifida). One distinguishes between spina bifida occulta (= hidden) and spina bifida aperta (= visibly present). Anencephaly: Anencephaly represents a special kind of spina bifida. It arises when the neural folds do not fuse in the cranial region of the neural tube. When the fusion fails for the entire neural tube, one speaks of craniorachischisis totalis. Both anomalies are always fatal.

Secondary abnormalities are due to the influence of teratogenic factors. There are numerous teratogenic factors including: Infectious agents Medications, hormones and chemical products Physical agents (ionizing radiation) Other factors (metabolites, toxic substances) Studies of potentially teratogenic substances usually performed in two ways, epidemiologic or animal experiments. However, the examination of the teratogenic potential of a substance is made more difficult by the fact that most congenital abnormalities are multifactorial including the genetic makeup of the individual. This is why a teratogenic substance can have catastrophic consequences for one individual while there may be no effect in another. A few examples of teratogens: Thalidomide: Thalidomide is an example of a catastrophic teratogenic effect of a medication that was not detected despite animal trials. Thalidomide was introduced in 1956 as a medication against nausea for pregnant women. The abnormalities that resulted in children included effects to the limbs, the heart, the kidneys, the intestines and the external ear. Vitamin A and retinoic acid: Retinol is highly teratogenic in animals, but exhibits no effects in humans. Nevertheless, the daily intake of Retinol should not exceed 6,000 UI. Retinoic acid, on the other hand, which is often employed in dermatology for healing acne, (Roaccutane Isotretinoin) is responsible in 20% of the cases of a polymalformative syndrome that includes cranio-facial abnormalities and those of the CNS and the circulatory system. The critical period, for effects on the embryo, range from the 3rd to the 5th week. Utilization of retinoic acid during pregnancy is forbidden. Diethylstilboestrol: Diethylstilboestrol is a teratogenic substance that leads to anomalies of the vagina and the uterus in female embryos. Three kinds of abnormalities are seen: a vaginal adenosis, erosions of the cervix and transversal cleavages in the vagina. Antibiotics: During pregnancy, antibiotics must be taken with caution. Tetracycline, for example, leads to a coloration of the teeth. Streptomycin derivates lead to

damage of the 8th cerebral nerve which, in turn, leads to hearing problems. Penicillin, on the other hand, can be taken during pregnancy. Spontaneously, around 2 to 3% of children are born with a visible abnormality. At the time of delivery many anomalies are not yet recognizable. One assumes that up to 10% of the newborns may have a congenital anomaly. The distribution of these abnormalities and their causes may be as follows: Multifactorial, inherited origins: 10 - 20% Chromosomal origins: 3 - 5% Connected with irradiation: >1% Connected with medications or chemical substances: 4-5% Unknown origin: 65 - 70%

MATERNAL ADAPTATIONS TO PREGNANCY Arun Jeyabalan, MD, MSCR February 2009 Learning Objectives: To understand the maternal adaptations to pregnancy with emphasis on: o Cardiovascular system o Uterine blood flow o Respiratory system o Renal system o Water and solute metabolism To describe the implications of these changes in the clinical management of the pregnant woman

Recommended reading: Maternal Physiologic & Immunologic Adaptation to Pregnancy. Koos BJ, Newayhid BS, Moore JG. In th Essentials of Obstetrics & Gynecology 4 edition. Eds: Hacker NF, Moore JG et al. Elsevier Saunders, Philadelphia , PA , 2004. References: 1. Maternal Physiology. In Williams Obstetrics, 22nd edition. Eds: Cunningham FG et al. McGraw- Hill Co, NY, 2005. Chapter 5, p.121-150. 2. Maternal Physiology in Pregnancy; Michael C. Gordon. In Obstetrics: Normal and Problem Pregnancies 4th edition. Eds: Gabbe SG et al. Churchill, Philadelphia, PA. 3. Chesleys Hypertensive Disorders of Pregnancy. 2nd edition. Eds: Lindheimer M et al. Appleton and Lange, Stamford CT, 1999. (Detailed review of cardiovascular and renal adaptations in normal and hypertensive pregnacies) Maternal Adaptations to Pregnancy I. Goals of Pregnancy A. B. C. D. II. Effectively carry a fetus to full term Achieve an uncomplicated delivery Result in a healthy mother and infant Return of these changes to nonpregnant baseline such that conception can occur

Important concepts in achieving these goals: A. B. C. Changes need to occur without impairing the mothers health Fetal benefit may or may not correspond to maternal benefit Strategies to achieve changes 1. 2. 3. 4. 5. initiate preconception occur early gradual bound by certain limits revert to baseline without permanent damage


Physiological Adaptations of the Uterus A. Uterine Changes 1. Diploid cell 3.5 kg fetus 2. Contains placenta amniotic fluid, fetus 3. Increase in size a. b. Nonpregnant (70g, capacity of 10ml) Pregnant (~1100g, capacity averages 5L at term)

4. Increase in blood flow a. b. c. d. Nonpregnant 50-100 ml/min Pregnant 500-1000 ml/min Occurs gradually of the course of pregnancy Relatively late event compared to changes in cardiac output and renal blood flow


Implantation of Placenta 1. Oxygen and nutrient transport to the developing fetus 2. Multi-purpose organ respiratory, digestive, excretory, metabolic, endocrine functions 3. Marked changes in feto-placental side and maternal vasculature to ensure proper growth and function 4. Abnormal implantation can lead to problems preeclampsia and fetal growth restriction


Changes in the nonreproductive organs 1. Cardiovascular system a. Blood volume changes i. ii. Increase total blood volume by 40-50% Increase plasma volume by ~40-50% (~1100-1600cc) - peaks at 28-32w iii. Increase RBC volume by 20-30% iv. Progressive hemodilution

From: Obstetrics: Normal and Abnormal pregnancy by Gabbe (original from Peck and Arias) 1979

Physiologic anemia of pregnancy (hematocrit atocrit nadir of 15-20% below nonpregnant)

v. Blood volume changes How? Maternal luteal hormones (Progesterone, Relaxin) & placental hormones vi. Blood volume changes changes Why? Decreased PVR, increased systemic vasodilation, underfilling of vascular tree

Decreased renal perfusion

Increased aldosterone

Increased intravascular volume


to meet the progressively increasing demands of the fetoplacental unit to decrease blood viscosity (decrease resistance to flow facilitating placental perfusion and lowering cardiac work) Increased reserve in anticipation of blood loss at delivery (500cc for a vaginal delivery and 1000cc for a c-section) Absence appears harmful

Anatomic changes of the heart i. ii. iii. Heart displaced to the left and upward Apex is moved laterally Apparent cardiomegaly on chest x-ray

iv. Grade II-III systolic flow murmurs at left lower sternal border is normal b. Systemic hemodynamic changes i. ii. Mean arterial pressure (MAP) Peripheral vascular resistance (PVR)

iii. Cardiac output (CO) iv. Heart rate (HR) v. Stroke volume (SV) vi. Definitions (reminder) Cardiac output = CO (liters/minute) = stroke volume x heart rate Stroke volume = SV (milliliters/beat) Heart rate = HR (beats/minute) Blood pressure Systolic BP/Diastolic BP Mean arterial pressure = MAP = (2DBP + SBP)/3

Total peripheral vascular resistance = TPVR = MAP/CO

vii. Hemodynamic Changes in pregnancy PVR & MAP Mechanisms Hormonal regulation (progesterone, relaxin) Vasorelaxation via nitric oxide and ?other pathways Blunted response to vasopressors (Angiotensin II, norepinephrine)

From: Moore et al. Gynecology and Obstetrics 1993

viii. Clinical Correlation Decrease in BP during second trimester 120/80 not normal in second trimester 140/90 of concern in pregnancy HR by ~20% (10-15 bpm; >100 bpm is not normal) SV by ~40% (~66 to 86 cc) SV is major contributor to CO cardiac output by ~50%

ix. Hemodynamic Changes -

x. Clinical relevance preload leads to CO Cardiac lesions that limit diastolic flow (stenotic and restrictive lesions) through ventricles are poorly tolerated in pregnancy Mitral stenosis, aortic stenosis, pulmonary stenosis, cardiomyopathy and primary pulmonary HTN

Periods of highest risk Early pregnancy 28-32w (peak of vascular volume increase) Immediate postpartum

xi. Distribution of cardiac output Uteroplacental blood flow 15X Renal perfusion 40% heart skin breasts No change in : brain (autoregulation), GI, musculoskeletal

xii. Postural changes i. Lateral and knee-chest positions associated with highest cardiac output

From: Gabbe et al. Obstetrics (original from Clark S et al AJOG 1991)

xiii. Clinical relevance Ideal positions in labor Management of fetal distress in labor by changing maternal position

Bedrest improves fetal growth? Supine hypotensive syndrome Compression of inferior vena cava by gravid uterus preload CO

xiv. Central hemodynamic assessment Clark et al (AJOG 1989) performed pulmonary catheterization of 10 normal pregnant women at term and ~12 weeks post partum (see table)

1112 Weeks Postpartum Cardiac output (L/min) 4.3 0.9 Heart rate (beats/min) Systemic vascular resistance (dynecmsec-5 ) Pulmonary vascular resistance (dynecmsec-5 ) Colloid oncotic pressure (mm Hg) 71 10.0 1530 520

3638 Weeks Gestation 6.2 1.0 83 10.0 1210 266

Change from Nonpregnant State +43% +17% -21%

119 47.0

78 22


20.8 1.0

18 1.5 90.3 5.8 7.5 1.8

-14% **NS** **NS**

Mean arterial pressure 86.4 7.5 (mm Hg) Pulmonary capillary wedge pressure (mm Hg) Central venous pressure (mm Hg) Left ventricle stroke work index (gmm-2 ) 6.3 2.1

3.7 2.6 41 8

3.6 2.5 48 6

**NS** **NS** Clark S. AJOG 161; 1989.

xv. Labor and puerperium CO & MAP increase with pain labor With epidural, baseline decrease in CO, MAP; but CO with ctx persists (300-500cc) Maximal CO is 10-30 minutes after delivery Nonpregnant CO = 3-4 L/min Pregnant = 6-7 L/min

Williams Obstetrics: adapted from Ueland and Metcalf 1975

xvi. Clinical correlates Important in managing women with cardiac abnormalities - Control cardiac output with epidural - Close observation during periods of highest risk - Avoid pushing - Vaginal delivery usually preferable to C-section because of fewer hemodynamic shifts (e.g., less blood loss) - Cardiac conditions can be unmasked during labor and delivery

Signal(s) of maternal and/or fetoplacental origin

vasodilation of peripheral arterial renal sodium and water retention total peripheral vascular resistance

Blood volume

Heart rate

Cardiac output
Systemic oxygen transport

Uterine blood

Oxygen delivery to fetoplacental unit


Coagulation changes i. Procoagulation state ii. Venous stasis Vessel wall injury Increase in practically all coagulation factors (except Factors XI and XIII), including fibrinogen

Why? protect against hemorrhage

iii. Disadvantages - increased risk of blood clots (deep venous thrombosis and pulmonary embolism. 2. Respiratory system a. Pulmonary adaptations i. ii. Anatomic changes Diaphragms elevated (2nd trimester) Chest diameter wider Cardiac silhouette enlarged and shifted to left

Hormonal & mechanical etiologies Progesterone-induced relaxation Large gravid uterus

Functional residual capacity and residual volume decrease by 20% (elevation of diaphragm) Vital capacity is not changed


tidal volume increases by ~40% RR no change Results in increase in minute ventilation

Effects of increased minute ventilation i. Increases carbon dioxide gradient between fetal and maternal circulation; thereby, facilitating removal of carbon dioxide from fetus

ABG pH pO2 pCO2 Base excess HCO3

Nonpregnant 7.40 100 40 +2 24

Pregnant 7.44 100 28-32 +2 21

Fetal (UV) 7.35 35-40 45 -2 24


Clinical relevance i. Decreased lung reserve important in the management of pulmonary conditions ii. Changes in arterial blood gas parameters are important in ICU decision making - decision to intubate and management of ventilator in pregnancy - Important in newer strategies for adult respiratory distress syndrome/ICU management

3. Renal changes in pregnancy i. Anatomic Increase in size of the kidneys by 1-2 cm Dilation of the collecting system (right > left) Secondary to progesterone smooth muscle relaxation and compression by gravid uterus ii. Physiologic Volume regulation increased plasma volume by ~50%, gradual sodium and water retention Sodium, water and osmolality i. Overall Na retention increases 900 mEq(+6-8L water) ii. Enhanced tubular reabsorption of sodium iii. Plasma osmolality decreases 10 mOsm/kg


90 80 70 60 50 40 30 20 10 0

Renal hemodynamic changes in normal pregnancy

Percent Increase






Weeks of gestation


Altered renal hemodynamics very dramatic change Increase in glomerular filtration rate by 40-60% Increase in renal blood flow by 50-85%

ii. d.

Acid-base regulation respiratory alkalosis, decrease bicarbonate, difficulty in compensating for acidotic state.

Clinical correlates i. iii. Change in lab values - Decrease creatinine, blood urea nitrogen, increase in creatinine clearance Glucosuria not necessarily abnormal

iv. Increased risk of pyelonephritis v. Decrease in serum bicarbonate reduced ability to buffer with acidosis

It is important to remember that alterations occur in virtually every organ system during pregnancy. We have chosen to focus on a few of the major organ systems that have marked relevance during pregnancy. A fundamental knowledge of these normal changes in pregnancy is imperative to the proper counseling and management of the gravid woman.

Lecture 15 Parturition Wednesday, February 18, 2009, 2:00 2:50 p.m. Hyagriv Simhan, M.D.
INTRODUCTION The precise sequence of events initiating human parturition, which is defined as "the act of bringing forth or being delivered of young," is unknown. We recognize that there is a complex interplay and interaction between fetal, placental, and maternal factors. Sequential, integrated changes must occur within the myometrium, cervix, fetus, and mother. They occur gradually; these processes play out over months. Parturition requires the uterus to develop coordinated contractility, the cervix to soften/dilate to allow passage of the fetus, (and to be truly successful) maturation of fetal organ systems necessary for extrauterine life and the mother to have undergone changes necessary for lactation postpartum. The mean duration of singleton human pregnancy is 40 weeks (i.e., 280 days from the first day of the last normal menstrual period). Term gestation is usually referred to as 259 to 293 days (i.e., 37 to 41.9 weeks). During pregnancy, the uterus undergoes tremendous growth resulting in an increase in weight from 40 to 70 grams in the nonpregnant state to 1100 to 1200 grams at term. Similarly, intrauterine volume increases from 10 mL in the nonpregnant state to as much as 5 (or more) L at term. With 2 or 3 fetuses, the intrauterine volume increases even more. Uterine blood flow increases from a pre-pregnancy rate of 50 to 70 ml/min to a rate of 500 to 700 ml/min at term. The percent of cardiac output changes from 2% to 17% (most of this - 80 to 90% - to the placenta). This summary provides a broad overview of the current state of knowledge of the processes of parturition. MECHANISM OF UTERINE CONTRACTILITY Uterine contractility is controlled by both free intracellular calcium concentration as well as the phosphorylation of the contractile protein, myosin. Figure 1 shows the cellular pathways controlling myometrial cell contractility. In resting myometrium, free intracellular calcium is maintained at a very low concentration by two mechanisms: a membrane cyclic adenosine monophosphate (cAMP)-dependent calcium-magnesium ATPase which transports calcium out of the cell and a cAMP-dependent calcium ATPase which sequesters calcium in the sarcoplasmic reticulum. A voltage dependent calcium channel allows calcium to enter the cell following depolarization. Intracellular calcium stores in sarcoplasmic reticulum are released by increased intracellular inositol trisphosphate (IP3). The oxytocin receptor (OT-R), PGE2 receptor (EP), and PGF2 lpha receptor (FP), activate phospholipase C resulting in hydrolysis of membrane bound phosphatidyl inositol to IP3 and diacylglycerol (DAG). DAG activates protein kinase C as well as cellular lipases resulting in the release of arachidonic acid from membrane phospholipids. Arachidonic acid is the precursor for prostaglandin synthesis. Contractions in myometrium result from the binding of the two contractile proteins, myosin (thick filaments) and actin (thin filaments). Increased free intracellular calcium binds to the regulatory protein, calmodulin. This calcium-calmodulin complex activates myosin light chain kinase (MLCK). The myosin light chain protein is phosphorylated by MLCK and binds to actin. This increases myosin heavy chain ATPase activity resulting in conformational energy for contractions. The binding of actin to myosin is broken by the dephosphorylation of the myosin by its own ATPase activity. Intracellular concentrations of cAMP (in addition to calcium) control contraction activity. cAMP is produced by the enzyme adenylate cyclase and degraded by the enzyme phophodiesterase. Adenylate cyclase activity is increased by the beta-adrenergic receptor (BAR). Binding of agonist to BAR results in increased cAMP through G-protein mediated activation of adenylate cyclase. cAMP inhibits contractions through phosphorylation and inactivation of MLCK and through a decrease in intracellular free calcium. The beta-adrenergic receptor agonists, terbutaline and ritodrine (used to treat preterm labor), inhibit contractions by this mechanism. Nitric oxide generated by nitric oxide synthase is also a

uterine relaxant. Nitric oxide acts through the conversion of GTP to cyclic GMP. Cyclic GMP inhibits uterine contractions by the same mechanism as cAMP, as discussed previously.

Cell-cell coupling is critical for coordinated myometrial activity. This coupling (electrical and chemical) is enabled by gap junctions (intercellular channels). Gap junctions consist of proteins called connexins (especially connexin-43). Six connexin proteins align to make up pores gap junctions have up to thousands of these pores! Gap junctions allow current and molecules up to 1000 Daltons to pass through. There is a massive increase in the number of gap junctions in late pregnancy and with labor onset a consistent finding across species. This enables the electrical synchrony and effective coordination of contractions necessary for efficient labor. This gap junction increase rapidly disappears following labor.

NORMAL PHASES OF PARTURITION As impressive as myometrial growth is, the physiologic adaptation of the uterus to the growing conceptus is even more impressive. The small, compact, muscular nonpregnant organ is transformed into a large accommodating sac, which is relatively unresponsive to any uterine stimulus. The uterus provides a safe and secure home for the fetus, shielding it from the external environment. Myometrial activity during most of pregnancy can be characterized by poorly coordinated, long lasting, low frequency contractures (often call Braxton-Hicks contractions, in humans). Then suddenly (around term), this quiescent and flaccid organ is transformed into a myometrium that is excitable and generates high frequency, high-intensity contractions. This uterus is spontaneously active and responds readily to a variety of exogenous stimulants (uterotonins).

Thus, it is useful to think of the uterus in terms of phenotypes. Parturition can be divided into four phases. Phase 0 (quiescence) begins at conception and is characterized by uterine muscle tranquility and unresponsiveness to stimuli, along with cervical integrity. The start of parturition is differentiated from the process of labor. In Phase 1 (activation), the uterus and cervix undergo biomolecular changes that prepare for the birth process. The myometrium transitions from a quiescent to an active state. It can then undergo stimulation from agonists. Phase 2 (stimulation) begins with the process of labor itself and ends with the delivery of the fetus. Phase 3 (involution) encompasses those processes by which the uterus involutes and lactation commences. Phase 0 encompasses approximately the first 35 to 36 weeks of pregnancy. Phase 1 lasts for 2-6 weeks (i.e., from 38-42 weeks), whereas Phase 2, labor and delivery, usually lasts less than 24 hours. Phase 3 lasts for 4-6 weeks in the woman who is not breastfeeding. In women who are breastfeeding, fertility can be delayed for several months to years.

Phase 0 is characterized by: (1) myometrial cell hyperplasia, hypertrophy, and unresponsiveness; (2)
limited availability of uterotonins, the agents that cause myometrial contractions; (3) limited propagation of contractile signals; and (4) cervical rigidity and non-distensibility. Uterine growth is attributable both to muscle cell hyperplasia and hypertrophy. The effects of estrogen mediate these processes, primarily. Early on, there is a marked increase in myometrial cell number. Thereafter, there is a significant increase in cell size. A single myometrial cell increases in length from 50 microns to over 500 microns. Progesterone plays a role in allowing increased myometrial cell size by reducing gene expression of a variety of myocyte stretch-induced contraction-associated proteins (CAPs) a cassette of proteins that includes the OT-R, FP, connexins, etc.. This myocyte growth attenuates development of muscular tension that might result from myocyte stretch. Another characteristic of Phase 0 of parturition is the limited availability of uterotonins. An uterotonin may be formed in uterine tissues and may act in an autocrine, paracrine or endocrine fashion. Oxytocin and prostaglandins are examples of uterotonins. The production and secretion of all uterotonins are limited during Phase 0 of parturition. Contractile signal propagation from cell to cell is also limited. Transmission of signals in the pregnant uterus is thought to occur primarily through gap junctions. The formation of gap junctions is inhibited by progesterone and stimulated by estrogen, oxytocin and prostaglandins. During Phase 0 of parturition, the quantity of gap junctions is at a minimum and the area of the muscle cell membranes that is occupied by gap junctions does not increase substantially until just prior to labor. Also, during Phase 0 parturition, the cervix is rigid and non-distensible. The cervix is composed of some smooth muscle but the primary components are collagen and connective tissue or ground substance. In early Phase 0, collagen is predominant with little ground substance. In pregnancy, ground substance, particularly hyaluronic acid, is increased. In late Phase 0 and in Phase 1, cytokine-induced increase in hyaluronic acid content retains water and expands the cervix tremendously, which leads to dispersion of the collagen fibers. Water retention gives the cervix an edematous/soft appearance but changes in the collagen matrix, due to increasing exposure to uterotonins, in particular, prostaglandins, at the end of Phase 0 and in Phase 1, lead to large increases in distensibility at term. All the changes described for Phase 0 of parturition can be explained by the presence of progesterone. It is known to allow myocyte hypertrophy, decrease the number and permeability of gap junctions,

decrease stretch-induced CAPs gene expression, decrease uterotonin receptor gene expression, increases gene expression for enzymes involved in uterotonin degradation, etc. In all mammalian species, including the human, pregnancy cannot be maintained without progesterone. Thus, progesterone is the most likely candidate accounting for myometrial quiescence.

Phase 1, the initiation of parturition, is characterized by a gradual increase in uterine responsiveness.

Phase 1 ends when active labor begins. Two characteristics of Phase 1 parturition are increased spontaneous muscle contractility and increased uterine responsiveness to uterotonins. Spontaneous contractions increase as term approaches and the uterus becomes more responsive to oxytocin near term. These observations can be explained by two biomolecular changes in the myometrium that are characteristic of Phase 1 of parturition and both are gestational-age related. First, the number of gap junctions increases. Second, the number of oxytocin and prostaglandin receptors in human pregnancy also increases as pregnancy advances. Phase 1 of parturition is also characterized by cervical ripening. Cervical collagen is broken down and rearranged and the quantity of various glycosaminoglycans is altered, increasing cervical compliance. What is responsible for the transition from Phase 0 to Phase 1 of parturition is not entirely known, but it seems clear, in virtually every species investigated, that the origin for the activation signals (physical growth and maturation of the hypothalamic-pituitary-adrenal [HPA] axis) is the fetal genome. These signals regulate the relative and functional concentrations of adrenal steroids, estrogen, progesterone, and prostaglandins and enhance the dynamic biochemical dialogue (paracrine and endocrine) between the fetus and its mother. That the fetus controls human parturition carries great teleological appeal. In every species studied (including humans), increases in the concentration of the major adrenal glucocorticoid product (cortisol for humans) are seen in the fetal circulation in late pregnancy. This ensures that fetal maturation (e.g., lungs, etc.) occurs synchronously with other parturitional processes. As the fetal HPA axis matures, there is increased release of ACTH from the pituitary. This stimulates release of cortisol and DHEAS from the fetal adrenal. In the human, this DHEAS serves as the substrate for placental conversion (aromatization) to estrogen. Many lines of evidence support that the ontogeny and maturation of the fetal HPA axis are the regulators of the onset of parturition: (1) Corticotrophin Releasing Hormone (CRH). This is a 41 aa product made in the hypothalamus and in the reproductive tract (trophoblast in the chorion and placenta as well as from the amnion and decidua). Maternal plasma concentration of CRH dramatically increases in the second half of pregnancy (much from the placenta) and peaks in labor; this is coupled with a decrease in its binding (inactivating) protein (CRH-BP) resulting in an increase in bioavailable CRH at parturition onset. Placental CRH gene expression is increased by glucocorticoids and stress; it is reduced by progesterone. Thus, while glucocorticoids decrease hypothalamic CRH release, they enhance CRH expression by trophoblast, amnion, and decidua. This results in a feed forward or positive-feedback loop (endocrine, here) between adrenal cortisol and placental CRH. In fact, maternal and fetal cortisol production correlate best with placental CRH expression and suggests that placental CRH production drives fetal HPA activation.

A possible explanation for this paradoxical cortisol stimulation of placental CRH expression may rest with the amounts of progesterone receptor (PR) expression in trophoblasts and progesterones weak antagonistic effects on the glucocorticoid receptor (GR). That is, before term, increased amounts of progesterone compete with decrease amounts of cortisol for GR binding. As maternal/fetal cortisol increases at term, this overcomes progesterones tonic inhibition of cortisol/GR-mediated CRH expression. CRH also enhances prostaglandin production by amnion, chorion, and decidua along with expression of prostaglandin receptors. Prostaglandins, in turn, stimulate placental CRH production. Thus, another feed forward loop exists (paracrine, this time). (2) Cortisol. As mentioned, there are increasing fetal and amnionic fluid concentrations of cortisol as term approaches. This increases production of prostaglandins (E and F, in particular) and prostaglandin H synthase 2 (PGHS2, COX2) activity in the fetal membranes and decidua. Cortisol also decreases prostaglandin degradation (by prostaglandin dehydrogenase, PGDH) in the chorion. (Progesterone inhibits this cortisol effect). In turn, these prostaglandins augment local cortisol production in the membranes. This further drives membrane COX2 activity and decrease in PGDH activity, thus resulting in a net increase in membrane prostaglandins. This local cortisol also augments placental CRH production, with its previously mentioned effects. Thus a third feed forward loop is evident (autocrine and paracrine). (3) Estrogen. Fetal HPA axis activation is accompanied by enhanced fetal DHEAS synthesis. This serves as the primary precursor for placental estrogen synthesis. Estrogens facilitate parturition by increasing the transcription of a variety of uterine-activating protein genes: connexins, OT-R, COX2, calcium channels, etc. Changes in circulating estrogen concentrations are accompanied by dramatic changes in myometrial cell expression of sex steroid receptor isoforms (e.g., PR-A form with its antagonistic anti-progesterone effects; ER alpha form with its agonistic pro-estrogen effects). In human myometrium, there are increases in PR-A isoform, PR-A/PR-B ratio, and ER alpha mRNA in labor.

As mentioned before, progesterone is the hormone largely responsible for maintaining uterine quiescence in Phase 0. In all species, except in humans, there is a decrease in serum progesterone at the time of parturition. In humans, however, there is a functional, rather than systemic, withdrawal of progesterone action in late pregnancy. Progesterone action is mediated largely through its receptor (PR a nuclear binding transcription factor). Proposed mechanisms include decrease in PR expression, change in PR isoforms, increase in expression of endogenous antagonists of progesterone or of PR, etc. PR exists in three main isoforms: PR-B, which is the activating or agonistic form, and PR-A/PR-C, both of which are antagonistic forms (represses PR-B function). It has now clearly been shown that there is a differentially increased expression of the A isoform in late pregnancy, resulting in an increase in the A/B isoform ratio. This results in a reduced progesterone effect, allowing the activating parturitional changes to take effect.

Phase 2, stimulation, starts with the onset of labor and ends with delivery of the conceptus. Characteristics of Phase 2 parturition include increased production of uterotonins (prostaglandins, oxytocin, etc.) and increased gap junction formation. The uterus undergoes spontaneous forceful rhythmic contractions. During the labor process, there is progressive cervical dilation and thinning (effacement) with expulsion of the fetus and expulsion of the placenta. Uterotonins function to enhance uterine contractility during labor and to assure that the uterus stays contracted after the fetus has delivered.
There is overwhelming evidence for the role of prostaglandins in the initiation of both pre-term and term human labor. Human uterine tissues are selectively enriched with arachidonic acid (the main precursor molecule), prostaglandins increase in concentration in the amnionic fluid, maternal plasma, and maternal urine before labor, and if you give prostaglandins you can initiate labor in ALL species, at any EGA!! As mentioned above, they also contribute during the transition from Phase 1 to 2. They function predominantly in autocrine and paracrine fashions. Prostaglandins are implicated in the three events most temporally related to labor onset: the onset of synchronous contractions, cervical maturation, and increases in myometrial sensitivity to oxytocin due to increases in OT-R and in gap junctions. Prostaglandins are produced in the amnion, chorion, decidua and myometrium. COX2 is the enzyme isoform most involved with prostaglandin output with labor onset there is a major increase in expression in amnion and chorion with labor. The amnion produces PGE2, while chorion and decidua produce both PGE2 and PGF2 lpha. PGDH is expressed primarily in the chorion. Progesterone decreases COX2 expression and increases PGDH expression; glucocorticoids work in a reverse fashion. Thus, as term approaches and glucocorticoids increase coupled with the known change in PR isoforms, there is an enhanced production (and decreased degradation) of prostaglandins in the fetal membranes, placenta, and maternal decidua. Oxytocin clearly plays a role in the maintenance if not initiation of both term and pre-term labor. There is little evidence to suggest that serum oxytocin concentrations increase prior to the onset of labor in humans, however oxytocin may be produced locally in decidua. Oxytocin mRNA is increased 5 fold in decidua coincident with the labor. Using a more sensitive assay of oxytocin, investigators have demonstrated higher pulsatile concentrations of oxytocin in women in spontaneous labor versus no labor. Increasing sensitivity (> 100 fold) to oxytocin occurs at term largely as a result of increased receptors. Uterine OT-R increases in sheep, rat, rabbit, and human pregnancy immediately prior to the onset of labor as well. This increased uterine sensitivity to oxytocin is also observed in patients who deliver preterm. In a retrospective analysis of oxytocin contraction tests among complicated pre-term pregnancies, uterine sensitivity to oxytocin increases prior to the onset of pre-term labor and delivery. An increase in uterine oxytocin receptors has also been found in patients delivering pre-term.

Oxytocin most likely has a dual role in the initiation of labor both through direct action on myometrium stimulating contractions and through the stimulation of prostaglandin synthesis in the decidua. Decidual prostaglandins not only increase myometrial sensitivity to oxytocin but also stimulate uterine contractions. Oxytocin receptors are present on decidual and endocervical cells. These receptors have the same affinity for oxytocin as myometrial receptors. The concentrations of receptors are highest in pre-term labor and in early labor. Oxytocin stimulation of receptors in endocervical and decidual cells leads to PGE2 and PGF2alpha synthesis. The components of Phase 3 (involution) are: placental expulsion, uterine involution, milk letdown, and restoration of fertility. Oxytocin is largely, if not predominantly, involved with the former three processes. The return of fertility depends on the duration of breastfeeding. In the woman who continues to breastfeed full-time, fertility can be delayed for several months to a year. In the woman who chooses not to breastfeed, fertility can be re-established in 4-8 weeks. References: 1. Lockwood CJ: The initiation of parturition at term. Obstet Gynecol Clin Nor Am 2004, 31: 93547. 2. Norwitz ER: Physiology of parturition. Up-To-Date 2007 3. Characteristics of Parturition, by JRG Challis and SJ Lye, Chapter 4, pp. 79 87, in Maternal Fetal Medicine: Principles and Practice, 5th edition (Saunders 2004) , eds. RK Creasy, R Resnik, and JD Iams. 4. Parturition, Chapter 11, pp. 251 90, in Williams Obstetrics, 21st edition (McGraw-Hill 2001), eds. FG Cunningham, NF Gant, KJ Leveno, LC Gilstrap III, JC Hauth, and KD Wenstrom. 5. Smith R: Parturition. NEJM 2007, 356(3): 271 83. 6. Simhan HN, Caritis SN Prevention of Preterm Delivery. N Engl J Med 2007;357:477-87

The Challenges of Pharmacotherapy During Pregnancy Steve Caritis, MD Lecture 15 February 18, 2009 3:00 pm
A. Background The pregnant or soon to become pregnant woman poses many challenges to health care providers who are considering pharmacotherapy. Whether the patient is currently taking medication for a preexisting condition or will require medication during pregnancy, the health care provider must consider not only the impact of the administered medication on the fetus but also whether the prescribed agent is safe for the mother and whether any dose adjustment is required. The typical pregnant woman takes an average of 4-5 medications during pregnancy and countless other natural foods and supplements which might affect her or her fetus. Remarkable physiologic changes occur during pregnancy and these changes can affect every aspect of how the body handles xenobiotics including absorption, metabolism, and elimination. In addition to the physiologic changes which accompany pregnancy, the hormonal milieu during pregnancy is unique and modifies many aspects of drug handling. This review will summarize the pharmacological changes commonly associated with pregnancy. We will also review those issues which are of relevance to pharmacotherapy in pregnancy focusing on both the fetus and the mother. Finally, we will focus on two drugs used during pregnancy to describe how limited pharmacologic data can lead to pharmacological missteps. B. Pregnancy Effects on Drug Absorption, Distribution, Metabolism and Elimination Drug absorption in pregnancy may be altered due to changes in gut function. Gastric emptying and gut motility are delayed from 30-50% in large part due to the effect of progesterone, a smooth muscle relaxant. Gastric acidity is also reduced potentially affecting drug absorption. A slowing of intestinal transit increases gut metabolism of those drugs metabolized by cytochrome (CYP) 3A, the most abundant cytochrome P450 enzyme in the gut. Drug distribution is dramatically affected by the physiologic changes of pregnancy. Plasma volume increases 40-50% in singleton gestation and even more so in multifetal pregnancy. Extracellular fluid space and total body water are also increased. The entire fetal compartment is available to many drugs in the maternal circulation further increasing the total volume of distribution. Thus, total body water increases by up to 8 liters in pregnancy. The binding of drugs to plasma proteins also affects the volume into which a drug may be distributed. Large molecules are commonly confined to the intravascular space whereas smaller molecules may be distributed throughout the body. Drug metabolism is altered in pregnancy primarily as a consequence of pregnancy hormone effects on the liver metabolic enzymes. Drug metabolizing enzymes (DME) are generally divided into two groups. Oxidative DMEs include the cytochrome P450 enzymes and the flavin monooxygenases. These oxidative enzymes introduce an oxygen ion into to the substrate drug generally resulting in hydroxylation or demethylation. These are considered Phase I metabolic enzymes. The Phase II enzymes are conjugating enzymes and include UDP glucosyltransferases (UGTs), glutathione transferases (GSTs), sulfotransferases (SULTs) and N-acetyltransferases (NATs). These Phase II enzymes catalyze the coupling of endogenous small molecules such as sulfates and glucuronides to xenobiotics. This results in the formation of soluble compounds that are readily excreted. Although there are more than fifty CYP450 isoenzymes, a small number of these enzymes metabolize the majority of xenobiotics. For example, over 80% of all hepatic CYP450 enzyme activity is accounted for by CYP450 1A2, 2D6, and 3A. These as well as other metabolizing enzymes are also present in other tissues including the gut, lung, kidney, and placenta. The expression as well as activity of these enzymes is affected differentially by pregnancy associated hormones. A well recognized effect of pregnancy is the 50% reduction in caffeine metabolism due to inhibition of CYP1A2. On the other hand, other DMEs demonstrate increased activity in pregnancy such as CYP2D6 which is primarily responsible for the metabolism of beta adrenergic receptor blockers

such as metoprolol. Clearly these effects on drug metabolizing enzymes will have a substantial effect on women who consume caffeine or are treated with a beta blocker. Drug elimination is profoundly affected by the 50% increase in renal blood flow and glomerular filtration that occurs in pregnancy particularly those drugs that are excreted unchanged. C. Placental Transport Most xenobiotics cross the placental barrier by simple diffusion. Small lipid soluble molecules cross the placenta easily and rapidly given that uterine blood flow is dramatically increased during pregnancy reaching rates in the third trimester of 500 ml/minute. Molecular weight needs to exceed 1000 before size becomes a factor in transport. Most xenobiotics have molecular weights less than 500. Placental transport is also affected by the binding of the drug to albumen in the maternal blood, the degree of drug ionization (increased transport) and the degree of lipid solubility (increased transport). The placenta also possesses several drug metabolizing enzymes which reduce the amount of drug reaching the fetus. The placenta is also richly imbued with efflux proteins such as p glycoprotein and breast cancer resistance protein (BCRP). These efflux transporters limit fetal exposure to xenobiotics. D. Pharmacotherapy During Pregnancy 1. Fetal Considerations Any medications taken by the mother may impact her fetus. The drug effects may lead to malformations (teratogenicity), toxicity or to functional alterations in fetal development or maturation. Although fetal malformations secondary to xenobiotics have received the most attention, the number of xenobiotics known to be teratogenic is very small (Table 1). Table 1: Teratogens Agent Androgens Angiotensin-converting enzyme (ACE) inhibitor and angiotensin II receptor antagonists Warfarin Critical Period 8th 13th week 2nd and 3rd trimester Effect Labial fusion , clitoral hypertrophy, masculinization of female fetus Renal impairment, renal tubular dysplasia, anuria, oligohydramnios Fetal Warfarin Syndrome (facial anomalies and epiphyseal stippling) CNS defects Neural tube defects, cardiac defects, cleft lip and palate, microcephaly, craniofacial defects Hemorrhage in the newborn (vitamin K) Cardiac defect (Ebstein anomaly) Newborn toxicity Vaginal adenocarcinoma, abnormalities of lower mullerian tract Bilateral amelia or phocomelia Abortion, CNS malformations, cardiac facial dysmorphism, etc. Fetal hypothyroidism

6th 9th week Throughout pregnancy 1st trimester


3rd trimester Lithium Diethylstilbestrol (DES) Thalidomide Isotretinoin Iodine 1st trimester 3rd trimester 10-13 week 20 -36 days post conception 6th 13th weeks 2nd and 3rd trimester

The phocomelia associated with prenatal exposure to thalidomide makes an indelible impression. Thalidomide was developed to treat morning sickness and was sold worldwide during the 50s and early 60s but inadequate testing was done to assess its safety. Approximately 10,000 babies were born with severe malformations before the teratogenic link was made. In part this was due to the small window of risk as exposure only during the 20th 36th day post conception placed the fetus at risk. This tragedy led Congress to pass legislation mandating drug testing for safety during pregnancy before any drug can be approved for sale in the US. Problems such as the thalidomide tragedy help lead to the FDA classification of drugs based on their teratogenic history or risk. The FDA drug classification which is listed in Table 2 is thus of very limited value to the health care provider as most drugs prescribed would fall into the C category. Human data are lacking or animal studies are positive or not done. Table 2:

Teratogenesis is primarily a first trimester issue since most organogenesis is completed by the 8 weeks of gestation (6 weeks post conception). Drugs administered after the period of organogenesis can adversely affect organ maturation and functions. Drugs can also be toxic to the fetus just as they can create toxic effects in adults. Some toxic fetal/neonatal effects are listed by drug category in Table 3. Table 3: Drug Toxicities Agent NSAIDS Sulfonamides Paroxetine (Paxil) Toxicity Oligohydramnios, ductal closure Hyperbilirubinemia Cardiac malformations, persistent pulmonary hypertension


Beta adrenergic blocker Sulfonurea Ketamine Narcotics Sedative/hypnotics 2. Maternal Considerations

Growth delay, bradycardia Hypoglycemia CNS depression Addiction, withdrawal Hypotonia, sedation, withdrawal

The dramatic changes in maternal physiology and alterations brought about by the hormonal milieu of pregnancy affect many aspects of drug handling in pregnancy. The changes outlined earlier lead to dramatic changes in pharmacokinetics and drug elimination often necessitating a change in dosage. When drug concentrations are monitored, dose adjustments can be made to achieve therapeutic levels. When an easily measurable end organ response, such as blood sugar is used to titrate dosing, the dose can be adjusted to achieve the desired response. This is particularly important since the doses of insulin required to achieve euglycemia are dramatically higher than those needed in non-pregnant diabetics. Many drugs however are not monitored by concentration measurements or do not have a discrete measurable effect that can be quickly and precisely measured (for example, proton pump inhibitors, selective serotonin re-uptake inhibitors, asthma medications, and some antibiotics are dosed based on standard regimens developed in men or non-pregnant women. These dosage recommendations commonly lead to overdosing or more commonly underdosing the pregnant woman. Another dosing conundrum is faced when the desired response is measurable but the lack of response is due to drug resistance or lack of responsiveness to the drug. In this scenario, increasing dosage will lead to side effects and the lack of effect is not due to an inadequate dosage. This situation is particularly evident in pregnant women with preterm labor or those whose labor is being induced. 3. Labor Inhibiting and Labor Stimulating Drugs Not Enough Drug or Too Much Drug a) Tocolytics The Case of Ritodrine Ritodrine is a beta adrenergic receptor agonist that was specifically developed to treat preterm labor. It gained FDA approval in 1980 based on evidence that it inhibited spontaneous oxytocin and prostaglandin induced myometrial contractions. Clinical trials demonstrated efficacy in treating preterm labor with minimal side effects. Unfortunately, the initial dose ranging studies for both oral and intravenous drug administration regimens were performed in non-pregnant women and men. The recommended dosage regimen called for an escalating IV infusion until labor was inhibited. The starting IV dose of 100 g/minute was to be increased by 50 g/minute every 10 minutes until contractions stopped up to the maximum dose of 350 g/minute. Once contractions were stopped, the patient was switched to an oral ritodrine preparation of 20 mg every 4 hours. These dosing regimens proved catastrophic. The first case of maternal death with ritodrine therapy was reported within a year of FDA approval. Over the next 3 years, several more cases of maternal death were reported with beta adrenergic agonist therapy. Ritodrine was subsequently removed from the US market. What went wrong with this FDA approved drug? First, the pharmacokinetic data which were used to develop dosing regimens were based on men and non-pregnant women. Second, the studies focused only on acute events during short term labor inhibition therapy. Third, clinicians knowing nothing about the potential ill effects of the drug, gave more drug when they did not see the desired effect i.e labor inhibition. They equated an inadequate response to an inadequate dose. Fourth, there was inadequate post marketing surveillance. The lack of adequate pharmacokinetic data clearly explains the side effects seen with IV therapy. With a constant infusion of 50 g/minute plasma concentrations vary five fold (Figure 1).

Figure 1: Variation in plasma concentration with an infusion of 50ug/min ritodrine

Ritodrine is primarily metabolized by hepatic Phase 2 enzymes (sulfation glucuronidation) so differences between subjects is attributable to genetic expression of Phase 2 enzymes. Figure 2 demonstrates that women who rapidly conjugate and eliminate ritodrine (Pts C&D) never achieve labor inhibiting concentrations (90 ng/ml estimated) even at the highest infusion rate. Figure 2 : Variation in PK of beta-agonist and impact on labor inhibition

A (200ug/min) B (100ug/min) Inhibition

C (350ug/min)

D (350ug/min)

Poor metabolizers (Pts A&B) achieve therapeutic levels at infusion rates of 200 & 100 g/minute respectively. By utilizing an infusion protocol that increases every 10 minutes until contraction stop, and maintaining that infusion for a prolonged duration, desired plasma concentrations are exceeded by 100%. This occurs because plasma concentrations during a constant infusion increase until steady state is reached this generally requires 4-5 half lives. In the case of ritodrine with a half-life of 156 minutes, steady state is not reached for 600-800 minutes. Thus, the full impact of a given infusion rate is not appreciated in 10 minutes yet the infusion rate is increased. A regimen (MWH) that reduces the infusion rate by 50 g/minute every 30 minutes once inhibition is achieved keeps the drug concentration near the desired therapeutic level and reduces the risk of side effects. (Figure 3)

Figure 3: Ritodrine concentrations with two protocols of drug administration

Errors in the oral dosing regimen also created problems with ritodrine. The oral regimen was intended to sustain myometrial quiescence but the dose recommended was far too low because of the differences in drug elimination between pregnant and non-pregnant subjects. Figure 4 shows the differences in pregnant and non-pregnant women after a 20 mg oral ritodrine dosage. Figure 4: Ritodrine concentrations in pregnant and non-pregnant women after 20 mg oral ritodrine

Figure 5 shows that the plasma concentrations achieved with the lowest IV dose averages about 25 ng/ml where as the concentration achieved with an oral dose of 20 mg peaks at 8 ng/ml and in a matter of 4 hours is barely detectable. Figure 5: Comparison of ritodrine plasma concentrations with IV and oral administration

Consequently, women receiving the recommended dose clearly had inadequate drug to inhibit contractions and therefore treatment failed. This is in contradistinction to the IV dosing regimen which overdosed women leading to pulmonary edema and occasionally even death. b) Oxytocics The Case of Oxytocin Induced Labor Oxytocin is used to induce labor or to enhance labor when uterine contractility is less than desired. The drug is administered intravenously with initial low rates followed by escalating infusion rates. Most infusion regimens were not based on pharmacokinetic properties of oxytocin but rather were based on uterine response. Generally the infusions were started at 1 milliunit (mu)/minute and were doubled every 10 minutes up to a rate of 40 mu/minute or higher. Frequently, no response was seen early so in many cases very high infusion rates were achieved quickly. This approach has two fundamental flaws. First, the rapidly increasing infusion rates ignore the fact that the half life of oxytocin is about 10 minutes. Steady state concentrations are not achieved until 4-5 half-lives have elapsed 40 minutes in the case of oxytocin. Thus, the infusion rate that changes every ten minutes leads to an excessive infusion rate. The clinical manifestation of this is uterine hypercontractility which can cause fetal distress and lead to emergency cesarean section. The second flaw this infusion strategy is that it ignores the responsiveness of they myometrium which is affected by the number of myometrial oxytocin receptors. Therefore, like high and low metabolizers of ritodrine, the response to oxytocin is highly varied. E. Obstacles in Optimizing Pharmacotherapy in Pregnancy Pregnancy poses considerable challenges to health care providers considering pharmacotherapy for preexisting or a new pregnancy related condition. For the most part, few drugs are teratogens and the most common problems faced related to dosing medications in pregnancy. Information about proper dosing is extremely limited in large part because the pharmaceutical industry has little incentive to undertake studies in pregnant women. The potential litigation risks are enormous when one considers the fetal implications. There is also little motivation for the pharmaceutical industry to study drugs in pregnant women since the drugs approved for various maladies in nonpregnant women will be used in pregnant women by prescribing physicians even if the proper dosage in pregnancy is unknown. Thus, any money invested on studies of pregnant women will not likely increase market sales. Another major challenge in pregnancy is the lack of adequate post marketing surveillance. Without closely monitoring adverse events after a drug has gained FDA approval, an opportunity to quickly identify problem medications is lost. This is particularly relevant in pregnant women whose fetuses may not demonstrate serious adverse outcomes until they reach adulthood. Finally, the dictum of avoiding all medications during the first trimester is a sound one in general, but may not make sense in the women with a preexisting disease such as diabetes, lupus or ulcerative colitis. Reading Mattison D, Zajicek A, PharmD. Gaps in Knowledge in Treating Pregnant Women. Gender Medicine 2006; (3) 3:169-182. Morgan DJ. Drug Disposition in Mother and Foetus. Clinical & Experamental Pharmacol & Physiol. 1997; 24: 869-873. Frederiksen M. Physiologic Changes in Pregnancy and Their Effect on Drug Disposition. Seminars in Perinatology. June 2001; (25) 3: 120-123. Dawes M, Chowienczyk P. Pharmacokinetics in Pregnancy. Best Practice & Research Clin Obs & Gynecol. 2001; (15) 6: 8-19-8-265. Anderson G. Pregnancy-Induced Chages in Pharmacokinetics; A Mechanistic-Based Approach. Clin Pharmacokinet. 2005; (44) 10: 989-1008.

Teratology: Background and Overview Marta Kolthoff, M.D. Lecture 16 - February 18, 2009 4:00 pm

I. A.

HISTORICAL PROSPECTIVE Thalidomide 1. Prior to this disaster placenta was thought to be an effective barrier to adverse effects of drugs. 2. Demonstrated the concept of critical periods. 3. High rates (20-30%) of unique malformations. (Lenz, Knapp, 1962) B. Bendectin 1. 2. 3. 4. Combination of doxylamine and pyridoxine. Used by 40-50% of all pregnant women. Withdrawn from market in 1982 following wave of lawsuits. Rate of hospitalization for severe nausea and vomiting increased 2-fold after its withdrawal. 5. No decrease in any category of malformations following its withdrawal from the market (Ornstein et al). 6. Still marketed in Canada and other countries without adverse effects. Best classified as a litogen. C. Isotretinoin (Accutane) 1. Despite animal studies suggesting risk, only limited package insert warnings. 2. Retinoid Pregnancy Prevention Program a. Implemented in 1989 with detailed warnings and a signed consent acknowledging the need for two effective methods of contraception. b. 30% of women with exposed fetuses were using no form of contraception (Pastuszak, et al).


INTRODUCTION Teratology is defined as the study of malformations, but is more commonly used in the context of the study of the deleterious effects of environmental agents on the developing embryo. Included among these agents are drugs, infections, and irradiation. The teratogenicity of a particular agent is difficult to prove, or disprove, in humans, principally because of mans genetic heterogeneity, but also because the effect of the agent is dependent on a number of other factors that include: A. Stage of embryonic development. Foremost among these factors is the timing of exposure. During the first 2 weeks after conception the embryo is relatively resistant to teratogenic insults. An insult may be significant enough to be lethal, but if the embryo survives, no organ-specific anomalies occur. In humans the process of organogenesis occurs between weeks 3 and 8 after conception. This period is the time of maximum susceptibility to teratogens. The pattern of anomalies often will depend upon which systems are differentiating at the time of exposure. Following organogenesis, embryonic differentiation is characterized by an increase in organ size more than continued organogenesis. During this stage, a teratogen may affect the overall growth of the fetus or the size of a specific organ, but it is not likely to produce a visible malformation. The two organs that are possible exceptions to this rule are the brain and the gonads. Both continue to differentiate throughout gestation. B. Dosage. An agent may be teratogenic only at specific dosages, and the dosage may vary depending on the stage of embryonic development. The route of administration also plays a role if absorption is a factor. Finally, administration of small doses or a teratogen over several days may produce a different effect than administration of the same total dose at one time. Drug Interactions. The simultaneous administration of two agents may have one of three effects. First, they may have the same effect as when either of the two is administered separately; that is, their actions are independent. Second, one may provide a protective effect against the action of the other. Possible mechanisms for this interaction include induction of enzyme systems, and competition for binding sites. In the third case, one agent may enhance the teratogenic potential of the other. This synergistic effect could be the result of enzyme inhibition or saturation of binding sites, which might inactivate a teratogen.



Genotype. There appears to be a genetically determined susceptibility to teratogenicity. These differences in susceptibility may be explained by differences in maternal ability to absorb and metabolize a teratogen, by differences in rates of placental transfer, and by variation in fetal metabolism. Specificity of the agent. An agent may be teratogenic in one species but not another. Therefore, animal testing may not provide information on the effect of an agent used in human pregnancies. 1. High dose of glucocorticords or benzodiazepines cause oral cleft in animals, but not in humans (Shioni and Mills; Rosenberg et al.; Fraser, Sojoo). 2. Salicylates causes cardiac malformations in animals, but not humans (Werler et al.).



PROOF OF TERATOGENICITY A. Teratogenicity is difficult to prove in humans. A large sample is necessary in most cases, and usually only retrospective data are available. It also must be kept in mind that most congenital anomalies occur in individuals who are not exposed to a teratogen. Types of Studies 1. Case Report Useful only if drug is taken by a relatively small number of women, or causes a rare malformation. 2. Retrospective Studies a. Cohort: whether women who took drug have more birth defects than those that do not take the drug. b. Case-Control: whether mothers of children with a specific malformation took a drug more often than mothers of children without the malformations. 3. Drug Registries more likely to have negative (bad) outcomes reported. 4. Prospective Observational based on calls to teratogen hot lines. Common Methologic Issues 1. Sample size very few drugs would increase risk of malformations by a factor of 2. 2. Effect of maternal disease many maternal conditions may increase the risk of fetal malformations (epilepsy, diabetes). 3. Recall bias women giving birth to malformed child are more likely to remember drug exposure.




4. Observational studies may not be comparing women with same type and severity of a disorder. 5. Voluntary reporting risk of lithium was 30% based on registry studies and 1% based on prospective observation. 6. Meta-Analysis negative studies are less likely to be published.


SCOPE OF THE PROBLEM A. B. C. Pregnant women take an average of almost four drugs, excluding nutritional supplements, during pregnancy. Only 20% of pregnant women abstain from drug usage. Of those taking medications, 40% take the drug during the first trimester (Forfar and Nelson).


SPECIFIC AGENTS (Briggs et al.) A. Anticonvulsants 1. Hydantoin. A recognized syndrome is seen in approximately 10% of exposed infants (Hanson et al.). Effects include digital and nail hypoplasia, depressed nasal bridge, mental retardation, and slightly increased incidence of congenital heart disease. 2. Valproic acid. This is associated with an increased risk of neural tube defects, approximately 1% (Bjerkedal et al.). 3. Carbamazepine. Associated with approximately a 1% risk of neural tube defects (Rosa). 4. Phenobarbital. There is no increased risk of malformation with the use of phenobarbital alone. Antimicrobial agents 1. Sulfonamides. These may be a hazard to the newborn because of the competition with bilirubin for albumin-binding sites. However, they present little danger as a teratogen (Heinonen et al.). 2. Penicillin. This has been widely used during pregnancy for the last 20 years without an implication of teratogenicity. 3. Cephalosporins. Although chemically similar to penicillin, there has been no systematic epidemiological study of their prenatal effects. 4. Erythromycin. There is no reported teratogenic effect of this group. 5. Aminoglycosides. These have the potential to be toxic. Nine of three hundred ninety-nine infants exposed to kanamycin had a hearing loss (Jones). 6. Doxycycline. Analysis of 32,804 infants without birth defects and 18,515


with birth defects showed more doxycycline use in groups with malformations, but no difference in first trimester exposure. Thought to be safe (Czeizel and Rockenbauer). C. Antifungal/Antiviral Agents 1. Fluconazale. Associated with malformations in animal studies. Human studies (prospective, observational) showed no increased risk (Mastroiacova et al). 2. Metronidazole. Meta-analysis of 7 published studies found no increased teratogenic risk for women exposed in first trimester (Burtin et al.). D. Progestogens (including birth control pills). There is no association with limb anomalies or cardiovascular defects as previously reported. Recent data suggest no substantially increased rate of malformation over that expected in non-exposed pregnancies (Simpson; Bracken; Raman-Wilms et al.). Accutane (isotretinoin). This is used in the treatment of acne. It is a potent teratogen in the first trimester, resulting in fetal malformations that include microcephaly, ear abnormalities, cardiac defects, and major central nervous systems lesions. It has a very long half-life (Lammer et al.). No evidence that topical tretinoin causes birth defects (Shapiro et al.). Corticosteroids. Although cortisone is a potent palatal teratogen in rodents, no increased risk has been found in humans. Psychotropic drugs 1. Lithium. Possibly slightly increased risk (approximately 1%), specifically Ebstein's anomaly (Jacobson et al.). 2. Valium. Recent well-controlled studies indicate no increase in clefting among fetuses exposed to Valium (Shioni and Mills; Rosenberg et al.). 3. Tri-cyclic antidepressants. No increased risk of birth defects or longterm developmental abnormalities (Nulman et al.). 4. Fluoxetine (Prozac). No increased risk of malformations (Goldstein et al.) or long-term effects (Nulman et al.).





Anticoagulants 1. Warfarin (coumadin). First-trimester exposure appears to result in nasal hypoplasia, chondrodysplasia punctata, and possibly mental retardation. Dandy-Walker malformations found in 1-2% of exposed fetuses. Second- and third-trimester exposure may result in mental retardation, optic atrophy, and microcephaly (Hall et al.). 2. Heparin. This does not cross the placenta and is the drug of choice. Miscellaneous 1. Vitamin A a. One study suggested that as little as 10,000 IU of Vitamin A per day might be teratogenic (Rothman et al.). b. A subsequent study found no difference in rate of malformations between women consuming greater than 10,000 IU per day and those consuming less than 5000 IU per day (Mills et al.). c. No studies have shown a risk from -carotene. 2. Alcohol a. Most common major teratogen . b. Clinical features (Jones; Jones et al.): -pre/post natal growth deficiency. -IQ average 63, poor fine motor coordination, irritability in infancy, hyperactive in childhood. -microcephaly, small eyes and jaw, short nose, smooth philtrum, thin smooth upper lip, abnormal joint position, small ends of fingers. -VSD, ASD c. Risks (Briggs et al.): -2 drinks/day = slightly smaller birth weight. -6 drinks/day = 6% have fetal alcohol syndrome. -8-10 drinks/day = 30-50% incidence of fetal alcohol syndrome. 3. Cocaine a. Literature has a bias toward studies that show an association between cocaine abuse and adverse pregnancy outcomes (Koren et al, 1989). b. Defects seen appear to represent vascular disruption. c. Increased frequency of CNS infarctions and disruptions. d. Other lesions that have been reported include: limb-body wall complex, limb reduction defects, gastroschisis (MacGregor et al; Chasnoff et al.).


e. An increased frequency of congenital heart defects was noted in

one study (Lipshultz et al) but not substantiated in a meta-analysis of 6 other studies (Lutiger et al.). f. Some studies have shown increased incidence of urinary tract abnormalities, but others did not find the association (Chavez et al; Rosenstein et al). g. Two major effects appear to be increased risk of placental abruption and intrauterine growth retardation.


CONCLUSION The obstetrician is often in a quandary regarding drugs in pregnancy. Only a few have been studied well enough to define the hazards or pronounce them "safe". Unfortunately new drugs are marketed daily, and there is often a lag time in reporting, even if an agent is found to be teratogenic. The information is often not readily accessible to obstetricians; a recent review of teratology found only 21% of its references in obstetrics journals. Optimal preventions can be achieved only if the patient is seen for counseling prior to pregnancy.


TABLES A. B. C. Table I Drugs with Proven Teratogenic Effect Table II Drugs/Agents Not Documented As Teratogenic Table III Risk Information Databases


REFERENCES Bjerkedal T, Czeizel A, Goujard J, et al. Valproic acid and spina bifida. Lancet. 1982;2:1096. Bracken MB. Oral contraception and congenital malformations in offspring; a review and meta-analysis of the prospective studies. Obstet Gynecol 1990;76: 552-7. Briggs GG, Freeman RK, Yaffe SJ. Drugs in Pregnancy and Lactation. 4th Edition. Baltimore: Williams & Wilkins; 1994. Burtin P, Taddio A, Ariburnu O, et al: Safety of metronidazale in pregnancy: A metaanalysis. Am J Obstet Gynecol 172:525-529, 1995. Chasnoff IJ, Burns WJ, Schnoll SH, Burns KA: Cocaine use in pregnancy. N Engl J Med 313:666-669, 1985. Chavez GF, Mulinare J, Cordero JF: Maternal cocaine use during early pregnancy as a risk factor for congenital urogenital anomalies. JAMA 262:795-798, 1989. Czeizel AE, Rockenbauer M: Teratogenic study of doxycycline. Obstet Gynecol 89:524-528, 1997. Forfar JO, Nelson MM. Epidemiology of drugs taken by pregnant women: Drugs that affect the fetus adversely. Clin Pharmacol Ther. 1973;14:633. Fraser FC, Sojoo A: Teratogenic potential of corticosteroids in humans. Teratology 51:45-46, 1995. Goldstein DJ, Corbin LA, Sundell KL: Effects of first-trimester fluoxetine exposure on the newborn. Obstet Gynecol 89:713-718, 1997. Hall JH, Pauli RM, Wilson KM. Maternal and fetal sequelae of anticoagulation during pregnancy. Am J Med 1980;68:122-40. Hanson JW, Myrianthopoulos NC, Harvey MAS, Smith DW. Risks to the offspring of women treated with hydantoin anticonvulsants with emphasis on the fetal hydantoin syndrome. J Pediatr. 1976;89:662. Heinonen OP, Sloan D, Shapiro S. Birth Defects and Drugs in Pregnancy. Littleton, Colo: Publishing Sciences Group; 1977:296-313.

Jacobson SJ, Jones K, Johnson K, et al. Prospective multicentre study of pregnancy outcome after lithium exposure during first trimester. Lancet 1992;339:530-3. Jones HC. Intrauterine ototoxicity: A case report and review of literature. J Natl Med Assoc. 1973;65:201-203. Jones KL, Smith DW, Ulleland CN, Streissguth AP: Pattern of malformation in offspring of chronic alcoholic mothers. Lancet 1:1267-1271, 1973. Jones KL: Fetal alcohol syndrome. Pediatr Rev 8(4):122-126, 1986. Koren G, Graham K, Shear H, Einarson T: Bias against the null hypothesis: The reproductive hazards of cocaine. Lancet 2:1440-1442, 1989. Lammer EJ, Chen DT, Hoar RM, et al. Retinoic acid embryopathy. N Engl J Med. 1985;313:837. Lenz W, Knapp K: Die Thalidomide Embryopathic. Dtsch Med Wochenschr 87:1232-42, 1962. Lipshultz SE, Frassica JJ, Orav EJ: Cardiovascular abnormalities in infants prenatally exposed to cocaine. J Pediatr 118(1): 44-51, 1991. Lutiger B, Graham K, Einarson TR, Koren G: Relationship between gestational cocaine use and pregnancy outcome. A meta-analysis. Teratology 44:405-414, 1991. MacGregor SN, Keith LG, Chasnoff IJ, et al.: Cocaine use during pregnancy; Adverse perinatal outcome. Am J Obstet Gynecol 157:686-690, 1987. Mastroiavoca P, Mazzone T, Botto LD, et al: Prospective Assessment of pregnancy outcomes after first trimester exposure to fluconazole. Am J Obstet Gynecol 175:1645-50, 1996. Mills JL, Simpson JL, Cunningham GC, et al: Vitamin A and birth defects. Am J Obstet Gynecol 177:31-36, 1997. Neutel CI, Johansen HL: Measuring drug effectiveness by default: the case of Bendectin. CAN J Public Health 68:66-70, 1995. Nulman I, Rovet J, Stewart DE< et al: Neurodevelopment of children exposed in utero to antidepressants. N Engl J Med 336:258-262, 1997.

Ornstein M, Einarson A, Koren G: Bendectin/Dielectin for morning sickness: A Canadian follow-up of an American tragedy. Reprod Toxicol 9:1-6, 1995. Pastuszak AL, Koren G, Rieder MJ: Use of the Retinoid Pregnancy Prevention Program in Canada: Patterns of contraception use in women treated with isotretinoin and extretinate. Reprod. Toxicol 8:63-68, 1994. Raman-Wilms L, Tseng AL, Wighardt S, Einarson TR, Koren G. Fetal genital effects of first-trimester sex hormone exposure: a meta-analysis. Obstet Gynecol 1995;85:141-9. Rosa FW. Spina bifida in infants of women treated with carbamazepine during pregnancy. N Engl J Med 1991;324:674-7. Rosenberg L, Mitchell AA, Barsells JL, et al. lack of relation of oral clefts to diazepam use in pregnancy. N Engl j Med. 1983;309:1282. Rosenstein BJ, Wheeler JS, Heid PL: Congenital renal abnormalities in infants with in utero cocaine exposure. J Urol 144:110-112, 1990. Rothman KJ, Moore, LL, Singer MR, et al: Teratogenicity of high vitamin A intake. N Engl J Med 333:1369-1371, 1995. Shapiro L, Pastuszak A, Curto G, Koren G: Safety of first trimester exposure to topical tretinoin: Prospective cohort-study. Lancet 350:1143-44, 1997. Shioni PH, Mills JL. Oral clefts and diazepam use during pregnancy. N Engl J Med. 1984;311:919. Simpson, JL. Relationships between congenital anomalies and contraception. Adv Contracept. 1985;1-3. Werler MM, Mitchell AA, Shapiro: The relation of empiric use during the first trimester of pregnancy to congenital cardiac defects , N Engl J Med 321:1639-42, 1989.



Aminopterin, methotrexate Angiotensin-converting-enzyme inhibitors Anticholinergic drugs Antithyroid drugs (propylthiouracil and Methimazole) Carbamazepine Cyclophosphamide Danazol and other androgenic drugs Diethylstilbestrol Hypoglycemic drugs Lithium Misoprostol Nonsteroidal anti-inflammatory drugs Paramethadione Phenytoin Psychoactive drugs (e.g., barbiturates, opiods, and benzodiazepines) Systemic retinoids (isotretinoin and etretinate) Tetracycline Thalidomide Trimethadione Valproic acid Warfarin

CNS and limb malformations Prolonged renal failure in neonates, decreased skull ossification, renal tubular dysgenesis Neonatal meconium ileus Fetal and neonatal goiter and hypothyroidism, aplasia cutis (with methimazole) Neural-tube defects CNS malformations, secondary cancer Masculinization of female fetuses Vaginal carcinoma and other genitourinary defects in female and male offspring Neonatal hypoglycemia Ebstein's anomaly Moebius sequence Constriction of the ductus arteriosus, necrotizing enterocolitis Facial and CNS defects Growth retardation, CNS deficits Neonatal withdrawal syndrome when drug is taken in late pregnancy CNS, craniofacial, cardiovascular, and other defects Anomalies of teeth and bone Limb-shortening defects, internal-organ defects Facial and CNS defects Neural-tube defects Skeletal syndrome and CNS defects, Dandy-Walker

The drug is not currently in clinical use *Koren et al, 1998 (16)


TABLE II Agents Not Documented Teratogens*

Drugs and Chemicals Acetaminophen Acyclovir Antiemetics (eg, phenothiazines, trimethobenzamide) Antihistamines (eg, dosylamine) Aspartame Aspirin Caffeine Hair spray Marijuana Metronidazole Minor tranquilizers (eg, meprobamate, chlordiazepoxide, fluoxetine) Occupational chemical agents Oral contraceptives Pesticides Trimethoprim-sulfamethoxazole Vaginal spermicides Zidovudine


TABLE III Computer Teratology and Reproductive Risk Information Databases

Micromedex, Inc. REPRORISK (REPROTEXT, REPROTOX, Shepards Catalog of Teratogenic Agents and TERIS) Englewood, CO 800-525-9083

National Library of Medicine, MEDLARDS Service Desk GRATEFUL MED (TOXLINE, TOXNET and MEDLINE) Bethesda, MD 800-638-8480

Reproductive Toxicology Center REPROTOX Columbia Hospital for Women Medical Center Washington, DC 202-293-5137

Shepards Catalog of Teratogenic Agents University of Washington Seattle, WA 206-543-3373

Teratogen Information System TERIS and Shepards Catalog of Teratogenic Agents Seattle, WA 206-543-2465


Burhan Mahmood M.D, FAAP
Thursday, February 19, 2009 8:30 10:00 am

Upon completion of this session the student will: 1. Understand key aspects of fetal circulation that differentiate fetal and adult circulations; 2. Recognize prenatal events required to ensure an uncomplicated transition; 3. Comprehend postnatal processes which contribute to normal transition; 4. Be able to give examples of abnormal transition.

Framing Concept: Fetal and neonatal development is a continuum of anatomic and

physiologic maturational processes in every organ system. Birth is a sentinel event in the developmental continuum A) The mother and placenta provide nearly total support of fetal physiologic functions. During fetal life, the placenta allows maternal support of fetal respiratory, gastrointestinal, hepatic, renal, and immunologic functions. Multiple anatomic and physiologic features of circulation in the fetus optimize placental support of these vital functions. B) The process of preparing the fetus for the neonatal transition begins before birth. i) Cortisol increases exponentially. This surge of cortisol turns on (via DNAbinding and transcription, and MRNA translation) processes that regulate surfactant production and excretion, glucose homeostasis, and thermoregulation, among others. ii) Catecholamine levels rise just prior to birth and turn on the enzymes that help reabsorb fetal lung fluid, so that the baby can breathe air, and that help mobilize glycogen so that glucose levels do not drop after birth. iii) NOTE that these hormone changes are dependent upon the normal labor process. When babies are born prematurely or by elective Cesarean section without labor, these processes may not occur and the baby may be inadequately prepared for extra-uterine life. C) Inadequate preparation for the transition from fetus to neonate can result in morbidity or mortality of the newbornspecific examples will follow each organ system discussion below.

The Neonatal Transition

1. CARDIOVASCULAR FUNCTION: A. Fetal Circulation i) The placenta: The functions of the circulatory system are uptake of oxygen in the respiratory system, the distribution of nutrients and oxygen to the tissues, and the elimination of waste products. In utero, the placenta is the organ functioning for nutrient and gas exchange. The presence of the low resistance placenta explains differences between fetal and adult circulations. Pulmonary circulations differ by the presence of high fetal pulmonary vascular resistance and right-to-left vascular shunts. Cardiovascular circulatory differences are a greater fetal cardiac output/unit weight, lower fetal systemic vascular resistance (placental resistance is low), lower fetal systemic blood pressure, greater fetal hemoglobin concentration, and greater fetal hemoglobin-oxygen affinity. Respiratory differences include lower fetal arterial blood pO2 and oxygen saturation, and greater fetal arterial blood pCO2. Metabolic differences are greater fetal oxygen delivery, greater fetal oxygen utilization/unit weight, greater fetal nutrient delivery and utilization per unit weight, and a metabolically and hormonally active placenta. ii) Shunting to create a parallel circuit: The unique features of the fetal circulation include a high pulmonary vascular resistance, a low resistance placental circulation, and 3 fetal shunts - the ductus arteriosus, the foramen ovale, and the ductus venosus. The two cardiac ventricles function in parallel rather than in series. (see diagram on next page). From the placenta, blood reaches the fetus via the umbilical vein, through either the liver circulation or through the ductus venosus to join the inferior vena cava. Inferior vena cava return from the placenta is directed preferentially through the foramen ovale into the left atrium, whereas virtually all of the inferior vena cava return emanating from the lower body flows through the right atrium to the right ventricle. Thus, placental blood is preferentially directed into the left ventricle and aorta from which it is distributed to the brain, heart, and other upper body structures. Conversely, inferior vena cava and superior vena cava blood enters the right ventricle, bypasses the pulmonary circulation via the patent ductus arteriosus, and is directed preferentially down the descending aorta to supply the abdominal structures and lower body and the umbilical circulation. Normally about 2/3 of total cardiac output is ejected by the right ventricle.

B. Neonatal Circulation i) Changes in vascular resistance: In the newborn under normal conditions, postnatal respiration of oxygen (i.e., FiO2 = 0.21) leads to profound decreases in pulmonary vascular resistance and constriction of the ductus arteriosus. With clamping of the umbilical cord, the low resistance umbilical circulation ceases abruptly, raising systemic vascular resistance and blood pressure. The result is that systemic pressure becomes slightly greater than pulmonary, which changes the direction of blood shunting across the atrial septum from right-to-left to left-to-right. Anatomically this results in a tissue flap covering the foramen ovale. This functional closure of the foramen ovale converts the two ventricles from a parallel-pumping heart to a series system. ii) Potential shunting: The changes in relative resistances and changes in shunts are dynamic, not structural, changes in the newborn period. When systemic blood pressure is on the rise and pulmonary resistance is on the decline, there is a transitional period when the pressures are approximately equal. At this time, blood either ceases to flow through the ductus arteriosus, or flows back and forth through it. When pulmonary

resistance falls further, blood flows from left-to-right briefly. The high oxygen content of this blood and the loss of dilating prostaglandins (most of which had come from the placenta, and the metabolism of which occurs in the now perfused lung) cause constriction of the muscular ductus arteriosus. Final closure occurs by longer-term replacement of the muscle/endothelium with fibrous tissue. C. Child/Adult Circulation: i) Absence of extrapulmonary shunts with a fixed series circuit ii) Pulmonary vascular resistance decreases during the first weeks of infancy. iii) Systemic vascular resistance and blood pressure increase during childhood. iv) There is no further possibility of shunting between the atria or through the ductus, which has become the ligamentum arteriosum.

Cardiovascular Transition Abnormalities:

1. Congenital Heart Disease (CHD) a. Acyanotic CHD In some forms of congenital heart disease the blood is well oxygenated through normal pulmonary circulation. With a ventricular septal defect, the postnatal shunt through the hole in the ventricular septum becomes left-to-right as the pulmonary resistance falls (and, thus, pulmonary pressure < systemic pressure). The blood in the systemic circulation is fully oxygenated (unless the shunt becomes large enough to cause congestive heart failure). With a coarctation of the aorta, blood flows through the heart and pulmonary circulation in a normal series pattern, and is fully oxygenated. Blockage of the systemic flow at the site of the coarctation usually results in heart failure and shock, and death if not corrected surgically. b. Cyanotic CHD Cyanosis due to congenital heart disease occurs with inability to oxygenate blood completely because of either 1) an abnormal pulmonary circulation or 2) mixing of oxygenated and deoxygenated blood. In fetal life these problems are not apparent because fetal oxygenation occurs via umbilical circulation and because of fetal vascular shunts (see above). For example, a common and severe form of cyanotic congenital heart disease is Hypoplastic Left Heart Syndrome, in which the left ventricle is too small to function normally. If the left ventricle were not present during fetal life, 100% of the blood would be ejected by the right ventricle through the ductus arteriosus. A portion could flow retrograde across the aortic isthmus to supply the brain and heart while the remainder would follow the usual distribution of right ventricular blood. The absence of the left ventricle in fetal life does not adversely affect fetal growth, development or oxygen delivery. Similarly, fetal shunts remain open for

several hours (to days) after birth, allowing ongoing mixing of right (pulmonary) and left (systemic) circulations. Cyanosis is often mild or absent in an otherwise healthy infant during this time. The clinical exam changes dramatically when the fetal shunts close in an infant with cyanotic congenital heart disease. The systemic circulation then contains only deoxygenated blood. Untreated, a defect such as Hypoplastic Left Heart Syndrome is lethal once the ductus arteriosus closes. Another example is Transposition Of The Great Arteries, where the embryonic twist of the conotruncal root did not occur. The great vessel leaving the right ventricle is thus the aorta (rather than the pulmonary artery), and takes right ventricular blood to the systemic circulation. The vessel connected to the left ventricle is the pulmonary artery, and recirculates oxygenated blood to the lungs. Because the venous return to the heart is not affected, the blood going out the abnormally connected left ventricle-to-pulmonary artery goes to the lungs, and returns to the left atrium, only to go out the left ventricle-pulmonary artery again. The only way this oxygenated blood can get to the body (to the right ventricle-aorta) is through intracardiac and/or ductal shunting. The blood reaching the systemic circulation is therefore hypoxemic, and the baby appears blue. These babies may require emergency cardiac procedures to re-open the foramen ovale if it has already closed. This will allow some mixing of oxygenated and deoxygenated blood in the ventricles, so that the blood in the Aorta is only mild-to-moderately (rather than severely) deoxygenated . 2. Persistent Pulmonary Hypertension (PPHN) Also called persistent fetal circulation (PFC), persistent pulmonary hypertension is a critical neonatal illness in which there is an inadequate transition from fetal to infant circulation at birth. Pulmonary vascular resistance remains abnormally elevated after birth, which allows bi-directional or even rightto-left (fetal) shunting through the foramen ovale and patent ductus arteriosus. In the absence of the umbilical circulation to the placenta, however, the right ventricular blood is deoxygenated. Thus, right-to-left shunting after birth permits deoxygenated blood to bypass the lung and proceed into the left heart and aorta. No matter how much oxygen you give to a baby with PPHN, you do not improve the systemic oxygen saturation unless the blood can get to lungs to pick up that oxygen. The circulatory pattern in infants with PPHN is extremely labile and sensitive to changes in blood oxygen, pH, cardiovascular status, and thermal and environmental stressors. Therapy consists of oxygen, assisted ventilation, cardiovascular support, correction of metabolic abnormalities, and intensive supportive care. Inhaled nitric oxide to dilate the pulmonary vasculature was approved by the FDA early in 2000 for the treatment of infants with PPHN. Although oxygen is the strongest pulmonary vasodilator, some infants require nitric oxide in addition. If this does not help, then extracorporeal membrane oxygenation (ECMO), which is a form of cardiopulmonary bypass, can be life saving. The baby goes on by-pass until the underlying lung condition is treated

and the pulmonary vascular resistance returns toward normal. This abnormal circulatory transition may occur as an isolated condition or in association with infection, meconium aspiration, asphyxia, or congenital anomalies. Persistent pulmonary hypertension is most common in full-term infants.

2. RESPIRATORY FUNCTION: A. Anatomy and Embryology: Embryonic stage (3-7 weeks post conception) Lung bud arises from foregut Trachea and esophagus separate Pseudoglandular stage (5-17 weeks) Formation of tracheo-bronchial tree Lymphatics, cilia, goblet cells, etc differentiate Canalicular stage (17-28 weeks) Alveolar type I and II cells differentiate Lamellar bodies form (early surfactant) Saccular stage (29-35 weeks) Distal air spaces branch and grow Surfactant formation escalates Fetal lung fluid and fetal breathing Alveolar stage, 36 weeks years Alveolar septal walls thin and alveolar surface area increases A. Lung fluid secretion: The fetal lung epithelium secretes fluid into the lung lumen from midgestation until near delivery. Chloride is actively transported across the pulmonary epithelium into the lung lumen, establishing a negative electrical potential gradient. The volume of luminal lung fluid present during fetal development is critical to differentiation and proliferation of pulmonary and vascular tissues. The balance between lung liquid production and efflux out the trachea and into the amniotic cavity maintains normal lung expansion. Presence of fetal lung liquid in the amniotic fluid is the rational for amniocentesis to estimate lung maturity in pregnancies at risk for premature delivery. At birth, epinephrine, -adrenergic agonists and surfactant cause absorption of fetal lung fluid by stimulating Na+,K+-ATPase driven sodium uptake by the pulmonary epithelium. Lung liquid moves into the interstitium, then to the blood or lymphatic circulation. Corticosteroid and thyroid hormones stimulate early maturation of epinephrine-induced absorption of lung liquid. Other agents, such as atrial natriuretic factor, prostaglandin E2 and nitric oxide, also reduce fetal lung fluid volume. Failure to absorb lung fluid correctly after birth causes the condition called Transient tachypnea of the newborn. (see below).

B. Surfactant: Normal pulmonary function requires the presence of surfactant in the alveoli. Pulmonary surfactant forms a lipid monolayer that blocks the interaction of air and water molecules to decrease alveolar surface tension, thus decreasing collapsing forces. Production of surfactant by the Type II pneumocytes typically begins in the latter part of the third trimester of gestation. Premature infants demonstrate immature cellular differentiation and are often unable to produce and/or secrete surfactant lipids or proteins. Fetal lung maturation and surfactant production can be accelerated by a number of hormones, such as corticosteroids and thyroid hormones, and growth factors. The catecholamine surge associated with birth results in a massive secretion of prenatally synthesized surfactant. Mechanical forces also are active in the control of surfactant release, via the establishment of tidal breathing and activation of stretch receptors. Surfactant is composed primarily of phospholipids (80-90%) and protein. Lecithin (dipalmitylphosphatidylcholine) accounts for ~80% of the lipid and is highly compressible. Phosphatidylglycerol (PG) is the second most common phospholipid (13% of lipid), but appears late in gestation. Surfactant proteins A, B, C, and D function to spread and stabilize surfactant phospholipids, in biosynthesis, in recycling of surfactant, and in host defense.

Respiratory Transition Abnormalities:

1. Transient Tachypnea of the Newborn (TTN): Decreased secretion and/or increased absorption of lung fluid begin 2-3 days before spontaneous labor at term. The stimulus during labor for switching from active chloride secretion into the lumen to active sodium transport into the interstitium is unknown. This switch is the most important factor in the removal of lung fluid in preparation for breathing. Mechanical compression of the chest during vaginal delivery may squeeze additional fluid through the trachea into the oropharynx. After birth and inflation of the lung with air, remaining fluid shifts into the perivascular spaces around large pulmonary blood vessels and airways. In lambs, clearance of lung fluid is complete in 6 hours after normal vaginal delivery. Clearance is prolonged after caesarian delivery, with prematurity and with hypoxemia. Prolonged retention of fetal lung fluid contributes to respiratory distress in the newborn. Signs include tachypnea (respiratory rate >60), retractions, nasal flaring, grunting respirations, hypoxemia, and hypercarbia. Resolution occurs within a few hours to a few days. Treatment is supportive. 2. Respiratory Distress Syndrome: Infants with insufficient surfactant production, secretion or recycling will have a low lung compliance disease called Respiratory Distress Syndrome (RDS). The most common cause of RDS is premature birth, because surfactant synthesis and the build up of surfactant stores is not complete. The pathological correlate of RDS is Hyaline Membrane Disease (HMD). Prevention of RDS, i.e., avoidance of premature delivery, is the most effective medical treatment. For unavoidable preterm births, treatment consists of the

combination of prenatally administered corticosteroid and postnatal respiratory support, including oxygen, positive pressure, assisted ventilation, and exogenous administration of bovine or synthetic surfactant. Within a few days after birth at any gestational age, surfactant synthesis is turned on and the baby will begin to improve.

3. NUTRITION: A. Nutrient storage during fetal life i. Protein accretion is steady throughout fetal life ii. Fat accretion occurs in the third trimester (brown fat last few weeks important for thermoregulation) iii. Glycogen storage occurs in the third trimester Glycogen store are available for immediate mobilization after birth. Allows for normal glucose homeostasis Umbilical cord cut sugar supply drops glucose drops counter-regulatory hormones turned on glycogenolysis and glucose mobilization B. Water content (%) decreases throughout gestation to about 75% in the term newborn. Babies will lose some of this water in the first few days after birth. Normal babies will lose 10% of their birth weight as water in the first 3 days of life. They will regain this weight (as nutrient stores) by around day of life 7. C. Mineral and vitamin reserves are related to maternal diet D. Exogenous nutrient supply Human Milk i. Colostrum Colostrum is a yellowish, thick fluid secreted by the breast during the first week postpartum. Colostrum is composed of material present in the mammary ducts and glands at delivery and, gradually, newly produced milk. Human milk production usually begins after parturition. On the first day, infant feedings are 2-20 cc each, and total up to 100 cc. Frequent feedings (i.e. every 2-3 hours) are necessary to stimulate prolactin and hence adequate milk production. Colostrum provides about 67cal/100 cc. In comparison to later milks, colostrum has a higher content of whey protein, electrolytes, fat soluble vitamins, and minerals. Contents of fat (2%) and lactose are lower. Colostrum is very rich in immunoactive components (see below).


Transitional milk As milk production increases, the composition changes. The transitional phase begins at the end of the first week postpartum and lasts one to three weeks. The composition changes are rapid for a few days, then more gradual. Changes are not consistent between mothers and between samples from any given mother. Mature milk The composition of mature human milk is relatively stable by one month postpartum. Water is the largest constituent in breast milk. Breastfeeding provides the entire water requirement for infants even in hot climates. Mature milk is comprised of up to 3.6% fat. Fats contribute about 50% of calories and are absolutely necessary for healthy growth and development. The longer the interval from last feeding, the lower the fat content. The lipid fraction is the most variable constituent in milk, changing during a feeding, during the day, from day to day, among individuals, and with maternal diet and nutritional status. Any fatty acid may be utilized by the infant for energy. However, essential fatty acids (linoleic C18:2, linolenic C18:3) are required for normal growth and development. Human milk also contains very long chain polyunsaturated fatty acids (PUFA, e.g., C20:2, C20 :3, C24:4, C22:3) required for brain and visual tissue growth. It has been proposed that the presence of PUFAs in breast milk, but not in previous breast milk substitutes, is causally related to higher neurodevelopmental performance (IQ) among breastfed infants. Newer formulas now contain PUFAs. Proteins constitute 0.9% of the content of human milk. The main forms of protein in milk, casein and whey, are species specific. Casein is a group of milk-specific proteins that form complexes with calcium carbonate and calcium phosphate. When milk clots or curdles due to heat, enzyme activity or pH changes, the casein-calcium salt clot becomes insoluble. Whey proteins are the water soluble -lactalbumin, lactoferrin, lysozyme, and immunoglobulins. The whey protein:casein ratio changes throughout lactation from 90:10 in early milk to 60:40 in mature milk. The whey:casein ratio of cows milk (and thus many cow milk based formulas) is 20:80. Hypoglycemia results from disordered glucose homeostasis at birth. Risk factors for hypoglycemia include: Prematurity, small size for gestational age at birth, large size for gestational age at birth (typically, infants born to mother with diabetes mellitus or gestational diabetes) and sepsis. Premature and small for gestational age infants develop hypoglycemia because they have poor glycogen stores, and thus are unable to mobilize glycogen to glucose in the immediate newborn period. Premature infants are often too ill to feed immediately after birth, and thus must be placed on intravenous glucose solutions until they are well enough to feed. Small for gestational age babies may be mature enough to eat, but may not be able to take in the volume of feedings necessary to maintain normal glucose levels. These newborns may also require IV fluids until regular feedings have been established, and glycogen stores replete.


The infant of the Diabetic women is a special case of a baby who is at high risk for hypoglycemia. Diabetic mothers often have high or fluctuating sugar levels during pregnancy. The excess glucose crosses the placenta into the fetus. Because the fetus can secrete normal amounts of insulin, the fetus maintains normal sugar levels, but often grows large due to the excess energy stores. At birth, clamping of the umbilical cord abruptly disrupts infants sugar supply, but does not change its level of insulin. The sugar begins to fall. When fed (especially if sugar water is used instead of human milk or formula), these infants may have an exaggerated insulin release relative to the amount of sugar ingested and become hypoglycemic. In many cases infants of diabetic mothers cannot take in enough milk/formula in the first day of life to prevent hypoglycemia. They are given intravenous glucose infusions, and often require 2-3 times the normal glucose infusion rate to prevent hypoglycemia. 4. GASTROINTESTINAL FUNCTION: A. Digestion and absorption Infants fed breast milk have a short gastric emptying time. Digestion of human milk is aided by the presence of digestive enzymes, such as mammary amylase, milk lipase, bile salt stimulated lipase, and proteases. Gastrointestinal levels of comparable enzymes are lower in infants than in adults. The enzymes in breast milk augment those in the infant GI tract. Milk also contains several growth factors, such as epidermal growth factor. EGF in breast milk functions to stimulate the proliferation and maturation of intestinal mucosal cells. Other hormones and growth factors in human milk are insulin, IGF-1, TGF-, and thyroid hormones. B. Immunity Human milk contains many specific and non-specific antimicrobial factors that protect the immunologically immature infant against environmental pathogens. The main immunoglobulin in milk is IgA. Colostrum has 1740 mg/dl and mature milk has 100 mg/dl of IgA. During the first two weeks of lactation much of the IgA is synthesized by the breast into secretory IgA. Secretory IgA resists digestion by proteases and survives gastric pH, to pass into the intestine and providing protection against viral and bacterial pathogens. The specificity of the IgA molecules is determined by the enteromammary pathway. Antigen in the maternal respiratory and gastrointestinal tract is transported to the maternal breast, presented to plasma cells that then produce specific antibodies for secretion into the milk. Nonspecific antimicrobial factors in milk include lactoferrin, lysozyme, oligosaccharides, mucins, and others. Anti-inflammatory factors also include antioxidants, antiproteases, platelet activating factor-acetylhydrolase, and epidermal growth factor. Immunomodulating agents include cytokines, nucleotides, chemokines, and others. Breast milk is rich in cellular content as well, particularly leukocytes, macrophages and monocytes. The highest number of immune competent mononuclear cells is in colostrums.

C. Liver function i. Hepatic enzymes a. P450 redox reactions immature ( Longer half-lives of methylxanthines, anticonvulsants, etc) b. Alternate metabolic pathways ii. Bilirubin metabolism: Jaundice is a yellow discoloration of the skin caused by elevated levels of bilirubin, a product of heme degradation. The newborn is unique because the serum bilirubin concentration frequently rises to levels not observed in health at any other age. This physiologic jaundice is noted in the majority (~60%) of full-term infants and nearly 100% of premature infants. Physiologic jaundice is a benign, self-limited condition in most infants. In contrast, pathologic jaundice also may occur in the newborn. Pathologic jaundice may cause higher serum bilirubin levels which can result in bilirubin staining not only of the skin and mucous membranes, but also of specific regions of the brain. Sensorineural hearing loss is the most common sequela of excessive hyperbilirubinemia. Bilirubin staining of the basal ganglia and specific subcortical nuclei is known as kernicterus and results in a characteristic bilirubin encephalopathy. Bilirubin encephalopathy presents clinically with a sluggish Moro reflex, opisthotonic posturing (arching), poor feeding, a high-pitched cry, hypotonia, and seizures. Long-term outcome is poor, with neurodevelopmental delay, hearing loss and choreoathetoid cerebral palsy. Clinical evaluation of all newborns for jaundice, appropriate diagnostic evaluation and treatment is the key to prevention of adverse outcomes of hyperbilirubinemia.

Causes of Pathologic Hyperbilirubinemia:

Production: Increased Hemolysis (accelerated RBC breakdown) -Fetal-maternal blood group incompatibility: Rh, ABO, minor Ag -RBC membrane defects: congenital spherocytosis -RBC enzyme deficiency: G6PD, pyruvate kinase -Hemoglobinopathy: thalassemia Extravasation of blood -Ecchymoses, hematomas (birth trauma) -Cerebral, pulmonary or occult hemorrhage Polycythemia ( RBC mass) -Maternal-fetal or fetal-fetal hemorrhage -Prenatal hypoxia -Delayed cord clamping with infant below placenta Swallowed maternal blood Clearance: Decreased Transport and/or Uptake Transport Impaired bilirubin transport -Hypoxia, acidosis -Drugs: sulfa (bactrim), aminosalicylic acid -Serum free fatty acids: breast milk, fat emulsions -Hypoalbuminemia Uptake Impaired hepatic uptake -Enzyme defect: Gilbert syndrome (9% of the population!!) -Decreased venous flow after birth Conjugation: Decreased Impaired bilirubin conjugation -Metabolic: hypoglycemia, hypothyroidism -Drugs: chloramphenicol -Enzyme defect: glucuonyl transferase deficiency, types I and II -High intestinal obstruction -Breast milk jaundice Excretion: delayed Enterohepatic circulation -Delayed passage of meconium: low intestinal obstruction, cystic fibrosis, Hirschsprungs Decreased intestinal motility: Ileus

Every major organ system is anatomically and functionally immature in the newborn. In that sense, successful newborn transition persists beyond the first few days of life. For example, the glomerular filtration rate of the newborn is consistent with renal failure in an adult, and increases over months. Diet and medications must be adjusted appropriately. The neurologic system is the slowest to mature. Attributes most relevant in the newborn are muscle tone, cranial nerve function (e.g. the gag reflex), primitive reflexes (suck, root), development of sensory pathways, and social interactions. Comprehensive care of infants therefore requires an understanding of developmental processes in anatomic, physiologic and neurologic domains.

References: Please read before class. Topic Rudolphs Pediatrics (20th ed) pp 1409-1413 Fetal circulation* pp 1168-1172 Fetal oxygenation* pp 1459-1462 Congenital heart disease pp 1512-1514 Abnormal transition pp 1598-1604 pp 1608-1611 pp 1133-1136 Hyperbilirubinemia

Rudolphs Pediatrics (19th ed) pp 1309-1313 pp 1091-1095 pp 1357-1361 pp 1411-1412 pp 1485-1492 pp 1495-1497 pp 1054-1057

Scientific Essentials of Repro Med pp 387 pp 376-383

pp 386-393

See also: Ch 9 in Essentials of Obstetrics and Gynecology, 3rd edition, 1998, pp.93-99. For breastfeeding: American Academy of Pediatrics (http://www.aap.org/policy/re9729.html) Policy statement on Breastfeeding and the Use of Human Milk (RE9729).

Breastfeeding for Health Debra Bogen Lecture 18 Thursday, February 14, 2008 10:10 am Learner will be able to: Describe benefits of breastfeeding for mother, infant and society Describe 3 difference between human milk and cow milk Describe 2 ways by which breast milk provides immunological protection to infants Use what you learned about lactational physiology to understand common breastfeeding problems Benefits to Society Health care cost savings (hospitalizations, outpatient visits, chronic diseases) Example: Health Care Costs of Formula-feeding in the First Year of Life. Ball and Wright, Pediatrics 1999;103(4)870-876. Health service utilization for 3 illnesses, 1st year of life (lower respiratory tract, otitis media, gastroenteritis) Per 1000 never-breastfed infants compared with 1000 infants exclusively breastfed for at least 3 months (adjusted for confounders) 2033 excess office visits 212 excess days of hospitalization 609 excess prescriptions for these three illnesses Extra cost: $331 to $475 per never-breastfed infant during first year of life missed days at work, garbage, pollution Benefits to Mother Postpartum bleeding (oxytocin acts on smooth muscle in breast and uterus; women experience menstrual or labor-like cramps during first few weeks of breastfeeding but stop after few weeks after oxytocin receptors off the unterus) Risk of breast and ovarian cancer Faster return to pre-pregnancy figure (~ 500 additional calories per day) Improved maternal-infant attachment Benefits to Infant Nutritional Infections Immunologic Developmental Preterm growth and development

Contents of Cow and Human Milk

Protein content in whole cows milk more than double that of human milk Amount of protein in milk linked to growth rate of animal
White: Protein Black: Carbohydrate Grey: Fat

Whole Cows Milk

Human Milk

Proteins in Milk Casein (curds) protein: forms calcium complexes Whey proteins: composed of -lactalbumin, lactoferrin, lyzozyme, immunoglobulins, albumin Ratio Casein:Whey - Human: 40:60 Cow: 80:20 Casein difficult to digest and linked to a range of diseases and allergies Infant formula contains more whey than casein (similar to ratio of human milk)

Fat content of milk

Whole cows milk and human milk - similar amounts of fat Types of fat vary
cow: saturated fat human: unsaturated fat

Higher level of unsaturated fatty acids in human milk reflects important role in brain development 2

Brain develops rapidly during first year of life; triples in size by age one year Grows faster than body Brain largely composed of fat Early brain development and function requires sufficient supply polyunsaturated essential FA Omega-6 fatty acid arachidonic acid (AA) and omega-3 fatty acid docosahexaenoic acid (DHA) both essential Both supplied in human milk, not in cows milk AA and DHA-enhanced infant formulas now standard but may not work the same because not packaged the same way in formula as human milk

Comparing Milk: Human, Cow & Commercial Infant Formula

Nutritive comparisons of milks based on the needs of 0-6 month old infants. *

Protein Carbohydrate Fat Water Calories (kcal)

HUMAN 1.0 6.9 4.4 87.5 70

COW 3.3 4.7 3.3 88.0 61

FORMULA 2.0 7.0 1.1 80 60


Complexity if Breastmilk Iron example Breast milk: 0.3 mg/L iron (>50% bioavailable) Infant formulas: 12 mg/L iron (< 10% bioavailable) Explained by several factors Human lactoferrin receptor on mucosal brush border postulated; efficient iron absorption, not bovine lactoferrin Diffusion-mediated absorption of citrate bound iron Ascorbic acid in human milk allows more efficient absorption High Ca in cow milk-based formulas may interfere Phytates in soy formulas interfere with absorption Health Risks Associated with not breastfeeding - increased incidence of Type 1 diabetes mellitus Type 2 diabetes mellitus Inflammatory bowel disease Childhood cancer Sudden infant death syndrome 3

Obesity Compromised oral-motor development Less optimal development of arch of mouth Increase in orthodontic problems

Human milk and Immunity Symbiotic with infants immature immune system Thousands of components in human milk Interact in complex fashion with one another and with infants body Many immunologically active factors White blood cell (B, T, macrophages) Immunoglobulins (sIgA) Growth factors Hormones Proteins that bind toxins

Secretory IgA

Enteromammary circulation allows maternal mucosal immune system to protect intestine of infant via human milk. When an oral inoculum of pathogen enters maternal gut, it is sampled from lumen by Peyers patches, and its antigens are presented to underlying lymphatic cells. IgA production is induced at basolateral side of mammary cell, and IgA traverses mammary cell to enter milk as sIgA. The sIgA enters gut of breast-fed infants, where it protects infant by binding to pathogen.

From: NEWBURG: Pediatr Res, Volume 61(1).January 2007.2-8.

Components of Human Milk Over 4000 components in breast milk Lactoferrin, major human milk protein o Binds iron to assist absorption; Bacteriostatic effects too o Partially digested to lactoferricin B antibacterial activity against gram +/- bacteria Triglycerides o digested by lingual and gastric lipases into FFA + monoglycerides in stomachs of BF infants o toxic to enveloped viruses and some parasites Glycans

10% maternal caloric input for milk used to generate non-nutritive complex carbohydrate

Human milk glycans

In infants fed artificial formula or milk lacking protective glycans, pathogens (eg. noroviruses) bind to their glycan receptors on mucosal surface, which is essential first step in their pathogenesis. For infants consuming human milk that contains glycans homologous to infant gut receptors, when pathogens such as noroviruses enter gut, they are bound to soluble glycans of milk, rendering them less likely to bind to mucosal receptors, thereby protecting infant.

From: NEWBURG: Pediatr Res, Volume 61(1).January 2007.2-8.

Breastmilk changes over time no two samples the same Colostrum (early milk) - protein, sIgA, sodium, lactose Mature milk by 10 to 14 days Within one meal, milk made de-novo as breast emptied, increase fat from fore- to hind-milk Between meals - change in milk content during day and across time

Any and exclusive breastfeeding rate by age (2002 NIS)

Li, R. et al. Pediatrics 2005;115:e31-e37

Copyright 2005 American Academy of Pediatrics

Racial/ethnic disparities in breastfeeding rates (percent and 95% CI; 2002 NIS)

Li, R. et al. Pediatrics 2005;115:e31-e37

Copyright 2005 American Academy of Pediatrics

Contradictions to breastfeeding Maternal infections o HIV, active tuberculosis, herpes on breast 6

Inborn errors of metabolism o Galactosemia o Tyrosinemia Maternal exposure to drugs of abuse, medications, and environmental agents o Medications and Mothers Milk, Thomas Hale, PhD o National Library of Medicine Website (http://toxnet.nlm.nih.gov/cgi-bin/sis/search)

Maternal Factors to Support Optimal Breastfeeding Normal breast anatomy o Breast reduction can interfere with nerve supply (old procedures) o Augmentation usually not a problem can contribute to engorgement Intact neuroendocrine reflex Good general health and nutritional status Effective support system

Pituitary releases prolactin and oxytocin

Stimulation of nerve endings in mothers nipple/areola sends signal to mothers hypothalamus/ pituitary

Hormones travel via bloodstream to mammary gland to stimulate milk production and milk ejection reflex (let-down)

Inadequate latch (often painful over time) Inadequate milk transfer Dehydration and Poor weight gain Low milk supply

Infant suckles at the breast

Copyright 2003 American Academy of Pediatrics

Case 1: A new mother comes to see you 3 weeks postpartum. She is concerned that her milk supply is not enough for her baby because he fusses and cries and still looks for food after she has nursed. What are some possible causes of low milk supply? Thyroid (hypo or hyper) Retained placenta Hormonal birth control started Polycystic Ovarian Syndrome Inadequate emptying of the breast over time Negative feedback decreases supply over time Breastfeeding Resources 7

Breastfeeding: A Guide for the Healthcare Professional (Ruth Lawrence and Robert Lawrence , 5th edition) Medications and Mothers Milk by Hale (paperback ~ $20, most recent edition 2006) AAP Red Book for ID & BF questions HHS Blueprint for Action on Breastfeeding: http://www.womenshealth.gov/Breastfeeding/bluprntbk2.pdf La Leche League: http://www.lalecheleague.org/ Academy of Breastfeeding Medicine: http://www.bfmed.org/ Dr. Jack Newman: http://www.bflrc.com/newman/articles.htm Breastfeeding handouts (multilingual): http://www.breastfeedingtaskforla.org/resources/breastfeeding-public-education.htm

Pathology of the cervix

Esther Elishaev, M.D. Department of Pathology, Magee Womens Hospital University of Pittsburgh School of Medicine

Overview of cervix development

Mesoderm derived Mllerian ducts fuse at day 54 post-conception and form uterovaginal canal, lined by mllerian columnar epithelium Uterovaginal canal joins endoderm lined urogenital sinus at mllerian tubercle, which becomes vaginal orifice at hymenal ring Epithelium stratifies at caudal uterovaginal canal to become squamous; epithelium proliferates to become almost purely squamous in vagina by day 77 Endocervical glands and vaginal fornices appear between days 91 and 105 Cervix responds to estrogenic stimulation by marked growth

Overview of cervix anatomy

Lower 1/2 to 1/3 of uterus, cylindrical, connects uterus to vagina via endocervical canal Consists of portio vaginalis (portion that protrudes into vagina) and supravaginal portion 2.5 to 3.0 cm long and 2.0 to 2.5 cm in diameter Anteriorly abuts on bladder; posteriorly is covered by peritoneum that forms lining of cul-de-sac Endocervix: endocervical canal Ectocervix (exocervix): vaginal portion of cervix External os: opening of endocervical canal to ectocervix Fornix: reflection of vaginal wall that surrounds ectocervix Internal os: indistinct upper limit of endocervical canal Transformation zone: (see histology) Cardinal ligaments: fibromuscular bands that fan out from lower uterine segment and cervix to lateral pelvic walls and provide main support for cervix Uterosacral ligaments: connective tissue surrounding cervix and vagina that extends towards vertebrae Lymphatics: cervix is drained by parametrial, cardinal and uterosacral ligament routes

Overview of cervix histology

Cervical stroma- fibromuscular tissue Epithelium: Endocervix: lined by columnar epithelium that secretes mucin; epithelium has complex infoldings that resemble glands or clefts on cross section; mucosa rests on inconspicuous layer of reserve cells Ectocervix (exocervix): covered by nonkeratinizing, stratified squamous epithelium, either native or metaplastic. Squamocolumnar junction (SCJ): where squamous and glandular epithelium meets. The position of this junction is variable due to both cervical anatomy and differentiation with squamous metaplasia of basal cells which results in cephalad migration of the SCJ in adults.

Forming the transformation zone which is covered by metaplastic squamous epithelium. Transformation zone (TZ): the area between original location of squamocolumnar junction and the border of metaplastic squamous epithelium; epidermalization and squamous differentiation of reserve cells transform this area to squamous epithelium; site of squamous cell carcinomas and dysplasia

The transformation zone area of the cervix

Menarche: ovaries produce estrogen, which stimulates glycogen uptake by cervical and vaginal mucosa which in turn promotes growth of endogenous acid producing vaginal microorganisms and drop in vaginal pH. Basal/reserve cells respond by proliferating, causing squamous metaplasia.

Infections of the Upper Genital Tract

Pelvic inflammatory disease (PID) Common disorder characterized by pelvic pain, adnexal tenderness, fever, and vaginal discharge; it results from infection by one or more of the following groups of organisms gonococci, chlamydiae, and enteric bacteria. Infections after spontaneous or induced abortions and normal or abnormal deliveries (called puerperal infections) are important in the production of PID. Pathology : inflammatory changes appear in the affected organ manifested predominantly by acute suppurative and some chronic inflammatory infiltrate 1. 2. 3. 4. 5. Acute suppurative salpingitis. Pyosalpinx. Salpingo-oophoritis. Tubo-ovarian abscesses. Hydrosalpinx.

Complications of PID include: Peritonitis Intestinal obstruction due to adhesions between the small bowel and the pelvic organs Bacteremia, which may produce endocarditis, meningitis, and suppurative arthritis Infertility, one of the most commonly feared consequences of long-standing chronic PID

Infections Confined To The Lower Genital Tract

Acute and Chronic Cervicitis Some degree of cervical inflammation may be found in virtually all multiparous and in many nulliparous adult women, and it is usually of little clinical consequence. Cervicitis due to Infections: (HPV, Trichomonas, Gonococci, Chlamydiae, Herpes Simplex Virus - mostly type 2) may produce significant acute or chronic cervicitis and should be identified for their relevance to upper genital tract disease, pregnancy complications, or sexual transmission. Anatomic Distribution of Common Female Genital Infections Location and Manifestations of Infection Organism Herpesvirus Molluscum contagiosum HPV Chlamydia trachomatis Neisseria gonorrhoeae Candida Trichomonas Source STD STD STD STD STD Skene gland adenitis Vulva Herpetic ulcers Molluscum lesions Genital warts, intrapeithelial neoplasia, invasive carcinoma Follicular cervicitis, endometritis, salpingooophoritis Vaginitis in children Acute cervicitis Acute endometritis and salpingitis Vagina Cervix Corpus Adnexa

Endogenous Vulvovaginitis STD Cervicovaginitis

STD sexually transmitted disease HPV - human papilloma virus.

Benign Lesions Of Cervix

Endocervical polyp 2-5% of adult women Usually multigravida age 30-59 years Produces bleeding or mucoid discharge Probably secondary to chronic inflammation and not neoplastic Endometriosis of cervix May cause abnormal uterine bleeding, post-coital bleeding Mean age 37 years, range 20 to 51 years. Leiomyoma of cervix Uncommon; only 8% of uterine leiomyomas occur in cervix Clinically may mimic an endocervical polyp

Microglandular hyperplasia (MGH Common cervical lesion associated with birth control pills or pregnancy in young women, although also in post-menopausal women Usually incidental, may grow as a polypoid mass Nabothian cysts A normal finding; due to obstruction of crypt openings containing mucus by squamous epithelium, causing acute and chronic cervicitis

Malignant and Premalignant Lesions Of Cervix

In the US in 2008, there were estimated to be 11,070 new cases of invasive cervical cancer, and 3870 cancer-related deaths are expected; this represents approximately 1 percent of cancer deaths in women Incidence and mortality associated with cervical cancer are higher among minorities, as illustrated by 2006 to 2008 American Cancer Society Statistics The incidence of cervical cancer is about 30 percent higher in African Americans (11.5 /100,000) than in whites, with about twice the mortality (5.0 versus 2.4 /100,000), representing 2 percent of cancer deaths in African American women Based on SEER data, in the US squamous cell carcinoma (SCCs) account for approximately 70% of cervical cancers, adenocarcinomas 25%, and adenosquamous carcinomas 3-5% (adenosquamous tumors exhibit both glandular and squamous differentiation). Mean age at diagnosis of invasive cervical cancer in the United States is 47 years. No form of cancer better documents the remarkable effects of prevention, early diagnosis, and curative therapy on the mortality rate than cancer of the cervix, due to effectiveness of the Papanicolaou cytologic screening test in detecting cervical lesions

Cervical intraepithelial neoplasia (CIN), adenocarcinoma, and squamous cell cancer of the cervix share many of the same risk factors The human papillomavirus (HPV) is central to the development of cervical neoplasia and can be detected in 99.7 percent of cervical cancers (squamous cell and adenocarcinoma). Most HPV infections are transient and the virus alone is not sufficient to cause cervical neoplasia. Molecular epidemiologic data has established the following risk factors for cervical neoplasia, all of which indicate a complex interaction between host and virus.

Early age at first intercourse Multiple sexual partners A male partner with multiple previous sexual partners The presence of a cancer-associated HPV The persistent detection of a high-risk HPV, particularly in high concentration (viral load) Certain HLA and viral subtypes Exposure to nicotine Genital infections (chlamydia)

Human papilloma virus (HPV) Papillomaviruses are double-stranded DNA viruses that belong to their own family, the Papillomaviridae Among the more than 40 genital mucosal HPV types identified, approximately 15 are known to be oncogenic. The two most common ones, HPV 16 and 18, are found in over 70% of all cervical cancers. The virus causes spectrum of changes ranging from condyloma accuminatum (flat, spiked and inverted condyloma and warty atypia), cervical intraepithelial neoplasia(CIN) and invasive carcinoma Koilocytosis / koilocytotic atypia: characteristic cellular changes seen on histologic tissue sections and cytology smears due to HPV infection Manifested by enlarged nuclei, nuclear pleomorphism, wrinkled (raisinoid) nuclei, hyperchromasia, binucleation (almost always present) perinuclear halos with distinct clear zone around nucleus and condensation of denser cytoplasm around the periphery. Low risk HPV subtypes (associated with genital condyloma and Low grade Squamous Intraepithelial Lesion =LSIL): 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 High risk HPV subtypes (associated with low and high grade SIL =LSIL and HSIL and invasive carcinoma): 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82; HPV 18: associated with lesions of glandular origin and small cell neuroendocrine carcinoma

Cervical squamous precancers classified as Cervical Intraepithelial Neoplasia (CIN).

CIN I (=low grade dysplasia of cervix) (histology)= LSIL (low grade squamous intraepithelial lesion) (cytology) (dysplasia disordered growth manifested by alteration in size, shape and organization of cell => expansion of immature cells) Micro: Epithelial disorganization (compared to surrounding epithelium) confined to the lower (basa)1/3 of the epithelial thickness. Koilocytotic changes on the surface CIN II (moderate dysplasia of cervix) (histology)= HSIL (high grade squamous intraepithelial lesion) (cytology) Micro: Epithelial disorganization in the lower 2/3 of the epithelial thickness. Increased N/C ratio, pleomorphic nuclei with hyperchromasia, loss of polarity, increased mitotic activity Koilocytotic changes on the surface can be seen. CIN III (severe dysplasia of cervix) (histology)= HSIL (high grade squamous intraepithelial lesion) (cytology) carcinoma in situ (CIS) Micro: Epithelial disorganization involves full epithelial thickness, pleomorphic nuclei with hyperchromasia, loss of polarity, increased mitotic activity with abnormal mitotic figures, increased N/C ratio 1-7% are associated with early invasive disease; 10-20% are estimated to progress to carcinoma if untreated

The peak incidence is about 30 years for high-grade neoplasia Precancerous lesion may exist in the noninvasive stage for as long as 15- 20 years and shed abnormal cells that can be detected on cytologic examination They do not invariably progress to cancer and the majority spontaneously regress, with the risk of persistence or progression to cancer increasing with the severity of the precancerous change They are associated with papillomaviruses, and high-risk HPV types are found in increasing frequency in the higher-grade precursor lesions

Cervical Glandular Precancers

Endocervical glandular atypia / dysplasia Not a reproducibly defined entity Adenocarcinoma in situ (AIS) Disordered growth in the glandular mucinous epithelium May be increasing in incidence Average age 35 to 40 years at presentation, range 27 to 74 years 30-60% have associated SIL HPV 16 or 18 are risk factors are present in 50-90% of cases Precursor to most cases of invasive adenocarcinoma Micro: hyperchromatic, enlarged, crowded nuclei with coarse chromatin, small single or multiple nucleoli, frequent mitotic figures the abnormal cells involving part or all of epithelium lining architecturally normal glands and/or forming the surface, apoptotic bodies common, abrupt transition to normal epithelium

Carcinoma of cervix
WHO classification of cervical tumors
Epithelial tumors
Squamous lesions Squamous cell carcinoma, not otherwise specified Keratinizing Nonkeratinizing Basaloid Verrucous Warty (condylomatous) Papillary (transitional) Lymphoepithelioma-like Squamotransitional Early invasive (microinvasive) squamous cell carcinoma Glandular tumors Adenocarcinoma Mucinous adenocarcinoma (endocervical, intestinal, minimal deviation, villoglandular subtypes) Endometrioid adenocarcinoma (may have squamous metaplasia) Clear cell adenocarcinoma Serous adenocarcinoma Mesonephric adenocarcinoma Early invasive adenocarcinoma

Other epithelial tumors Adenosquamous carcinoma Glassy cell carcinoma variant Adenoid cystic carcinoma Adenoid basal carcinoma Neuroendocrine tumors Carcinoid tumor Atypical carcinoid tumor High grade neuroendocrine carcinoma - small cell or large cell types Undifferentiated carcinoma Mesenchymal tumors and tumor like conditions Leiomyosarcoma Endometrioid stromal sarcoma, low grade Undifferentiated endocervical sarcoma Embryonal rhabdomyosarcoma Alveolar soft parts sarcoma Angiosarcoma Malignant peripheral nerve sheath tumor Leiomyoma Genital rhabdomyoma

Mixed epithelial and mesenchymal tumors

Carcinosarcoma (malignant mllerian mixed tumor) Adenosarcoma Wilms tumor

Melanocytic tumors
Malignant melanoma

Miscellaneous tumors
Germ cell tumors

Lymphoid and hematopoietic

Squamous Cell Carcinoma (SCC) Of Cervix

Reduction due to Papanicolaou screening test to detect premalignant lesions (1 million cases of SIL detected per year in US, and 11,070 new invasive carcinomas) Risk factors (see above pathogenesis for premalignant and malignant cervical lesions) Prognostic factors: clinical stage, nodal status, number of involved nodes, tumor size, depth of invasion, endometrial extension, parametrial involvement, angiolymphatic invasion Grading (degree of differentiation) does not correlate with prognosis Gross: tumor can be fungating (or exophytic) or ulcerating. Micro. malignant squamous cells are divided into 3 subtypes based on degree of differentiation. Well differentiated: predominantly mature squamous cells with abundant keratinization Moderately differentiated: less distinct cell borders and less cytoplasm than well differentiated tumors; also more nuclear pleomorphism and more mitotic activity Poorly differentiated: small primitive appearing cells with scant cytoplasm, hyperchromatic nuclei and marked mitotic activity; no/rare keratinization

Adenocarcinoma of cervix
Incidence increasing in US up to 25% of cervical cancers (due to decreasing rates of squamous cell carcinoma and difficulty in diagnosis of glandular lesion using current screening methods) Associated with in-situ adenocarcinoma (mean 5 year interval) Tumor grade of adenocarcinoma (for classical adenocarcinoma, not variants) Grade 1: well-differentiated Grade 2: moderately differentiated Grade 3: poorly differentiated

Staging of cervical carcinoma

AJCC prefers clinical staging (FIGO staging) of all patients for uniformity Clinical stage should be determined prior to start of definitive staging, and not be altered because of subsequent findings once treatment has started Pathologic findings should be recorded as pT, pN or pM, but should not change the clinical staging

Cervical cancer is staged as follows

Stage 0. Carcinoma in situ (CIN III) Stage I. Carcinoma confined to the cervix Ia. Preclinical carcinoma, that is, diagnosed only by microscopy Ia1. Stromal invasion no greater than 3 mm and no wider than 7 mm (so-called microinvasive carcinoma) Ia2. Maximum depth of invasion of stroma greater than 3 mm and no greater than 5 mm taken from base of epithelium, either surface or glandular, from which it originates; horizontal invasion not more than 7 mm Ib. Histologically invasive carcinoma confined to the cervix and greater than stage Ia2 Stage II. Carcinoma extends beyond the cervix but not onto the pelvic wall. Carcinoma involves the vagina but not the lower third. Stage III. Carcinoma has extended onto pelvic wall. On rectal examination, there is no cancer-free space between the tumor and the pelvic wall. The tumor involves the lower third of the vagina. Stage IV. Carcinoma has extended beyond the true pelvis or has involved the mucosa of the bladder or rectum. This stage obviously includes those with metastatic dissemination.

Pathology of the uterine corpus

Esther Elishaev, M.D. Department of Pathology, Magee Womens Hospital University of Pittsburgh School of Medicine

Overview of uterine corpus anatomy Corpus is upper 2/3 of uterus above level of internal cervical os
Uterus is hollow, pear shaped organ 40-80 g, 7-8 cm Peritoneal reflection is lower posteriorly than anteriorly Before puberty: endometrial tissue is inactive and composed of tubular glands, dense fibroblastic stroma, thin blood vessels After menopause: inactive (no proliferation or secretion), thin, often with cystic glands lined by flat or cuboidal cells, fibrotic stroma Anatomical divisions of uterus Fundus: cephalad to line connecting the insertion of fallopian tubes (FT) Cornua: lateral regions of fundus associated with intramural FT Isthmus/lower uterine segment: portion of corpus connecting with cervix Cervix: lower 1/3 of uterus; at and below level of internal cervical os Uterine cavity: 6 cm long, triangular shape, lined by endometrium (endometrial mucosa) then myometrium, then serosa Endometrium divided to Basalis layer is retained during menstrual cycle, does not respond to hormonal stimuli Functionalis layer (superficial 1/2 to 2/3) responds to menstrual hormonal changes and is shed monthly

Dating of endometrium
Must biopsy uterine corpus above the level of the isthmus, to include functionalis Proliferative phase Length varies, assumed 14 days in a 28 days cycle; during this phase, glands become more tortuous due to epithelial proliferation, in response to estrogen Micro Uniform tubular glands (test tubes) with mitosis in the epithelium and stroma Cross section uniform round (doughnutshape) glands evenly distributed in the stroma Secretory phase Traditionally assumed to be 14 days in a 28 days cycle Progesterone secretion inhibits endometrial proliferative activity and induces secretory activity Micro Early secretory Days 17 - piano key appearance; subnuclear vacuoles Day 18: luminal vacuoles, smaller size, nuclei approach base of cell Day 19: intraluminal secretion begins Days 20-21: maximal secretions Late secretory Day 22: maximal stromal edema, best time for implantation Day 23: prominent spiral arterioles (thickened walls, coiling, endothelial proliferation) Day 24: perivascular pre-decidualization, serrated / tortuous glands Day 25: predecidualization below surface endometrium Day 26: confluence of predecidual tissue; stromal granulocytes appear

Day 27: prominent stromal granulocytes; focal necrosis and hemorrhage Day 28: prominent necrosis and hemorrhage, aka shedding (glandular and stromal breakdown); condensed stroma, intravascular fibrin thrombi; stromal granulocytes and neutrophils.

Pregnancy related changes

Arias-Stella reaction endometrial glands with abundant clear or eosinophilic cytoplasm and marked nuclear changes (large, hyperchromatic, pleomorphic, smudged) with rare mitotic figures; decidualized stroma;

Acute endometritis Limited to post-delivery or miscarriage Due to retained products of conception or instrumentation Micro. Microabscesses( aggregate of neutrophils) , destruction of glandular epithelium and mixed inflammatory infiltrate Chronic endometritis In women with pelvic inflammatory disease (PID), postpartum, post-abortion (retained tissue), IUD, tuberculosis (miliary or TB salpingitis), symptomatic bacterial vaginosis Micro: spindly stroma with edema; weakly proliferative glands; plasma cells are characteristic, usually with histiocytes, lymphocytes and lymphoid follicles

Endometrial tissue (glands and stroma) outside the uterus; Women 20-30 years old, up to 10% of all women affected Consists of functional layers of endometrium that responds to the hormonal menstrual changes Causes pain, infertility (1/3 of women are infertile) Pathogenesis: 1. regurgitation (retrograde menstruation) 2. angiolymphatic dissemination (to lungs, lymph nodes); 3. metaplastic change of secondary mullerian system (pelvic mesothelium) Sites: Ovaries > uterine ligaments > rectovaginal septum > pelvic peritoneum May undergo malignant transformation (Endometrioid carcinoma, clear cell Carcinoma)

Abnormal Uterine Bleeding

During reproductive life- endometrium is constantly engaged in shedding and regrowth which is controlled by proper timing of hormonal release in both absolute and relative amounts. Alterations in this fine-tuning results in a spectrum of disturbances, including atrophy, abnormal proliferative or secretory patterns and hyperplasia. 1. Dysfunctional Uterine Bleeding = DUB bleeding due to a functional disturbances is the largest single group and is encompasses abnormalities in the menstrual cycle or systemic diseases. 2. Bleeding due to a well-defined organic abnormality comprising a smaller group of bleeding abnormalities in which the cause is organic (ex: chronic endometritis, submucosal leiomyomas, endometrial polyp, or endometrial neoplasms see below)

Causes of Abnormal Uterine Bleeding by Age Group Age Group Causes Prepuberty Adolescence Reproductive age Precocious puberty (hypothalamic, pituitary, or ovarian origin) Anovulatory cycle, coagulation disorders Complications of pregnancy (abortion, trophoblastic disease, ectopic pregnancy) Organic lesions (leiomyoma, adenomyosis, polyps, endometrial hyperplasia, carcinoma) Anovulatory cycle Ovulatory dysfunctional bleeding (e.g., inadequate luteal phase) Perimenopausal Anovulatory cycle Irregular shedding Organic lesions (carcinoma, hyperplasia, polyps) Postmenopausal Organic lesions (carcinoma, hyperplasia, polyps) Endometrial atrophy

Anovulatory Cycle
An excessive and prolonged unopposed estrogenic stimulation due to lack of ovulation (lack of progestational phase) The most frequent cause of DUB Most common at menarche and the perimenopausal period. In most patients the anovulatory cycles are unexplainable and are probably occurring because of subtle hormonal imbalances. Unscheduled breakdown of the stroma occurs ("anovulatory menstruation"), with no evidence of endometrial secretory activity More severe consequences of anovulation are hyperplasia and carcinomas. Pathogenesis (1) An endocrine disorder, such as thyroid disease, adrenal disease, or pituitary tumors (2) Primary lesion of the ovary, such as a functioning ovarian tumor (granulose-theca cell tumors) or polycystic ovaries (see section on ovaries) (3) Generalized metabolic disturbance, such as marked obesity, severe malnutrition, or any chronic systemic disease. Micro: (proliferative endometrium with glandular and stromal breakdown) proliferating endometrial glands with mitoses, however the tubular structure is focally distorted and dilated tubular glands of different sizes but relatively normal ratio of glands to stroma fibrin thrombi with hemorrhage and stromal crumbling (fragmented pieces with dense stromal aggregates) similar to the pattern seen in menstrual endometrium (=glandular and stromal breakdown) stromal edema

Endometrial glands and stroma deep in myometrium Causes menorrhagia, pelvic pain during menstruation 15% of uteri

Endometrial polyp
Sessile masses of variable size that project into the endometrial cavity. May be single or multiple and is usually 0.5 to 3 cm in diameter but occasionally large and pedunculated. Micro: Glands can be similar to adjacent cycling endometrium or hyperplastic (see hyperplasia), which may develop in association with generalized endometrial hyperplasia Covered by endometrial epithelium almost circumferentially (on a biopsy) Stroma usually fibrotric with spindle cells and centrally located vessels Rarely, adenocarcinomas may arise within endometrial polyps. Endometrial polyps have been observed in association with the administration of tamoxifen, frequently used in the therapy of breast cancer. Cytogenetic studies indicate that the stromal cells in endometrial polyps are clonal, with chromosome (6p21) rearrangements.

Uterine leiomyomas (commonly called fibroids) are perhaps the most common tumor in humans. Benign tumors may be present in about 75% of females of reproductive age, and each uterus harbors an average of 6.5 tumors. Each leiomyoma is a unique clonal neoplasm. May be asymptomatic or can cause abnormal uterine bleeding (submucosal leiomyomas) impaired fertility, compression of the bladder (urinary frequency) They can occur within the myometrium (intramural), just beneath the endometrium (submucosal) or beneath the serosa (subserosal). Infrequently involve the uterine ligaments, lower uterine segment, or cervix. In pregnant women - increases the frequency of spontaneous abortion, fetal malpresentation, uterine inertia, and postpartum hemorrhage. Malignant transformation (leiomyosarcoma) within a leiomyoma is extremely rare. Gross and Micro Sharply circumscribed, discrete, round, firm, gray-white tumors varying in size. On histologic examination, the leiomyoma is composed of bundles of uniform in size and shape smooth muscle cells that resemble the uninvolved myometrium. Mitotic figures are scarce.

Endometrial Atrophy
Normal in prepubertal girls, perimenopausal or menopausal women Micro: Low cuboidal or columnar epithelium with no mitotic figures Glands usually tubular or cystic, may be closely packed Stroma compact and inactive, without mitotic activity

Endometrial Hyperplasia (Endometrial Intraepithelial Neoplasia =EIN)

Definition: Proliferation of glands with an increase in gland to stroma ratio compared with proliferative endometrium (glands>>>stroma) Presents as abnormal bleeding. Numerous studies confirmed the malignant potential of certain endometrial hyperplasias and the concept of a continuum of glandular atypia culminating in carcinoma. Etiology: prolonged estrogen stimulation of the endometrium by anovulation or increased estrogen production. Conditions promoting hyperplasia include

1. 2. 3. 4.

menopause polycystic ovarian disease functioning granulosa cell tumors of the ovary prolonged administration of estrogenic substances (estrogen replacement therapy).

A key factor in the development of endometrial hyperplasia and related cancers is inactivation of the PTEN tumor suppressor gene through deletion and/or inactivation. PTEN inactivation is seen in 63% of premalignant endometrial hyperplasias and 50% to 80% of endometrial carcinomas. PTEN loss has been documented in some normal-appearing endometrial glands of 43% of premenopausal women, suggesting that loss of PTEN expression may be an early step in endometrial carcinogenesis.

Microscopic classification Depending on the complexity of the glands (compared to proliferative endometrium) the hyperplasia is classified as either simple (resembling proliferative endometrium) or complex (individual glands have cloverleaf shape). When hyperplasia composed of uniform nuclei (resembling the nuclei of proliferative endometrium) it is designated non atypical (=without atypia). When the nuclei are enlarged, irregular and stratified, with abundant mitoses the process is designated as atypical. 1. Simple hyperplasias without atypia Micro: architectural changes in glands of various sizes, the glands are usually round. epithelial growth pattern and cytology are similar to those of proliferative endometrium. these lesions uncommonly (~1%) progress to adenocarcinoma. 2. Complex hyperplasias without atypia Micro: glands have a complex architecture with scalloped surface, cloverleaf shape with gland crowding, enlargement, and irregular shape. ~3 % progress to carcinoma 3. Simple hyperplasia with atypia ~ 8% progress to carcinoma 4. Complex hyperplasia with atypia ~ 29 % progress to carcinoma A literature review noted the frequency of concurrent carcinoma among patients with atypical endometrial hyperplasia ranged from 17 to 52 percent across studies (Cancer. 2006 Feb 15;106(4):812-9.) Currently complex atypical hyperplasia is managed by hysterectomy or, in young women, a trial of progestin therapy and close follow-up. Nevertheless, the low rate of regression usually requires eventual removal of the uterus.

Carcinoma of the Endometrium

Endometrial carcinoma is the most common gynecologic malignancy in the US; approximately 40,100 cases are diagnosed annually and 7470 deaths occur. Incidence rates are higher in whites than in black, Hispanic or Asian/Pacific Islander women, however, mortality is almost two-fold higher in blacks than in whites (7.1 versus 3.9 per 100,000 women) Women have a 2.5 percent lifetime risk of developing endometrial cancer and it accounts for 6 percent of all cancers in women. Fortunately, despite their high frequency, most cases are diagnosed at an early stage (due to abnormal bleeding) when surgery alone may be adequate for cure, with five-year survival rates for localized, regional, and metastatic disease are 95, 67, and 23 percent, respectively.

Differences in epidemiology and prognosis suggest that two forms of endometrial cancer exist: those related (type 1) to and those unrelated (type 2) to estrogen stimulation and have different light microscopic appearance and clinical behavior.

Type 1 Comprising 80% of newly diagnosed cases of endometrial cancer in the United States. Associated with prolonged exposure to unopposed estrogen and are preceded by atypical endometrial hyperplasia a premalignant condition. Genetic abnormalities identified in endometrioid carcinoma include microsatellite instability, mutations in K-ras, PTEN or beta-catenin genes, or a defects in DNA mismatch repair. Usually endometrioid histology, characterized by a proliferation of back-to-back malignant endometrial glands without intervening stroma. A three-step grading system (degree of differentiation) is applied to endometrioid tumors grade 1 well differentiated - easily recognizable glandular patterns grade 2 moderately differentiated - well-formed glands admixed with solid sheets of malignant cells grade 3 poorly differentiated - solid sheets of cells with barely recognizable glands Peak incidence is in the 55- to 65-year-old woman. Higher frequency with (1) obesity, (2) diabetes (abnormal glucose tolerance is found in more than 60%), (3) hypertension, and (4) infertility (women who develop cancer of the endometrium tend to be nulliparous and to have a history of functional menstrual irregularities consistent with anovulatory cycles). The close relationship between hyperplasia and type 1 cancer of the endometrium in this setting is supported by the following: Both hyperplasia and cancer are also linked with obesity and anovulatory cycles. Women with ovarian estrogen-secreting tumors have a higher risk of endometrial cancer. Endometrial cancer is extremely rare in women with ovarian agenesis and in those castrated early in life. Estrogen replacement therapy is associated with increased risk. Prolonged administration of DES to laboratory animals may produce endometrial polyps, hyperplasia, and carcinoma. In postmenopausal women, there is greater synthesis of estrogens in body fat from adrenal and ovarian androgen precursors, a finding that may partly explain why there is increased risk of endometrial cancer with age and obesity. As discussed previously, inactivation of the PTEN gene is common to endometrial hyperplasia and cancer, as is microsatellite instability.

Type 2 Comprising 20% of newly diagnosed cases of endometrial cancer in the United States. Older population compared to the estrogen dependent group Hormonal risk factors have not been identified with this group, and there is no readily observed premalignant phase. Very commonly associated with p53 mutations The tumors in this group are poorly differentiated, non-endometrioid histology usually have papillary configuration covered by malignant serous or clear cells. Overall, these tumors have a poorer prognosis than estrogen-related cancers due to their propensity to exfoliate, undergo transtubal spread, and implant on peritoneal surfaces. No grading system, the tumors are high grade (grade 3).

- In general the endometrial carcinomas are usually polypoid tumors. - Spread generally occurs by direct myometrial invasion with eventual extension to the periuterine structures by direct continuity. - Dissemination to the regional lymph nodes eventually occurs, and in the late stages, the tumor may metastasize to the lungs, liver, bones, and other organs.

Staging of endometrial adenocarcinoma is as follows: Stage I. Carcinoma is confined to the corpus uteri itself. Stage II. Carcinoma has involved the corpus and the cervix. Stage III. Carcinoma has extended outside the uterus but not outside the true pelvis. Stage IV. Carcinoma has extended outside the true pelvis or has obviously involved the mucosa of the bladder or the rectum. Cases in various stages can also be subgrouped with reference to the three grades described above: G1. Well-differentiated adenocarcinoma G2. Differentiated adenocarcinoma with partly solid (less than 50%) areas G3. Predominantly solid or entirely undifferentiated carcinoma. Serous and clear cell carcinomas are automatically classified as grade 3. Prognosis Heavily depends on the clinical stage at presentation. In the United States, most women (about 80%) have stage I disease clinically and have well-differentiated or moderately well-differentiated endometrioid carcinomas. Surgery, alone or in combination with irradiation, gives about 90% 5-year survival in stage I (grade 1 or 2) disease. This rate drops to approximately 75% for grade 3/stage I and to 50% or less for stage II and III endometrial carcinomas. As mentioned, uterine papillary serous and clear cell carcinomas have a propensity for extrauterine (lymphatic or transtubal) spread, even when confined to the endometrium or its surface epithelium. Overall, fewer than 50% of patients with these tumors are alive 3 years after diagnosis and 35% after 5 years.

Clinical Pathologic Correlation #4 DISEASES OF THE CERVIX AND UTERUS Esther Elishaev, MD Thursday, February 19, 2009 1:00 4:00 p.m. I. OBJECTIVES The general objectives of this module are to: A. Review the gross and microscopic anatomy of normal cervix and uterus. Understand the taxonomy, pathogenesis and natural history of the major neoplastic and non-neoplastic diseases of the uterus and cervix and to apply this understanding to relevant clinical situations. Recognize the macroscopic and microscopic morbid anatomy of the major diseases of the uterus and cervix.




FORMAT This Thursday afternoon period will be divided into three time segments. The first hour of each three-hour period will be a lecture (lecture room 2). The second two hours will be an interactive laboratory session based on clinical scenarios and recognition of normal and morbid anatomy. The final hour will be an optional general review period (in lecture room 2). Items to be discussed include: A. Diseases of the uterus including carcinoma, and leiomyomata. endometrial hyperplasia,


In-situ and invasive neoplasia of the cervix.


Preparation should include review of Sections IV (Lecture outline) and V (Laboratory materials) of the student syllabus and a review of the required reading detailed at the end of this section. Also, a brief review of the normal microscopic anatomy of the uterus and cervix in an appropriate histology text is recommended. In addition, a pathology text reference is provided at the end of this section for background.



Clinical Scenario B1 Premise/Background: Abnormal uterine bleeding is defined as uterine bleeding other than physiologic menstrual bleeding and is a very common gynecologic problem. In addition to dysfunctional uterine bleeding, there are multiple anatomic causes of abnormal uterine bleeding.

Clinical Summary: Chief Complaint: History/ROS: 44-year-old G3P2 female with bleeding of five months duration. irregular uterine

Patient has had regular periods since age 13, two uneventful pregnancies and one elective pregnancy termination and has been in general good health until her periods became irregular five months ago. Her medical history is remarkable for cholelithiasis treated surgically six years ago. Family and social history and review of systems are non-contributory. Physical examination, including gynecologic exam, shows moderate obesity and is otherwise unremarkable. Laboratory tests included a fractional D&C, which shows complex endometrial hyperplasia without cytologic atypia.


Assessment/Discussion: 1. 2. 3. 4. Identify the photomicrograph corresponding to the diagnosis identified in this patient's endometrial curettage. What is the significance of this diagnosis for the patient with respect to symptom relief and prognosis? What is the relationship of this patient's age, medical history and moderate obesity to this endometrial hyperplasia? What are possible treatment options for this patient and what are the risks and benefits of each?

Clinical Scenario B2 Premise/Background: Early diagnosis of cervical neoplasia by screening exfoliative cytology has had a major impact on the natural history of cervical cancer. The appropriate management of patients with abnormal "Pap" smears is a significant issue in gynecology. 26-year-old G1P1 female returning for follow-up of abnormal cervical cytology obtained eight weeks ago during annual gynecologic examination. Patient enjoys excellent health and has had no significant medical problems except summer respiratory allergies. Family and social history and review of systems are unremarkable. Patient has had annual gynecologic examinations with "Pap" smears since age 18, which have all been unremarkable until eight weeks ago when a routine screening cervical cytology showed cell changes consistent with a high grade squamous intraepithelial lesion (CIN3). Physical examination, including repeat speculum exam, was unremarkable. Repeat "Pap" smear again showed changes consistent with CIN3. Colposcopically direct cervical biopsies showed focal CIN1 with an unremarkable endocervical curettage.

Clinical Summary: Chief Complaint:



Assessment/Discussion: 1. 2. Discuss the value of repeat "Pap" smear, colposcopy and cervical biopsy in this patient. Identify the photomicrograph of the CIN lesion identified histopathologically (CIN1) and the photomicrograph of the CIN lesion predicted on the basis of exfoliative cytology. How can this discrepancy be explained? Review of both cytology specimens confirmed the presence of a CIN3 lesion. Repeat biopsy also showed the CIN3 lesion. The endocervical curettage remained unremarkable. The patient was treated with cone biopsy which showed CIN3 without invasive carcinoma. The lesion appeared totally removed. Taking into account the anatomy and epidemiology of CIN, what is the significance of this diagnosis and treatment for the patient with respect to recurrence of the CIN lesion and her subsequent risk of invasive squamous cell carcinoma of the cervix?

3. 4.

Normal and Morbid Anatomy This portion of the laboratory will examine normal microscopic anatomy of the uterus and cervix and the microscopic and gross morbid anatomy of endometrial adenocarcinoma, uterine leiomyomata, intraepithelial neoplasia of the cervix, and invasive squamous cell carcinoma of the cervix. Microscopic and gross specimens necessary for the exercises below are available at stations within the laboratory. Exercises can be done in any convenient order. (Microscopes will be needed.) 1. Microscopic Set IV Identify the examples of normal uterus and cervix. Normal uterus Normal cervix A A B B C C D D


Microscopic Set V Identify the examples of the following uterine lesions. Endometrial carcinoma Uterine leiomyoma A A B B C C D D


Microscopic Set VI Identify the examples of the following cervical lesions. CIN1 CIN3 Invasive carcinoma A A A B B B C C C D D D


Macroscopic Set III Identify the examples of the following uterine and cervical lesions. Endometrial carcinoma Uterine leiomyoma Cervical carcinoma A A A B B B C C C


REVIEW Uterus 1. 2. Name the three major histologic types of endometrial hyperplasia. What is the relationship of endometrial hyperplasia to endometrial carcinoma? What clinical symptom is most common in both endometrial hyperplasia and endometrial carcinoma? How common is endometrial carcinoma? What are the peak ages for endometrial carcinoma and hyperplasia? The pathogenesis of endometrial carcinoma and hyperplasia is strongly related to what hormone? Differentiate between grade and stage in endometrial carcinoma, tell how they affect prognosis and why the prognosis of endometrial carcinoma is fairly good? What cells make up leiomyomas (fibroids)? Are leiomyomas benign or malignant? How common are leiomyomas? What are two common symptoms with leiomyomas?


4. 5.



8. 9. 10. 11.

Cervix 1. 2. 3. Why is the transformation zone important? Most carcinomas of the cervix are of what type? What is the most likely cause of the decline in death rate for cervical cancers over the past fifty years? Describe the concept of preinvasive neoplasia and the nomenclature used to describe it in the cervix.



What is the peak age for invasive squamous cell carcinoma of the cervix and how does this compare to the peak age for CIN? Squamous cell carcinoma of the cervix correlates with factors that suggest what mode of transmission? Name a possible viral vector in squamous cell carcinoma. How is tumor stage related to prognosis in invasive cervical carcinoma? Discuss the definition, incidence, locations, pathogenesis and clinical significance of endometriosis.


7. 8.


REFERENCES 1. 2. Naus G. Diseases of the cervix and uterus. Cotran RS, Kumar V, Robbins SL. Robbins Pathological Basis of Disease, Saunders, Philadelphia, Tumors of the Cervix; Endometrial Hyperplasia and Tumors of the Uterus. or: any suitable pathology textbook. 3. Appropriate sections in Comprehensive Gynecology, Herbst AL (editor).


GOAL The goal of this session is to discuss various contraceptive alternatives as they relate to the needs of each patient. Below are listed a few patient scenarios for group discussion. It is not important that all cases be discussed. It is important the all students appreciate what questions and issues are important to know before advising the patient. It is also important to keep in mind that multiple methods may be appropriate. During the course of the discussion please cover the following topics: What are the medical considerations for each patient? Is STD prevention an important issue? How and when does sexual behavior work into contraceptive decision-making? What personal issues for the patient are important to consider when discussing various forms of contraception? How would a pregnancy impact the patients life, (education, career, financial stability, etc.) at this point in her life? How would the age of the patient affect the contraceptive method selected? Discuss long-term vs. short-term contraceptive goals for the patient, including method reversibility. Have an appreciation for the ability of the patient to comply with the method. Patient 1: history 22 year old woman Positive for gonorrhea Multiple sex partners Smoker One prior pregnancy (unplanned) which resulted in a full-term delivery Does occasional drugs (heavy drinking and occasional cocaine)

Questions for consideration: What are the most important factors that should be considered for this person? Patient 2: history 29 year old woman Married, monogamous for 7 years Strict Roman Catholic Recent delivery 3 months ago; was breastfeeding until last month Mother of 3 children (2 of the pregnancies were planned, the last pregnancy was unplanned)

Patient 3: history 34 year old woman Has 1 child Obese, BP of 155/90 Smoker

Patient 4: history 15 year old; G0P0 Boyfriend pressuring her for more intimacy Doesnt have a clue about menstrual cycle or birth control

Worksheet 1 for Contraception Workshop #3 Please complete before attending the workshop FEMALE Level 1 Level 2 Level 3 Level 4 Level 5 Level 6 Level 7 Level 8 Level 9 Hypothalamus Pituitary Egg Release Fallopian tube Cervical mucus MALE Hypothalamus Pituitary Sperm Production Vas deferens Urethra/prostate/seminal vesicles

Sperm transport into female system Fertilization Implantation Post-implantation

Place a number indicating at what level each form of contraception PRIMARILY works in a heterosexually active couple. You may mark more than one level if you think it is appropriate. _______ Diaphragm _______ Sponge & spermicide _______ Male condom _______ Rhythm Method _______ Tubal sterilization _______ IUD (copper [Paragard]) _______ IUD (hormonal [Mirena]) _______ Combined oral contraceptives _______ Progestin-only pills _______ Depo Provera _______ Contraceptive patch (OrthoEvra) _______ Vaginal ring (NuvaRing) _______ Vasectomy _______ Contraceptive implants (Norplant) _______ Contraceptive implants (Implanon) _______ Cervical cap _______ Female condom _______ Lactational Amenorrhea _______ Withdrawal _______ Emergency contraception (Plan B)

Sara Hamel, M.D. Lecture 19 February 23, 2009 8:00 9:20 am.
Learning objectives: By conclusion of this reading and the accompanying lecture, you will be able to 1. Discuss the reasons for monitoring growth in neonates and children 2. Define intrauterine growth retardation 3. Generate an evaluation of a child with IUGR 4. Interpret patterns on growth curves Obtaining and monitoring growth variables are important components of the early assessment of the newborn. The mortality rate of term infants whose weight is substantially less than the mean (< 3rd percentile) is five to six times that of normally grown infants of a similar gestational age. Undergrown infants commonly have associated congenital anomalies, intrauterine infection, perinatal asphyxia or asphyxia-related complications, such as hypothermia, hypoglycemia, pulmonary hemorrhage, and meconium aspiration. Premature infants who are undergrown but free of infection, anomalies, and asphyxia in some respects may have an advantage over weight-matched premature infants who have appropriate weight for gestation. Recognition of the fetus that exhibits poor growth in utero continues to be an important means of identifying a neonate with an increased risk of asphyxia and death. Significant growth retardation is defined as weight less than the 3rd percentile for gestational age, as determined on a standard intrauterine growth curve. Growth retardation can be classified as asymmetrical or symmetrical. In asymmetrical growth retardation the physical findings suggest growth disturbance late in the pregnancy (i.e., the cause of the growth failure was short term and recent). Thus, weight is reduced, but length and head circumference are relatively preserved. Asymmetric growth retardation results in a small infant whose head and length are close to normal size for gestational age but whose weight-length ratio is reduced. These babies are often long and thin with alert expressions; decreased subcutaneous, subareolar, and thigh fat; prominent ribs; and long nails. Review of birth histories frequently reveals the presence of significant oligohydramnios, fetal distress, and perinatal asphyxia. Symmetric growth retardation produces a small infant with a proportionally small head and length. Symmetric growth retardation produces a short and light neonate with a normal ratio of weight and length. Symmetric growth retardation is believed to begin early in gestation, to be present through much of gestation, and therefore, to affect cell number in addition to individual cell size. Clearly, some infants achieving lengths and weights less than the 3rd percentile are healthy and their smallness is genetically determined; additionally some term infants weigh more than the weights represented in the 3rd percentile but are clearly malnourished as determined by their lack of subcutaneous tissue; these infants may exhibit long-term sequelae of malnutrition. Growth retardation is termed intrauterine growth retardation (IUGR) when the condition is identified in the fetus and the resultant newborn is undergrown for gestational age with

evidence of malnutrition. The prevalence of IUGR varies with the population studied but generally constitutes about 3% of all births in developed countries. Definitions of standards for normal growth should ideally include features such as maternal height, weight, age, ethnicity, geographic factors, birth number and socioeconomic background. It is important to differentiate infants on the basis of sex, since male infants weigh on average 200 g more than female infants by term. Significant maternal risk factors for IUGR include low socioeconomic level, primiparity and/or a history of having borne a previous IUGR infant. Maternal nutrition appears to influence fetal growth. High altitude; low maternal height and weight; poor pregnancy weight gain; smoking; heroin, methadone, phencyclidine, piperidine, cocaine, or alcohol use; and maternal diseases (found in 7 to 35 percent of IUGR pregnancies) such as hemoglobinopathies, hypertensive cardiovascular diseases, preeclampsia, or advanced diabetes place the fetus at increased risk. The nature of the mother's work and the stress she undergoes during pregnancy may be risk factors as well. Multiple births represent a special risk category; for example, approximately 20 percent of twin gestations are significantly growth retarded. Growth assessment of children beyond the newborn period relies on the use of growth charts. At the conclusion of this handout you will find the standard charts used in pediatric medicine.

Normal Child Development and Developmental Disabilities

Sara Hamel, MD Lecture 20 - February 23, 2009 9:30 10:50 a.m.
Learning objectives: By conclusion of this reading and the accompanying lecture, you will be able to 1. Discuss the similarities and differences between principles of physical and behavioral development 2. List the major domains of functioning 3. Discuss the normal patterns of development in each domain 4. Describe at least one developmental disorder of each domain. PRINCIPLES OF NORMAL DEVELOPMENT For ease of description and investigation, development is commonly discussed in terms of domains of function as follows: gross motor skills, the use of the large muscles of the body; fine motor skills, the use of small muscles of the hands; cognition, the use of higher mental processes, including thinking, memory and learning; language, the comprehension and production of meaningful symbolic communication; and social/emotional functioning, emotional reactions to events and interactions with others. Within each of the domains, developmental change is an orderly process, captured by a few fundamental principles adapted from the field of embryology. The first principle is of biological preparedness. The child is born with a set of innate basic capabilities that serve as building blocks of later developmental progress. In motor development, for example, the neonate demonstrates primitive reflexes. These organized patterns of response are triggered by specific stimuli and are executed without voluntary control. The second principle is differentiation and hierarchical organization. Developmental progress entails increasing differentiation of early global abilities which permits greater versatility and precision. For example, the newborn reflexively and firmly grasps with the whole hand when pressure is applied to the palm. In the course of development, the older child acquires the skill to isolate movements of individual fingers. The third principle is qualitative changes. The products of change appear discontinuous though the process of change is continuous. The qualitative changes within each domain serve as milestones for marking progress.


Early Reflex Patterns At birth the neonate's large muscle groups work together in involuntary reflex patterns that are triggered by specific maneuvers. The best known is the Moro response. This reflex occurs spontaneously following a loud noise and is typically elicited in the course of a physical examination by causing an abrupt change in the infant's head position. The first phase of the response consists of symmetric

abduction and extension of the arms and legs. The second phase is marked by adduction of the upper extremities, like an embrace, and is frequently accompanied by crying. With the emergence of voluntary control from higher cortical centers, primitive reflexes are replaced by reactions that allow children to maintain a stable posture if they are rapidly moved or jolted. The expected emergence and disappearance of some of these early reflex patterns is presented in Table 1. Antigravity Muscular Control The infant's earliest control task is that of achieving and maintaining stable posture against the influence of gravity. This control develops in an organized fashion, from head to toe, or in a cephalocaudal progression, paralleling neuronal myelination. The neonate shows minimal control of the neck flexors holding the head upright only briefly when supported in a sitting position. The neonate's head lags behind arms and shoulders when the infant is pulled to a sitting position. As the child reaches about 5 to 6 months of age, she anticipates the direction of movement on the pull to sit maneuver, and flexes her neck before the shoulders begin to lift. In the prone position, the newborn remains in a tightly flexed position and can simply turn faces from side to side along the bed sheets. Progressive shoulder and upper trunk in the first few months of life plus a decrease in flexor tone, enables the young baby to hold her chest off the surface with her weight supported on the forearms by 3 to 4 months of age. Evolution of trunk control down the thoracic spine can also be observed with the child in a sitting position. Infants can sit independently by 5 to 8 months. Balance and equilibrium reactions also emerge in a cephalocaudad sequence. As control moves downward, protective equilibrium responses can be elicited when the child is sitting by abruptly but gently pushing the child's center of gravity past the midline in one of the horizontal planes in space. The reflex response involves increased trunk flexor tone toward the force and an outreached hand and limb away from the force, usually emerges by 6 months of age. It protects the child from falling to the side. At 10 months of age, the child develops the parachute response, an outstretch of both arms and legs when the body is abruptly moved head first in a downward direction, a protection for falling forward. Development of Locomotion Gross motor milestones can be described in terms of locomotion. Rolling from tummy to back prone to suprine is usually accomplished after the child gains sufficient control of shoulder and upper trunk musculature to prop up on arms, at age 3 to 4 months of age. Rolling from back to front, supine to prone requires control of the lumbar spine and hip region as well as the upper trunk and usually is present by 5 to 6 months of age. By 6 to 9 months, as voluntary control moves to the hips and legs the child is capable of getting up on hands and knees, assuming a quadriped position and then of creeping. The next developmental milestone is supported standing also called cruising if accompanied by stepping. Increased control to the feet and disappearance of the plantar grasp reflex allow the child to walk independently. Walking three steps alone occurs at a median age of 11.7 months although the range is from 9 to 17 months of age. Development of Complex Gross Motor Patterns

Though dramatic changes in gross motor skills occur in the first year of life with the attainment of upright mobility, further progress in gross motor skills continues throughout childhood. The developmental sequence beyond walking incorporates improved balance and coordination and progressive narrowing of the base of support. Milestones and expected age ranges for some of these accomplishments are listed in Table 2. FINE MOTOR DEVELOPMENT

Grasp At birth, the neonate's grasps reliably and reflexively at any object placed in the palm and cannot release the grasp. The reflexive palmar grasp gradually disappears at about one month of age. From that point on, the infant gains control of fine motor skills in an orderly progression, from the midline to the periphery, or from proximal to distal.
The 3 month-old infant usually is able to hold an object in either hand if it is placed there, although he has limited ability to grasp voluntarily or release that object. At approximately 4 to 5 months of age, infants begin to use their hands as entire units to draw objects toward them for voluntary grasp. The child next develops the ability to bend the fingers against the palm (palmar grasp), to squeeze objects, and to obtain them independently for closer inspection. Differentiation of the parts of the hand develops in association with differentiation of the two hands. Between 5 and 7 months of age, the infant can use hands independently to transfer objects across the midline. Further differentiation of the plane of movement of the thumb allows it to move from adduction to opposition. The site of the pressure of thumb against fingers moves away from the palm toward the fingertips. Between 9 to 12 months, the fine pincer grasp, allowing opposition of the tip of the thumb and the index finger develops. This milestone allows for the precise prehension of tiny objects smaller than the child's fingers. Development of Complex Fine Motor Skills Early in the second year of life, the young child uses the grasp to master tools and to manipulate objects in new ways. Dropping and throwing games, stacking objects, and putting objects in and out of receptacles become favorite pastimes for children of this age. Advancements in fine motor planning and control can be seen in the child's ability to copy a variety of drawings. By age 3, a child can copy a circle; by 4 a square, by 5 a triangle.

Early Sensory Processing Innate sensory capabilities serve as the building blocks of cognitive development. Even at birth the healthy neonate responds to visual and auditory stimuli. These responses, like the primitive reflexes, take the form of integrated patterns of activity. Even at birth, it is possible to get the full-term infant to fix on faces at 9 to 12 inches from her face and to track objects horizontally at least 30 degrees. Newborns also respond to sound, typically quieting to human voice or to gentle inanimate sounds such as rattles or music.

Development of Sensori-motor Intelligence A summary of cognitive, language and social development is in Table 3. The first 2 years of life are the sensori-motor period of development. The world of the newborn appears to be one in which things that are out of sight are also out of mind. Toys that are partially covered or seen from a different view may not be recognized by the infant. The 3 month old will cease reaching for an object if it is covered by a towel during the reach. Early progress in the development of object permanence is indicated by the infant's continued though brief gaze at the site where a familiar toy or face has disappeared. Between 4 and 8 months of age, infants demonstrate interest in changes in position and appearance of toys. They can track an object visually through a vertical fall, and search for a partially covered toy. At approximately 9 to 12 months of age, babies are able to locate objects that have been hidden. This accomplishment is the beginning of object permanence. At this point peek-a-boo becomes a favorite pastime. Later the baby can crawl away out of view of mother and recall where to return to find her. By 18 months of age, children can deduce the location of an object even if they have not seen the objects hidden from view. If a piece of candy is hidden in an adult's hand and then placed under a hat, the infant does not have strategies needed to seek the multiply displaced object beyond the first hiding place. The early toddler, however, first searches in the hand, and then, upon not finding the sweet, persists in searching other sites. This older child has developed a fully developed notion of object permanence. Parents learn that they no longer can get away with hiding "no-no's" out of the child's view or distracting a child who really wants something. The child maintains a mental image of the desired object and a plan for obtaining it. Development of Symbolic Capabilities In the second year of life, the child demonstrates mental activity independent of sensory processing or motor manipulation. For example, the child observes a television superhero performing a rescue mission and hours later reenacts the scene with careful precision. The child had a mental image of the event which she used to generate the delayed imitation. As children develop the capacity for pure mental activity, they use objects to stand for other objects or for ideas. Genuine pretending begins; the child engages in playful representation of commonplace activities using objects for their actual purposes but accompanied by exaggerated sounds or gestures. This use of the object not for functional purposes but to demonstrate that it stands for something else is symbolic activity. The period is referred to as the period of symbolic play. The next stage in development allows the child to plan pretend activities in anticipation of the play theme to come and combines many steps into the play. Preparing for play indicates an advance in pretending beyond that of improvising with the objects at hand. Development of Logical Thinking

The preschool child has well developed capabilities for mental representation and symbolic thinking. However, limited life experience and lack of formal education lead to a unique and charming logic in this period. For example, preschoolers assume that all objects are alive like themselves. A car and a tricycle are alive, they claim, perhaps because they are capable of movement. Preschoolers usually have not sorted out the differences between reality and fantasy, believing explicitly or implicitly in magic, Santa Claus, and the Tooth Fairy. The logic of the preschooler is in large part influenced by the appearance of objects. Since an airplane appears to shrink as it takes off from the airport, the preschooler assumes that all of the people on the airplane shrink while they are on it. The idiosyncratic logic of the preschooler is gradually replaced by conventional logic and wisdom. School age children follow a logic akin to adult reasoning, at least in so far as the stimuli are concrete. They know that the airplane just looks smaller because it has moved further from the viewer and they giggle at the suggestion that the people on the plane might have shrunk. The limitations in their thinking become obvious only when they must reason about the hypothetical or abstract. They cannot, for example, devise scientific experiments because they cannot systematically consider all of the possible outcomes or reasons for a phenomenon. Adolescents, at least in western societies and with the benefits of formal education, tend to extend logical principles to increasingly diverse problems. They can systematically generate multiple logical possibilities when faced with scientific experiments. They can consider hypothetical problems. They apply these principles in reasoning not only to school work but also to their social situations. For example, the adolescent may think about who will go with whom to the prom. "She thinks that I think that she wants to go with him, but I know that she wants to go with me."

Early Skills in Speech Perception and Production Language skills are subdivided into two realms: receptive skills - the ability to comprehend communication, and expressive skills - the ability to produce the communication. The neonate demonstrates skills that are useful in receptive language. Even before birth, fetuses detect sounds and show preferences for some sounds over others. Pregnant women report that their unborn child may kick after sudden loud noises, and that they may kick harder with rock than classical music. At birth, the infant is particularly attuned to human voice and may turn toward a parent who is gently whispering. Children remain interested in sounds as they grow older and turn to the source of sound by 3 to 4 months of age. By 2 to 3 months of age, children begin to coo or make musical sounds spontaneously. This is the first step toward the development of expressive language. The sounds are undifferentiated vowel sounds lacking consonants and syllables. Babies use these gentle noises in a reciprocal exchange with their care givers as the earliest sign of shared vocal communication. The ability to coo is most likely an innate capacity. Hearing impaired

children begin to coo at the same age as children with normal hearing but gradually lose the ability to generate sounds unless they receive some kind of intervention. The sounds babies make undergo dramatic differentiation in the first year of life. By about 6 months of age, children place consonant sounds with vowel sounds creating what is known as babble. In this period, the infant says ma-ma or da-da without necessarily referring to a parent. By 9 to 12 months of age, they integrate babble with intonational patterns consistent with the parent's speech. This is called jargon. Development of Language In the second half of the first year the child develops early skills in true receptive language. By 6 months of age the child reliably responds to his name, and about 9 months can follow verbal routines: wave bye-bye, and show how big the baby is. Receptive language can be demonstrated as children follow increasingly complex commands. For example, one-step commands such as "throw the ball" will be understood by approximately 1 year of age. Understanding labeling of pictures begins after 1 year of age. The ability to choose between two pictures when asked "show me the... " should be consistent between 18 and 24 months of age. By 2 years of age, receptive language skills have advanced beyond the understanding of simple labels. The child is able to identify objects by their use. Continued advances in receptive language occur during the preschool years and are highly susceptible to environmental stimulation or deprivation. Expressive language initially develops slowly. The child's first meaningful words, often ma-ma or da-da referring specifically to the parents, are produced around the first birthday. Over the next 6 months the child may only master an additional 20 to 50 words. These early words come and go from the child's vocabulary; "dog" may be used endlessly one week and not used again for months. Early forms may be idiosyncratic child-forms: "bow-wow" may be the initial term for dog, baba for bottle. At approximately 18 months, word usage grows dramatically, increasing at an average of 4 or 5 words per day. Standard forms replace baby talk and simultaneously, the child begins to combine words into short sentences. The child's earliest two-word sentences typically contain important content words but lack prepositions, articles, and verb tense markings. This two word phase has been called telegraphic speech; the child's early sentences are vaguely similar to telegrams. By age 3, the child has developed complex language with the use of pronouns and prepositions, questions, and negatives. By age 5, the child uses all parts of speech, as well as clauses and complex sentences. In terms of the structure of the language, the productions of the kindergarten student are indistinguishable from the sentences of an adult.

Early Capabilities: Social Responsivity The earliest social-developmental task of the newborn is to establish a mutually satisfying relationship with his care givers. Neonates begin the social-developmental process by

visually fixing on faces in preference to other sights, a skill that is evident during the first few days of life. The responsive smile develops soon thereafter. Although the social smile may not appear until the child 4 to 6 weeks of life, it is another innate behavior. Smiling appears in infants from all cultures at about the same age. Visually impaired infants who cannot appreciate a smile on the faces of their care givers, nonetheless smile at ages comparable to sighted children. By 3 to 4 months of age, normally developing infants respond differently to different tones and may become distressed or excited by angry voices. Cooing in response to smiling or nodding also emerges. Development of Attachment The first major accomplishment in social development is attachment to familiar care givers. During the first 6 months of life, the infant is rather indiscriminate, engaging in these early social behaviors with any one willing to play. Evidence of a special relationship between parent and child may be subtle. For example, when the infant's play is disturbed, crying may only be calmed by only a parent's voice. The child's development of object permanence is primitive at this stage, although it is more quickly applied to a parent who is out of view than to an object out of view. By 6 to 8 months of age, children protest when their parents leave the room even if they lose interest when a toy is covered. As infants begin to recognize faces of familiar care givers, they may squirm and cling in the company of unfamiliar people, exhibiting stranger awareness. The severity of the reaction varies with infant's temperament and with experiences in child care. By 1 year of age, most children have experienced some periods, from moments to hours, of separation from a parent. Infants who have developed a secure attachment with their parents show signs of recognition and pleasure at reunion. In fact, as the child progresses in gross motor development, he initiates separation by walking away independently and exploring at greater distances from his parents. Typically, the infant regularly returns to the parent for some verbal encouragement, eye contact, or hugs and then ventures farther. In contrast, infants who have not developed a secure attachment may show indifference, ambivalence or disorganization at reunion with their parents. Their exploration of the environment during the toddler years is limited. These children are at risk for troubled social relationships with adults and peers as they get older. Development of Social Play Infants and young toddlers tend to line up and do similar activities simultaneously. Play is parallel in nature. By 2 years of age, with the development of symbolic capabilities in cognitive development, children begin pretend play. Play allows children to explore social roles as well as to practice what they have recently learned about the functions of various objects. Near 3 years of age, children begin to include one another in their pretending games. At first both children may select the same role (two mothers, for example) while later the roles will become more interactive.

The child's abilities to share are shaped by his social experiences. Children who attend day care may share successfully earlier than children raised at home. Taking turns is challenging for the preschooler who possesses a very limited concept of time. However, taking turns can also be achieved through consistent experience. Impulse control is just developing in the preschool years. By 4 to 5 years of age peer interactions grow increasingly cooperative and complicated; pretend play involves themes requiring greater feats of imagination and experience such as trips or parties. Older preschoolers enjoy helping with household tasks and are frequently more interested in participating in gender-specific activities than they were at an earlier age. This development may relate to cognitive as well as social development. As children understand that they are in the same category as their same sexed parent, either men or women, they become interested in the implications of the category membership. Strict adherence to the rules of category membership reflect the concrete and inflexible thinking of the preschooler. Preschoolers do not often play games with rules. The reason for this preference is simple. For the preschooler, with primitive logical capabilities, rules are variable, and can be made and broken at the discretion of the players. It is often a challenge to get through a board game with a preschooler who decides not to follow the rules once he discovers that they are not working in his favor. Children become capable of playing by rules when they reach school age. School age children, with superior logical capabilities, realize that rules are invariant, and must be followed regardless of the implications for the self. Children play games with rules first with some supervision. As they progress through the elementary school years, board games and sports become preferred activities for groups of peers. Development of Sense of Self Self awareness and independence develop gradually throughout life. It is exceedingly difficult to determine when the process begins. The earliest indications of an emerging identity occur at 6 to 9 months of age when the baby displays interest in his own mirror image. The 7 to 8 month old may prefer grabbing the cup or spoon than accepting a passive role in eating. This same infant may resist pressure to do something that he would rather not do; for example, he may fuss to stand when he has been placed in sitting. Beyond a year of age, toddlers rapidly expand their sense of self. They explore their environment with ease. They are increasingly able to operate independently; they feed themselves and manage a cup and a spoon. They develop clear ideas about what they want to have and to do. One to two year old children also enjoy their own accomplishments and can clap for their own successes. An emerging sense of self and the thrust for independence make discipline challenging in the toddler era. No longer can distraction and gentle redirection eliminate the need for the parent to take a position. If a parental prohibition contradicts the child's wants then the child may dig in and protest. Temper tantrums begin. The child has clear ideas in mind but cannot always appreciate the parent's point of view. Reasoning fails and discipline becomes necessary.

As the child reaches 2 to 3 years of age, toilet training can begin. substantially in their interest in achieving bladder and bowel control.

Children differ

By mid to late elementary school, with progress in cognitive development toward abstract and hypothetical thinking, children develop the ability to reflect self-consciously about themselves and others. First or second graders struggle to understand the causes of conflict or their emotional reactions to it. Older elementary school children and adolescents can analyze situations, reasons, reactions. They begin to understand their own motivations and features of the environment that trigger their responses.


Evaluation of developmental problems proceeds in the same manner as evaluation of other medical concerns: history, physical examination (including neurological and developmental evaluation) and laboratory testing. Important in establishing a diagnosis is consideration of the pattern of development across the various domains. CEREBRAL PALSY (CP) Cerebral palsy is a disorder of movement and posture resulting from injury to the motor areas of the brain and is an important cause of delayed and deviant development of gross motor skills. The type of cerebral palsy is a function of the location of the injured area. The insult to the brain may occur prenatally, during labor and delivery, or postnatally up through the preschool years. Most important in making the diagnosis of cerebral palsy is establishing that motor problems are static, not progressive. Regression of motor skills suggests a different set of diagnostic possibilities including surgically treatable lesions of brain or spinal cord or inherited neurodegenerative diseases. The definitive diagnosis of cerebral palsy should not be made until the child is at least 1 year of age. Abnormalities of tone are of particular significance in the diagnosis of cerebral palsy. Children with CP may present initially with hypotonia, and then develop increased tone over the first 12 to 18 months demonstrating clearly rigid or spastic hypertonia by age 2. Some children with CP demonstrate increased extensor tone beginning in early infancy. Further evidence of abnormally increased tone will be found when the supine child is pulled to an upright position and extends at the hips and knees, coming to stand on pointed toes rather than the appropriate sitting posture. This child, when placed in ventral suspension, will scissor the lower extremities as a result of hypertonia of the leg adducters and internal rotators. Evaluation of primitive reflexes is helpful in making the diagnosis of cerebral palsy. Damage to the central nervous system prevents high levels of control from superseding and inhibiting the influence of the early reflexes. Thus, obligate or persistent primitive reflexes are signs of cerebral palsy. For example, in the normal variant of the asymmetric tonic neck reflex (ATNR), the infant can move out of the reflex for example, as the gaze is directed to the other side of the body. An obligate ATNR, however, will cause the infant to remain in the fencer position until the head position is passively moved. This finding is not normal in a child of any age and is highly suggestive of cerebral palsy. Children with cerebral palsy

may have additional developmental concerns. They require comprehensive evaluation and treatment plan.


Delays in Cognitive Development Delays in cognitive development that persist to school age may indicate intellectual disability (mental retardation). According to the American Association on Mental Retardation, (whose members voted in June 2006 to change the name to the American Association on Intellectual and Developmental Disability), intellectual disability (mental retardation) is defined as "significantly sub-average general intellectual functioning existing concurrently with deficits in adaptive behavior and manifested during the developmental period." In this definition, significantly sub-average functioning refers to scores, obtained on standardized intelligence tests, that are at least two standard deviations below age-group norms; adaptive behaviors refer to the broader areas of functioning such as self-care, community survival skills, such as using the telephone, making change, using public transportation, and social interactions; the developmental period refers to the period from birth to 18 years of age. The ability to predict intellectual performance and academic achievement from developmental testing during infancy is quite limited. However, as the child approaches school age, particularly if he has had optimal educational support, the ability to predict later difficulties improves. By the time a child is 6 to 7 years of age, particularly if he has had good environmental conditions and early intervention programming, then intellectual limitations as measured on an IQ test typically characterize the individual's abilities throughout his lifetime. At that point, the term intellectual disability (mental retardation) is more specific and accurate than developmental delay. The vast majority of children classified as intellectual disability (mentally retarded) function in the mildly impaired range. These children typically have a normal physical examination, with no apparent evidence of malformation or deformity. Children with mild intellectual disability are likely to present with normal motor milestones and delays only in adaptive areas such as cognitive skills, self-care, language acquisition, or play. The detection of disability in these mildly affected youngsters may not occur until the child experiences school performance difficulties. The etiology of mild intellectual disability is generally felt to be multi-factorial, a combination of multiple genetic contributions and of limited social enrichment. The more significant the degree of intellectual disability is, the more likely that a specific etiologic factor will be found. Children who score in the moderate, severe or profound range of intellectual disability are likely to have congenital malformations of the central nervous system, severe neurological insults in the prenatal or perinatal period, an inherited disorder, or another specific diagnosis. In a society that prizes intellectual accomplishments, the identification of cognitive delays is very upsetting for a family. Children with mild intellectual disability can be educated to read and write and do simple mathematics. As adults they can often live independently and hold down jobs. The extent of their disability will be most prominent during the school years or during times of life crisis beyond school age. Children with moderate intellectual disability probably may not learn to read phonetically and write. Nonetheless, their abilities in

language, self-care and adaptive skills may allow them to live and work in semi-independent supervised settings. Children with severe and profound mental retardation require substantial life long support.


Language Development Delays and disturbances in language development are most frequently associated with mental retardation, hearing impairment, childhood autism, or environmental deprivation. However, language difficulties may occur in a child with normal hearing, normal intellectual functioning, normal capacity to relate to others and opportunities to learn; in those cases they are labeled specific language disabilities. The reason for specific language disorders remains unknown. There are no specific physical signs associated with specific language disorders. The physician's role in large part is to rule out other disorders with different etiologies and prognoses. Many children with specific language disorders during the toddler era will develop adequate speech and language and communication skills by mid-elementary school. At present, there are no variables consistently associated with good prognosis; however, prognosis for communication clearly is improved through early communication therapy. Many toddlers and preschoolers with selective problems in language acquisition develop reading difficulties during the school age years.

Childhood autism is a behaviorally defined syndrome characterized by deviant social development, absent or very abnormal speech and language development, and very unusual, inflexible, concrete cognitive abilities. Although the pathogenesis of autism is poorly understood, it is now clear that this disorder is found in all parts of the world, in all races, and in all socioeconomic groups. A higher rate of autism and other neurodevelopmental disorders in the families of children with autism suggests a heritable disorder. A precise mechanism has not been discovered. In the physical examination, one of the earliest warning signs for the physician may be the baby's lack of social responsiveness marked by absent smiling, poor eye contact, and little response to parental or physician attention. History and examination in the second 6 months of life may reveal a baby who is not babbling, who is disinterested in waving, imitating, engaging in baby games, or in playing with baby toys. Some children with autism may have a relatively normal developmental course into the second or third year and then suffer regression in social, behavioral and communication development. Parents give a history that the children lose interest in social contact and lose verbal language skills that they had previously demonstrated without demonstrating regression in motor abilities. Parents of toddlers may also report and the physician may observe a preoccupation with fingers and repetitive motor activities such as whirling, rocking, or hand flapping. It is also during the second and third years that unusual language patterns emerge, such as the lack of meaningful speech or gestures, the noncommunicative use of sounds as with echolalia, or use of unusual verbal rituals such as counting, particularly when the child is stressed. Between 2 and 3 years of age, the child

with autism may appear to be withdrawn and prefer to play alone; The child may use toys in idiosyncratic ways, such as spinning them or lining them up in rigid patterns. It should be noted that many children with autism demonstrate very uneven development, with minimal speech or language skills, but with excellent memory or fine motor puzzle-solving skills. In the past, only the most extreme variants of the disorder were diagnosed as childhood autism. Under those clinical definitions, about 75 percent of autistic children score in the range of mental retardation on tests of intellectual functioning. More than 90 percent require lifelong social support systems because of continuing symptoms and severely handicapped development. Current thinking favors the idea that autism is a spectrum or a continuum of disability. With a broader definition of the disorder than in the past, as well as with early identification and educational remediation, the prognosis for many affected children may be better than previously thought.

The tasks of developmental surveillance, identification of children with variations in the usual developmental patterns, and referral for appropriate developmental services, especially for children during infancy and in the preschool years, falls largely, and often exclusively, to primary care physicians. Examining physicians need estimates regarding the expected chronology of development. It must be stressed however that these developmental milestones are to be viewed as guidelines rather than as fixed time frames within which behavior acquisition may be judged as normal or abnormal. In evaluating a child, the physician must employ these guidelines in the light of his or her own clinical judgment, taking into account the child's own personality traits, experiences, and degree of cooperation. Recommendations for further assessment and treatment should be made in close consultation with the child's family because of their key role in assessment and treatment. Identification of developmental variations, delays and disorders will invariably be a difficult issue for parents. Identification of developmental disorders arouses grief, guilt, and fear about the future. Physicians must be sensitive to these family issues. Moreover, raising a child with a handicapping condition may stress usual coping mechanisms of even the most competent family. Hence, these families need continuous support and compassion. It is rare that the physician can cure a developmental disorder. However, that in no way means that the physician has nothing to offer. Families look to their doctor for a thorough diagnostic evaluation, for direction toward therapeutic and educational services that can help their child, for support through difficult times, and for hope that the affected child can reach his or her own potential, whatever that may be. Parents greatly appreciate physicians who stay actively involved once the diagnosis of a developmental disorder is made and who integrate health care issues with educational and social goals.

Table 1. PRIMITIVE REFLEXES AND PROTECTIVE EQUILIBRIUM RESPONSES Reflex Moro Hand grasp Crossed adductor Toe grasp ATNR Head righting Protective equilibrium Parachute Appearance Birth Birth Birth Birth 2 weeks 4-6 months 4-6 months 8-9 months Disappearance 4 months 3 months 7 months 8-15 months 6 months Persists voluntarily Persists voluntarily Persists voluntarily

Different sources may vary on the precise timing of the appearance and disappearance of these primitive and equilibrium responses.

Table 2: HIGHER GROSS MOTOR MILESTONES Age by Which 75% of Children Task Performed Task Walks well Throws ball overhand Pedals tricycle Balances on one foot for 1 second Hops on one foot Heel to toe walks (75% of time) Catches ball bounced (75% of time) 13 months 22 months 32 months 36 months 48 months 4-4/5 years 4-7/8 years

Table 3a: Developmental Milestones (Ages birth 2 years)

NOTE: mean ages for some milestones vary slightly from one authority to another (What follows on the next several pages are from several different sources.)


Piagetian Stage I II Age Birth to 1 month 1 to 4 months Object Permanence Shifting images Stares at spot where object disappeared (looks at hand after yarn drops) Visually follows dropped object through vertical trajectory (tracks dropped yarn to floor) Causality Generalization of reflexes Primary circular reactions (thumb sucking) Play Receptive Language Turns to voice Searches for speaker with eyes Expressive Language Range of cries (hunger, pain) Cooing Vocal contagion


4 to 8 months

Secondary circular reactions (recreates accidentally discovered environmental effects, e.g., kicks mattress to shake mobile) Coordination of secondary circular reactions

Same behavioral repertoire for all objects (bangs, shakes, puts in mouth, drops)

Responds to own name and to tones of voice

Babbling Four distinct syllables


9 to 12 months

Finds an object after watching it hidden

Visual motor inspection of objects Peek-a-boo

Listens selectively to familiar words Responds to "no" and other verbal requests Can bring familiar object from another room Points to parts of body

First real word Jargoning Symbolic gestures (shakes head no) Many single words -- uses words to express needs Acquires 10 words by 18 months Telegraphic twoword sentences

12 to 18 months

Recovers hidden objects after multiple visible changes of position

Tertiary circular reactions (deliberately varies behavior to create novel effect)

Awareness of social function of objects Symbolic play centered on own body (drinks from toy cup) Symbolic play directed toward doll (gives doll a drink)


18 months to 2 years

Recovers hidden object after invisible changes in position

Spontaneously uses nondirect causal mechanisms (uses key to move wind-up toy)

Follows series of two or three commands Points to pictures when named

Table 3b: Developmental Milestones (Ages 2 5 years) Selected Language Guideposts

Skill Comprehension Age 2 Follows simple commands Identifies body parts Points to common objects Labels common objects Uses two or three word sentences Uses minimal jargon Age 3 Understands spatial relationships (in, on, under) Knows functions of common objects Uses three to four word sentences Uses regular plurals Uses pronouns (I, me, you) Can count three objects Can tell age, sex, and full name Intelligible to strangers 75% of time Age 4 Follows two-part commands Understands same/different Age 5 Recalls part of a story Understands number concepts (3-4-5-6) Follows three-part commands Speaks sentences five words Uses future tense Names four colors Can count 10 or more objects


Speaks four to five word sentences Can tell story Uses past tense Names one color Can count four objects


Intelligible to strangers 25% of time

Normal dysfluency (stuttering)

Dysfluencies resolved

Table 3c: Developmental Milestones (Ages 2 5 years) Motor/Language

Domain Gross Motor Age 2 Runs well Kicks ball Climbs well Steps: 2 feet per step Age 3 Steps: alternates feet going up Jumps from step Tricycles Balances 1 foot momentarily Copies circle Copies cross Age 4 Hops 1 foot Steps: alternates going down Balances 1 foot 5 sec Age 5 Stands 1 foot 10 sec Skips Bicycles

Fine Motor

Tower 6 cubes Imitates a vertical line

Copies square Draws person 2 to 4 parts Scissors Follows 2 part commands Same/Different Long sentences Tells story Colors Counts to 4

Copies triangle Draws person with body Prints Recall story Follows 3 part commands Long, correct sentences Counts to 20

Language Comphrension

Follows some commands Identifies body parts Labels objects Two to 3 word sentences

Spatial relation (in, on, under) Knows object functions Three to 4+ word sentences Plurals Pronouns Count


Table 3d: Developmental Milestones (Ages 2 5 years) Neuromotor Accomplishments

Domain Gross-Motor Skills Age 2 Runs well Kicks ball Climbs well Goes up and down stairs (one step at a time) Age 3 Goes up stairs (alternating feet) Jumps from bottom step Pedals tricycle Stands on one foot momentarily Age 4 Hops on one foot Goes down stairs (alternating feet) Stands on one foot (5 seconds) Age 5 Stands on one foot (10 seconds) May be able to skip

Fine-Motor Skills

Builds tower of six cubes Imitates vertical crayon stroke Turns book pages singly

Copies circle Copies cross

Copies square Draws person with two to four parts Uses scissors

Copies triangle Draws person with body Prints some letters

Additional Reading Materials: American Academy of Pediatrics Joint Committee on Infant Hearing: Position statement 1982. Pediatrics 70:496-497, 1982. Diagnostic and Statistical Manual of Mental Disorders, 3rd ed. Washington, DC, American Psychiatric Association, 1980. Fraiberg S: Insights from the Blind. Basic Books Inc, New York, 1977. Illingsworth RS: The Development of the Infant and Young Child: Abnormal and Normal, 7th ed. Churchill, Livingstone Inc, New York, 1980. Knobloch H, Pasamanick B (eds): Gesell and Amatruda's Developmental Diagnosis, 3rd ed. Harper & Row, Publishers Inc, New York, 1974. Levine M, Carey W, Crocker A, Gross R: Developmental-Behavioral Pediatrics. WB Saunders Co, Philadelphia, 1983. Louick D, Baland T: Psychological tests: A guide for pediatricians. Pediatr AnnALS 7:86-101, 1978. Opitz JM: Mental retardation: Biological aspects of concern to pediatricians. Pediatrics in Review 2:41-40, 1980. Smith DW, Simons FER: Rational diagnosis AND evaluation of the child with mental deficiency. Am J Dis Child 129:1285, 1975. Dixon SD, Stein MT (eds): Encounters with Children. Year Book Medical Publishers, 1986. Rutter M: Developmental Neuropsychiatry. The Guilford Press, 1983.

Domestic Abuse
Judy Chang, M.D., MPH
Assistant Professor Department of Obstetrics, Gynecology & Reproductive Sciences

Lecture 21 - Monday, February 23, 2009 11:00 am 12:00 pm

Reproductive & Developmental Biology Course University of Pittsburgh School of Medicine February 9 27, 2009

Endocrinology of Sexual Maturation Kathleen Ryan, Ph.D. Learning Objectives: The student will understand: 1. 2. 3. The endocrine differences between pre- and post-pubertal children The hormonal events coordinating the onset of puberty The differences between menstrual function in adolescents and adult women.

Further Reading: Ferin M, Jewelwicz R, Warren M, 1993 The Menstrual Cycle, Chapter 7; review Chapter 2 Hillier (Scientific Essentials) Chapter 2.8 Rudolph (Pediatrics), pp. 39-67

Key words and phrases: sexual maturation, hypothalamus, Tanner Staging, somatic growth, fertility onset, disorders of puberty onset

Outline: 1. Endocrine status of sexually immature child. a. testis and ovary b. hypothalamus and pituitary Hypothalamo-hypophyseal-gonadal function of neonate. Events associated with sexual maturation. a. patterns of gonadotropin secretion b. secondary sexual characteristics c. Tanner staging of maturation d. changes in growth rate Timing of the onset of puberty a. role of the central nervous system b. role of gonads, adrenals and thyroid gland c. role of somatic growth Characteristics of menstrual cycles in adolescents

2. 3.



Lecture notes: Sexual Maturation 1. The endocrine status of the immature human (ages 1- ~ 8 years) may be characterized as "selective hypofunction" of all aspects of the endocrine axis regulating gonadal function. a. Testis and ovary In both male and female children, the gonads are small and unstimulated. In the case of the male, gonads are structurally complete, but immature. Thus, Leydig cells are present, but few in number; they are probably secreting testosterone, but at very low levels. As a consequence of the low testosterone secretion, the testis tissue, although differentiated, is un-stimulated. That is, Sertoli cells and seminiferous tubules are present as are basal germ cells, but tubules are relatively short, lumens are small, and no mature spermatids or spermatocytes are present in the testis. The ovary, on the other hand, contains all of the ova which will ever be available to each individual, in the form, primarily, of primordial follicles, that is, an ovum surrounded by a single layer of flattened, epithelial cells. There is some degree of follicular growth in the ovary of the immature girl, reflecting the "gonadotropin-independent" phase of follicular growth (see notes from Gosman lecture). Thus, ovaries from young girls show follicles at various stages of preantral development, and some very early antral follicles, but no large antral or preovulatory (Graafian) follicles and no corpora lutea. The absence of mature or large antral follicles is the reason for the very low levels of estradiol secretion in girls. There is very low level secretion of estradiol occurring from the small follicles in girls, but it is orders of magnitude lower than that typical of women with adult endocrine function. b. Hypothalamus and pituitary It has been documented that if gonads in immature children (or monkeys) are exposed to stimulatory levels of LH and FSH, comparable to those observed in adults, full gonadal function will ensue i.e. testicular maturation of mature sperm and ovarian follicle maturation, ovulation and normal corpus luteum function. It is also well documented that while LH and FSH secretion occur in sexually immature primates, the amount of gonadotropin secreted in either sex is insufficient to stimulate adult gonadal function. Thus, the underlying cause of the gonadal hypofunction characteristic of children of both sexes is insufficient secretion of LH and FSH. Studies of pituitary gonadotropin secretion in juvenile primates have shown that, with appropriate (i.e. adult) levels of stimulation from the hypothalamic peptide, GnRH, fully adult patterns of secretion of LH and FSH will result. This is not to say that there is no developmental pattern to the onset of gonadotropin secretion at puberty (as described below), however, it is clear that while pituitary hypofunction is the proximal cause of the sexual quiescence of the juvenile period, the pituitary cells are entirely capable of adult function (as are the gonads), given the appropriate level of hypothalamic support. By the process of elimination, then, we arrive at the hypothalamic decapeptide, GnRH. It is required to stimulate pituitary gonadotropin secretion (FSH and LH); replacement of GnRH via injection stimulates LH and FSH secretion in juvenile primates, and individuals who do not, for some reason, possess GnRH neurons (e.g. Kallmans syndrome), do not ever achieve sexual maturation. The puzzle is that one can measure GnRH in hypothalamic neurons in brains of young monkeys and humans (autopsy specimens). This GnRH has been shown to be biologically active, does not increase in quantity with the onset of sexual maturation, and its release can be elicited by electrical stimulation or by pharmacological agents.

Remarkably, gonadectomy via surgical intervention or because of genetic mutation, (Turner's syndrome or other with streak gonads) causes no permissive increase in the secretion of LH or FSH in juvenile primates. Remember that in the adult, the secretion of LH and FSH are under negative feedback control by the gonads, and removal of gonads in mature individuals of either sex causes a marked increase in both gonadotropins which is sustained as long as plasma steroid levels remain low. The absence of such a castration response in young primates shows that the the prepubertal gonad, with its low levels of steroid secretion, is not causing the low LH and FSH in juveniles (Figure 1). Clearly, gonadotropins will be low whether gonads are present or not. Thus, even though there are normal levels of biologically active GnRH in the hypothalami of young primates, it is not being released. With the absence of GnRH stimulation to the pituitary gonadotrophs, LH and FSH remain low, thus the gonads remain unstimulated and immature.

Figure 1. Developmental changes in LH and FSH in intact and castrate monkeys from birth to three years of age.

At present, it seems likely that the GnRH neurons in juvenile primates are competent to function at adult levels, but they lack a critical stimulus or are under tonic inhibition by some other factors or neurons (or both). The reason for this view arises from studies of infant humans and monkeys (neonatal). In infants of both species, there is evidence of substantial activity of the hypothalamo-hypophyseal axis governing LH and FSH secretion. LH secretion is clearly pulsatile, pulses of LH elicit testosterone from the testis of infant boys/monkeys and orchidectomy (removal of both testes) results in a marked increase in secretion of both LH and FSH in both species. It is clear that if this level of activity continued, testicular development and sexual maturation would occur. However, in both species, within 6-18 months after birth, LH and FSH in plasma decline to very low levels, stimulation to the gonad ceases and this quiescent state persists for the several years of the "juvenile" period. The reactivation of the reproductive axis which leads to adult endocrine function is called "puberty", and represents the resumption of GnRH secretion and the end of the juvenile period. 3. Events associated with sexual maturation External manifestations of sexual maturation are due, in large part, to increasing levels of steroid hormones in the blood from the awakening gonads. The rate and order of changes differ according to sex, but in both cases, the initiating event in sexual maturation is an increase in secretion of GnRH from the hypothalamus. There is controversy about whether this is an increase in frequency or amplitude of GnRH pulses. Since GnRH cannot be measured directly in

peripheral serum, this will remain speculative in the human, but examination of very low levels of LH in boys has led to the preliminary suggestion that frequency of GnRH secretion may not change, therefore the "activation" of the hypophyseal-gonadal axis represents an increase in the amount of GnRH secreted per pulse. In either event, before there is any external sign of sexual maturation, peripubertal primates exhibit an increase in gonadotropin secretion. This increase occurs first only at night, and is correlated with sleep (Figure 2). If the sleep-wake rhythm of the child is reversed, the elevation in pulsatile LH secretion also reverses

Figure 2. Developmental changes in pulsatile LH secretion in boys.

The response of the pituitary to a bolus injection of GnRH exhibits a developmental pattern which can be used clinically to assess pubertal status in children. With the first few days of increased (nocturnal) stimulation of the pituitary, a given bolus of GnRH results in a large rise in FSH, and a small increase in LH. This is the GnRH response of a pituitary which has had only minimal or no exposure to GnRH. As the increased hypothalamic input continues, this ratio of gonadotropic response shifts to favor LH. Thus, in a post-pubertal person, that same bolus of GnRH per kg body weight, will cause a brisk, high amplitude elevation in LH and a small increase in FSH. The significance of this change is not known, but the alteration in response to GnRH at different times during the awakening of the reproductive axis is well described enough to serve as a useful diagnostic tool in the evaluation of patients for progress of sexual maturation (Figure 3). As the pubertal cascade progresses, more gonadotropin secretion occurs during the daytime also, so by the time sexual maturation is complete, the clear day/night difference in LH secretion is gone (in humans), and the overall level of LH and FSH has increased to levels supportive of full gonadal activity (Figure 2).

Figure 3. Changes in response to fixed bolus of GnRH in children at differing maturational stages.

"Tanner staging" refers to a systematic description of the order of appearance of secondary sexual characteristics in boys and girls from completely infantile, immature (Tanner 1) to fully adult appearance (Tanner stage 5). Tanner staging for girls is based on changes in the morphology of the breast (primarily estradiol dependent) and appearance and distribution of pubic hair (primarily adrenal androgen). Staging of boys is based on morphological changes in the penis and scrotum as well as distribution of pubic hair, all these are testosterone dependent.

Tanner Staging: Girls

Breast Maturation
Stage 1 (prepubertal) Stage 2 Elevation of papilla only Elevation of breast & papilla as small mound, Areola diameter enlarged. Median age: 9.8 yr Further enlargement without separation of breast and areola. Median age: 11.2 yr Secondary mound of areola and papilla above the breast. Median age: 12.1 yr Mature stage: projection of papilla only, due to recession of areola to the general contour of the breast Median age: 14.6 yr

Pubic Hair
No pubic hair sparse, long pigmented hair, chiefly along labia majora. Median age: 10.5 yr Dark, course, curled hair, sparsely spread over mons. Median age: 11.4 yr Adult-type hair, abundant but limited to the mons. Median age: 12.0 yr Adult in quantity and scope, distribution to medial aspect of thighs. Median age: 13.7 yr

Stage 3

Stage 4

Stage 5

[Data from Speroff, Glass & Kase (eds) Clin Gyn Endo & Infert, Chapter 13, p377, 1984]

Tanner Staging: Boys

Stage 1 (prepubertal) Prepubertal: testes, scrotum and penis ~same size and proportion as early childhood. Stage 2 Enlargement of scrotum and testes. Skin of scrotum reddens and changes in texture. Mean age: 11.6 yr Enlargement of penis with growth and development of glans. Further growth of testes and scrotum. Mean age: 12.9 yr Increased size of penis with growth in breadth and development of glans. Testes and scrotum larger; scrotal skin darkened. Mean age: 13.8 yr Genitalia adult in size and shape Mean age: 15 yr

Pubic Hair
Prepubertal: no pubic hair

sparse, long pigmented hair, chiefly along base of penis . Median age: ~12.5 yr

Stage 3

Darker, courser, more curled hair, spread over juncture of pubes. Median age: 13.9 yr

Stage 4

Adult-type hair, area covered is still less than the adult. No spread to medial surface of thighs Median age: 14.4 yr

Stage 5

Adult in quantity and scope, distribution spread up linea alba. Median age: 15.2 yr

[Data from Tanner, WA and Tanner, JM, Arch Dis Child, 1970, 45:13-23.]

In girls, the first menstrual period (menarche) is a relatively late event in development. At the time of menarche, breasts and pubic hair have often reached stage 4, and in just about all cases, the peak height velocity has been passed and growth rate is slowing. There is wide normal variation in the age at which the above progression occurs, but once commenced, most children complete the process of physical maturation within 2-3 years of the onset of Tanner stage 2. It is somewhat rare, but not abnormal for the progression from Tanner stage 2-5 to take as long as 5 years, more often in boys than in girls.

4. Regulation of Growth During Development Gonadal growth is exclusively the result of the actions of LH and FSH on the testes and ovaries. As reviewed in the lectures on the adult, some of the actions are directly on the gonadal tissue, e.g. FSH stimulation of Sertoli cells and granulosa cells, and some of the actions are a consequence of the steroid secretion which results from the actions of LH on the Leydig cells and FSH on the granulosa cells. Secondary sexual characteristics are almost all a consequence of the increased plasma levels of gonadal steroids, particularly estradiol and testosterone. Infancy is the phase of life in which the most rapid growth occurs. The rate of growth slows throughout life from the peak rate of up to 25 cm/yr during infancy until it ceases sometime in the late teens. However, as shown in the graph of growth rates throughout life, sexual maturation is accompanied by a brief period of accelerated growth that is often termed the adolescent growth spurt (Figure 4). During this period of time, girls gain from 4-6 inches in height and boys gain from 5-10 inches

The accelerated growth characteristic of this period is mediated by increased sex steroids, especially estradiol. The estradiol increases somatomedin production by the liver of the adolescent, and Growth Hormone secretion is augmented at night in concert with the nocturnal increases in GnRH secretion. The increase in Estradiol, stimulated by the pubertal rise in FSH and LH, is required for the growth spurt to occur. In individuals lacking steroid secretion, or if sexual maturation is blocked (intense exercise) or reversed (by treatment with a GnRH agonist) the accelerated growth does not occur although growth at the slower rate does continue.
Figure 4. Pattern of rate of somatic growth in humans.

. The diagrams in figure 4 show the timing of this accelerated growth with respect to the events of sexual maturation and chronological age.

Figure 5. Timing of major landmarks of puberty in humans.

The difference in timing with respect to other pubertal events and in duration of the growth spurt is most likely due to the same hormone, Estradiol, in both genders. The rising titers of Estradiol first stimulate accelerated activity at the growth plates in bone, but then, as titers continue to rise and the duration of exposure to estradiol continues, the growth plates close and the intensive skeletal growth slows markedly. The estradiol connection in males was discerned when a patient was discovered who was fertile, but whose epiphyseal plates had never closed (Fig 6). This man was found to lack estradiol receptor. Thus, the most likely reason that the male growth spurt starts later in development and lasts longer is that the level of estradiol produced by the testis is much lower than that typical of the ovary. In the male, Estradiol levels rise more slowly than in the female, so the stimulus period to bone growth is longer, it begins later, so the height at which the growth spurt begins is greater and it peaks and thus ends later (more area under the curve), resulting in a larger increment in height from the growth spurt in males than is the case in females.

Figure 6. Growth curve in male lacking estradiol receptors.

5. Timing of the Onset of Puberty There were two major candidates for glandular causes of sexual maturation: the adrenals, "adrenarchy", or the possible maturational effects of gonadal steroids to mature the CNS. Both of these have been excluded as causes of the onset of puberty, although both have been shown to participate in the progress of normal puberty. As stated above, the demonstration of a juvenile "hiatus" of gonadotropin secretion (period of low LH/FSH) in castrate or functional castrate primates suggested that the juvenile period was independent of the gonads. Further study of such individuals revealed that they also exhibited an increase in gonadotropin secretion at the age that intact individuals would be initiating early pubertal changes. This showed that the pubertal increase in GnRH secretion also occurred in the absence of the gonads. A causal role of adrenal steroids in puberty was ruled out with the observations of normal gonadal puberty onset in the presence of early activity of the adrenal axis, and in persons with adrenal insufficiency. It should be noted, however, that altered adrenal functions can affect the age of onset of pubic hair growth and distribution, in girls and boys, but without accompanying CNS maturation of GnRH neuronal function, true sexual maturation does not occur. The actual mechanism which results in the initiation of GnRH secretion in children which eventually drives the maturational process is not known. There is broad agreement that the primary organ of maturation is the central nervous system, but what the stimulus (or stimuli) are which result in the onset of this remarkable developmental cascade at the ages of about 9 yrs in girls and 10-11 in boys is not known. Recent exciting research has identified a key player in the regulation of GnRH activation at puberty in monkeys to be a G-protein coupled receptor-54 (GPR54) with regulation through its ligand, kisspeptin-1 and the GnRH receptor. In addition, other factors which are known to influence the onset of puberty in humans are: nutritional status (starvation and intense exercise may delay) (perhaps via leptin and NPY), percent body fat, brain lesions or tumors, and several endocrine disorders. Proteomics research is just beginning to dissect the biochemical pathways governing this process. The understanding of these events is in an exciting new stage of progress.

Disorders of Puberty Onset Onset of overt, physical sexual maturation > 2 standard deviations from the population mean age of puberty (either early or late) is clinically significant. Precocious puberty: onset of sexual development in girls less than 8 years of age and in boys less than 9 years. True, precocious puberty is associated with all the hallmarks of normal maturation, especially evidence of early activation of hypothalamic GnRH secretion as the cause of the maturation. True precocious puberty is also called Central (as in CNS) Precocious Puberty (CPP). Diagnostic work up of CPP is focused on ruling out gonadotropin independent sexual maturation. Generally,clinical presentation of CPP recapitulates the pace and order of events in normal puberty. Studies will reveal nocturnal increase in LH secretion, pituitary response to exogenous GnRH will be appropriate to bone age rather than to chronological age, etc. CPP is five times more common in girls than in boys Idiopathic CPP (no apparent causal pathology) is 8-10 times more common in girls than in boys. CPP can be associated with CNS insult of any kind: tumor, hamartoma, infection, hydrocephalus Idiopathic CPP usually occurs in very young children: 6 years or younger Treatment: CPP can be treated by administration of a long acting GnRH agonist. (See first lecture by Dr. Cameron.) This treatment will selectively inhibit FSH/LH secretion by the pituitary until the child reaches appropriate age. Cessation of treatment is associated with onset of puberty within one year in most cases. Gonadotropin Independent Precocious Puberty: Steroid responsive tissues stimulated by gonadal activation unsupported by the hypothalamopituitary axis. These patients have suppressed LH/FSH due to high estradiol or testosterone levels. The pituitary response to exogenous GnRH is still in prepubertal pattern. This is also termed pseudopuberty. Autonomous gonadal function: Ovarian cysts usually transient, often cause early thelarche (breast development). McCune-Albright Syndrome ovarian cysts, caf-au-lait spots on skin, characteristic lesions on bones that are apparent on X-ray. Caused by a mutation in the signal transduction G Protein (post LH receptor) rendering the pathway constitutively active. Can occur in both males and females although rare in males. Granulosa-theca cell tumors most common estrogen secreting ovarian neoplasms in childhood.

HCG secreting germ cell tumors of the testis Mutations of the LH receptor rendering it chronically active Diagnosis of these conditions often begins with a failure to suppress gonadal steroid production with a GnRH analogue or antagonist. Frequently, the sequence of maturational events is not quite normal: e.g. evidence of high testesterone with small testicular size can suggest either independent activation of LHR or hCG secreting tumor. Tubules of testes remain small due to lack of FSH, all events driven solely by testosterone appear normal Treatment: Tumors can be removed surgically if necessary. Diffuse hCG secreting cells will respond to chemotherapy or sometimes to radiation. Mutations in LHR or signaling pathways are usually treated with anti androgens or androgen receptor blockers combined with aromatase inhibitors. Such patients are usually fertile as adults, so gonadectomy is avoided if at all possible in these patients.

Workshop #4 Tuesday, February 24, 2009, 9:15 10:30 am Activity 1: The Developmental Milestone Match Game Learning objectives: By the end of this session, participants will be able to: Describe the progression of developmental milestones in gross motor and fine motor skills, language and cognition, and social-adaptive skills. Describe the typical behaviors of children at ages 0-3 months, 4-6 months, 7-9 months, 10-12 months, 13-15 months, 15-18 months, 20-24 months, 3 years, 4 years and 5 years old Evaluate children in terms of their growth and development

Activity: Each group will be given a stack of cards. The cards include ages and also skills. Your job will be to arrange the cards such that the developmental sequence within a domain is represented as a row and the skills of a child at a particular age are represented as a column. At the end of the activity, you will have a reference that looks like this:
Age in months Domain Of Function Gross Motor Fine Motor
Language/ Cognition











Social/ Adaptive

Within the room you will also find growth curves. You will then assess the growth and development of 5 children who present for wellchild care with a physician at one of the scheduled health maintenance visits. (In these case studies, information that is missing is either normal or irrelevant to the questions.) Use the grid to assist you in assessing the development of the children. Use the growth curves to evaluate height, weight and head circumference. Use this information to arrive at an assessment and plan.

Case 1: Iris Chief concern: A couple brings their 24-month old daughter to you for her regular well child check-up. When you ask if there are any particular concerns they would like you to address, they admit that they have become very concerned about her development. Relevant background information: Iris was adopted from China at age 18 months after living in an orphanage and foster home. No information is available on her biological family. Immediately after her arrival in the US, she has had all of the requisite immunizations for a child from China. She has been very healthy since arrival. Her mother has taken a leave of absence from her job to be with Iris for the first year of her life in the US. The parents tell you that Iris has become an affectionate child who seems to enjoy her circumstances. She particularly gets excited when her father returns from a days work. She spends 2 hours in a peer-play-group with her mother twice a week and smiles and plays with the other children. They note that Iris runs smoothly and enjoys outdoor play, particularly in the park where she climbs up the sliding board and giggles on her way down. She feeds herself competently with a spoon and fork and scribbles with crayons, usually making lines and occasionally circles. After a few months, she became interested in dolls. She now has several dolls and she plays with them by feeding and bathing them. She can get their clothes off but not on again. She makes very few sounds, occasionally a ma-ma or da-da but without any clear reference to her parents. She points at interesting things and has an expressive face, but does not seem to understand words or use words. Physical and Neurological evaluation: Iris appears to be a well-developed and well-nourished but small child. Weight is 10.7 kg, length is 81 cm, and head circumference is 46.5 cm. She has no unusual features. Her physical and neurological examinations are entirely normal. Assessment and Plan: What do you think about this child? What are areas of concern? What may be the cause of those concerns? What would you do to evaluate further and to manage?

Case 2: Zachary Chief concern: This 15-month old child was born prematurely. On previous examinations, his mother had been very concerned about his physical health. Today, she asks you about his developmental progress. Relevant background information: Zach was born prematurely at 28 weeks gestation weighing 1260 grams. Up until that point the pregnancy had been normal, but his mother developed a urinary tract infection that seemed to precipitate the delivery. He was treated with surfactant and had mild respiratory distress of the newborn, requiring mechanical ventilation for 5 days and supplemental oxygen for 16 days. He had no other major complications during the neonatal period. He required one hospitalization for bronchiolitis in his first year of life and has had 4 bouts of acute otitis media. His head circumference was increasing faster than height and weight but has been on the same percentile curve since he was 6 months of age. His single mother is working 32 hours per week and he is in a local day care center. Zachs mother reports that he cruises, walking holding on to the coffee table and couch. He seems just about ready to take steps independently. He uses a spoon but is rather messy. His mother is unaware of whether he can stack blocks or put on lids. He says bye-bye, ma-ma, da-da, and has a name for the family dog and for his sister. He likes to run toy cars and trucks back and forth making engine sounds. He is definitely getting easier for his mother to care for as he assists with dressing and is content to play alone. Physical examination: Zach now weighs 9.2 kg, is 74 cm in length, and has a head circumference of 47 cm. His face is long and narrow but has no dysmorphic features. His physical examination is normal. Neurological examination reveals intact cranial nerves, brisk but symmetrical reflexes, no clonus, normal tone, and absence of primitive reflexes. Assessment and Plan: What can you expect in terms of growth and development in a child born prematurely? How is this child doing?

Case 3: Sam Chief concern: Sam is currently 4 years 7 month old. He will turn 5 on September 2. As such, he is eligible to begin kindergarten but would be the youngest child in his class. His parents are debating whether to send him to kindergarten in the fall or whether to give him another year of preschool. They ask you if his development is appropriate for age. Relevant background information: Sam is a cheerful and active boy. He likes to run, climb, and kick balls. He has not yet learned to skip. He is quite competent on a 2wheel bicycle with training wheels, but cannot handle the bike without those wheels. He can print his first name legibly in all capital letters using the lines for orientation. However, he doesnt like to draw. He speaks well and is completely intelligible. His parents note he still makes mistakes with the sounds of some words such as fink for think, wellow for yellow, and pisgetti for spaghetti. He dresses himself but still puts shoes on the wrong feet and cannot tie the laces. He has two close neighborhood friends and together they build forts and play soldiers. His parents observe that he is particularly good at sharing with his friends. He has been in a preschool program for 2 years and his teachers note that he is a sociable and popular student. Physical examination: Sam is a well-developed child. His weight is 21.9 kg and his height is 113 cm. His physical and neurological examinations are entirely normal. Assessment and plan: What is your assessment of this childs development? How about his growth? What might you say to his parents?

Case 4: Madeleine Chief concern: Madeleine is a 4-year old child with Down syndrome. Her parents are interested in when they should begin to toilet train their daughter. Relevant background information: Madeleine was a 5 pound 2 ounce baby girl born at term to a 27-year old woman. The diagnosis of trisomy 21 was made shortly after birth based on the childs physical features and hypotonia. Her initial cardiac evaluation revealed a ventricular septal defect that closed without surgery. She has had annual check-ups with no other medical problems identified. Madeleine was enrolled in free and public early intervention services at 2 months of age. She and her parents received weekly home visits from a developmental specialist until Madeleine was 2 years old. She was then enrolled in a twice weekly toddler group. She is now in an inclusive preschool setting where she spends 5 hours 4 days per week with 3 other children with developmental disorders, 12 children developing typically, 2 teachers and a student-teacher aide. Madeleine is able to walk and run without difficulty. She balances briefly on one foot but cannot hop. She loves to draw and occasionally can make a circle. She plays house with the other children and with baby dolls, can stack 8 blocks, but is poor at puzzles. Initially she was taught sign language for communication and did not use single words until 18 months of age. However, she has recently shown a spurt in verbal language and now relies on it for communication. She uses short sentences of up to 2 to 3 words. Her intelligibility is only about 75% for parents and lower for strangers. She follows 2-step commands. She does not yet know colors, shapes or relational words such as front and back. She has adjusted well to her classroom and is invited on play dates with some of the other children. However, she has a short attention span for stories and circle time. Physical examination: Madeleine is 14 kg and 92 cm tall. Her blood pressure is 92/68. She has upslanting palpebral fissues, brachycephaly, epicanthal folds, and bilateral single palmar creases. Her physical examination is otherwise unremarkable. Her neurological examination reveals hypotonia, depressed reflexes, but strength for antigravity movements. She walks well, runs with arms raised to elbow height, kicks a ball with both legs, but does not hop. Assessment and plan: How would you assess this childs development? At what age do children typically begin to use the toilet independently? When might this child do so?

Case 5: Leslie Chief concern: Leslie is a 9-month old child who is new to your practice. His mother brings him in for a routine check-up. She has no major concerns that she wants to discuss. Relevant background information: Leslies was a 6 pound 3 ounce baby boy born at term to a 23-year old woman. This was her first pregnancy and delivery. The baby required vigorous stimulation and some blow-by oxygen at birth, but then made a reasonable transition. He was a slow feeder and sleepy infant. He and his mother stayed one additional hospital day so that the nurses could instruct the mother on feeding techniques. He has had 2 upper respiratory infections and ear infections but has otherwise been healthy. Routine developmental assessment reveals that Leslie is a happy and easy baby. He sleeps well overnight and takes 3 daytime naps. He is on the bottle but does not particularly like baby food. He eats some cereals and occasional pureed fruits. He smiles freely and laughs occasionally. He rolls from back to front and sits with support but not independently. He reaches and grabs but does not transfer objects from hand to hand. His mother cannot think of any examples of his tracking a falling object. He coos responsively but has no consonant sounds and does not use ma-ma or da-da even non-specifically. Family history is negative for developmental disorders. Physical examination: Leslie is a small and thin child. Growth parameters are as follows: 6.7 kg, 67 cm, 43 cm head circumference. He has puffy eyes, epicanthal folds, a long philtrum, and blue eyes with a stellate pattern of iris. His lungs are clear to auscultation. His heart has a regular rhythm and rate but you note a murmur that is heard not only on the left sternal border but also in the right axilla and in the back. He is mildly hypotonic with symmetrical reflexes and adequate strength for anti-gravity movement. You note that he still has a vestige of the asymmetrical tonic neck reflex and a distinct plantar grasp. Assessment and plan: What are your concerns about this childs development? How about growth? How can you develop a differential diagnosis in this case? What further assessments and referrals would be useful?

Notes on Growth and Development Relevance Evaluation of growth and development is central to health supervision and medical care of children Any problem within physiologic, interpersonal, and social domains can adversely affect growth and development Pediatricians monitor growth and development To evaluate children's overall well-being To provide meaningful anticipatory guidance and reduce biologic or environmental risks To identify delay or deviance To provide appropriate remedial intervention Importance of Social Environment Infants and children survive physically and psychologically only in the context of their family and social relationships For the purposes of assessing growth and development and intervening, if necessary, infants and children must be considered in social context Pediatricians must be comfortable with children and with their families Growth Definition: Linear changes in bone, muscle, connective tissue, and fat Operationally: Changes in height, weight, head circumference Velocity of these changes Definition of development Systematic changes in functional capacity Operationally: Increasing capacity in developmental domains Velocity of such changes Growth in first 5 years Age Wt Wt/mo g /d 0-3 mo 3-6 mo 6-9 mo 9-12 mo 1-3 yr 4-6 yr 30 20 15 12 8 6 2 lb 1_ lb 1 lb 13 oz 8 oz 6 oz

Length cm/mo 3.5 2.0 1.5 1.2 1.0 3 cm/yr

HC cm/mo 2.00 1.00 0.50 0.50 0.25 1 cm/yr

RDA kcal/kg/d 115 110 100 100 100 90-100

Adapted from National Research Council, Food and Nutrition Board: Recommeded Daily Allowances. Washington, DC, National Academy of Sciences, 1989; Frank D, Silva M, Needlman R: Failure to thrive: Myth and method. Contemp Pediatr 10:114, 1993.

Neonatal-infant growth Birth weight (BW) and height are based primarily on maternal factors Newborn's weight may decrease 10% BW 20 to loss of extravascular fluid and poor intake Infants regain BW by 2 wk of age and grow at 15-30 g (1/2 - 1 oz)/day during the 1st mo Best food for infants is breast milk Amount, type of protein, sugar, fat, salts Presence of infection-fighting immunoglobulins Milk sustains growth until 4-6 months Toddler-Preschool Growth Head circumference slows 18-24 months Height and weight slow at 2 years Nutritional requirements and appetite slow accordingly Between 2 and 5 yr, children gain ~ 2 kg in weight and 7 cm in height/y Toddler's prominent abdomen flattens, and the body becomes leaner. Physical energy peaks, Need for sleep declines to 11-13 hr/24 hr School age growth Averages 3-3.5 kg (7 lb) and 6 cm (2.5 in)/y Growth occurs discontinuously, in irregular spurts lasting on average 8 wk, 3-6 times/y HC grows only 2-3 cm in circumference throughout the entire period, reflecting slowed brain growth in this period Body habitus (endomorphic, mesomorphic, or ectomorphic) tends to remain relatively stable. Adolescence Growth acceleration begins in early adolescence, Peak growth velocities are not reached until late Growth spurt begins distally, with enlargement of hands and feet followed by the arms and legs and finally by the trunk and chest. Rapid enlargement of the larynx, pharynx, and lungs leads to changes in vocal quality Adrenal androgens stimulate the sebaceous glands, promoting the development of acne. Elongation of globe leads to near-sightedness. Hormonal basis of growth Fetal erainsulin Neonatal and infant periodthyroid Toddler to school agegrowth hormone Adolescencesex hormones Measuring growth The most powerful tool in growth assessment is the growth chart Growth dynamics Velocity more important than status Need measurements over time

Growth charts In the rooms Charts have 7 percentile curves, representing the distribution of weight, length, stature, or head circumference values at each age. Curve indicates % children at a given age (x-axis) wose value falls below the corresponding value (y-axis) 50th percentile is the median The weight-for-height charts are similar Length or stature in place of age on the x-axis. Growth norms NCHS cross-sectional sample, based on chronologic age Generally accurate for longitudinal analysis Most inaccurate in adolescence Lumps together subjects who are at different stages of maturation. Net result is artificially leveled off the growth peak, making it seem as though adolescent children grow more gradually and for a longer period than they do. Growth charts derived from longitudinal data are recommended for adolescents Development Principles of development parallel principles of embryology Biological preparedness: Infants are born with building blocks for future development Differentiation and Hierarchical organization

The development of abilities can described as greater refinement of the abilities--e.g., a

crude grasp of all five fingers to a fine and precise use of thumb and pointer

These skills become controlled by organized and planned patters of behavior

Qualitative changes from small quantitative change: Gradual changes accumulate until resulting behavior bears minimal resemblance to previous behaviors Domains of function Organization of changes over time Gross motor Fine motor Cognition Language Social-emotional Adaptive function Biological basis of development Brain development Brain is the most complex organ in the body Each division and subdivision of the brain has its own characteristic organized structure Unlike other organs, brain is immature at birth Most of the changes in brain development occur in the first 3 years of life Some changes continue into late adulthood

Building blocks of the CNS Neurons Synapses Neuroglia

Role is to take in information, process and pass it on Create networks that encode concepts and control functions Do not transmit but facilitate Neurons Number of neurons at birth is 10 - 100x109 After birth, no further proliferation, minimal migration Programmed cell death occurs in neurons that cannot make connections Synapses At birth, the number of connections is 50x1012 Synaptic connections grow 20 fold in the first few months of life to 1000x1012 Synapses initially grow exuberantly then pruned Redundant synapses are source of plasticity Over time, synaptic circuits exposed to systematic input and used frequently are those maintained, others eliminated Neuroglial cells Essential for normal functioning of the nervous system Astrocytes maintain the blood brain barrier Oligodendrocytes manufacture myelin Microglia repair injured tissue Glial cell density changes over time In adult cortex 10-20x109 neurons and 5-10x more glial cells Changes in brain composition In intrauterine life, brain grows to 350 g Most important period is first 3 years In adolescence, the number of cortical connections stabilizes and declines At 20 years, brain has mass of 1350 g Changes in surface area and number of glial cells change to age 70 - 80 years old Some plasticity may remain until old age Areas of major brain development Association areas Areas of the brain that integrate multiple modalities of information Located in the parietal and temporal lobes of the brain Executive control areas Areas that are involved in planning, considering alternatives, evaluating outcomes Located in the frontal lobes of the brain

Historical views on development

Nature=Primacy of genetic inheritance

Genes initiate and direct development Architectural plan is present in genetic constituents and comes into play automatically and

Maturation allows minimal input Development is the sum of the polypeptides

Nurture: Behavioral development was the result of environmental influences

No inherent structures limited or influenced the role of the environment The mind as a tabula rasa Limited ability to explain the organized structure of brain and constancy of development
Limitations of the nature position Human genome has 80-100,000 genes Approximately 50% of the genes are used in formation of the nervous system Approximately 30% of the genes are used in functioning of the nervous system Considering the number of cells, migration, and differentiation, development cannot be accounted for by this number of genes Current interactionist views Genetic unfolding environmental conditions that influences availability of substrate, rates of enzyme activity, etc. Must consider the interactions of tissue, organ, organism, and environment Development is indeterminate process whose regulation depends on circumstances and consequences Infant developmental skills Sensory abilities Ability to track an inanimate object or face 30 to 180 degrees Ability to turn to sound of rattle or voice Motor abilities Primitive reflexes: Moro, Asymmetric Tonic Neck Reflex, Gallant, Place, Walk Movements: large bicycling movements Head control Semi-organized movements: uncovering their face

HUMAN SEXUALITY Feb. 25, 2009 1:00 pm - 3:30 pm Melanie A. Gold, DO Clinical Associate Professor of Pediatrics Staff Physician, University of Pittsburgh Student Health Service magold@pitt.edu

OBJECTIVES: 1. Develop skills in conducting a sexual history with patients in a comfortable, non-judgmental manner. Know when to refer patients to a specialist, when needed. 2. Develop a sound foundation of current information concerning basic content areas in human sexuality. These include gender differences in sexual response, sexual dysfunction and its treatment, and sexuality in the context of intimate relationships. 3. Be able to identify and clarify your own values, beliefs, and perhaps biases regarding a wide variety of sexual behaviors and sexual/gender identities. This includes an understanding and respect for the variation in sexual norms and mores across cultures, religions and ethnic groups, and life stages. Be aware of overt and covert sexist, racist, ageist or homophobic attitudes that will interfere with interactions and treatment of patients. I. Sexuality as a continuum II. Taking a Sexual History ----------------- Observe taking a sexual history ---------------------------------III. Normal Sexual Function, Sexual Response Cycle - Desire - Excitement - Orgasm - Resolution Break for 15 minutes IV. Sexual Dysfunctions, Paraphilias, and Gender Identity Disorders Sexual Desire Disorders - Hypoactive Sexual Desire Disorder - Sexual Aversion Disorder Sexual Arousal Disorders - Female Sexual Arousal Disorder - Male Erectile Disorder

Orgasmic Disorders - Female Orgasmic Disorder - Male Orgasmic Disorder - Premature Ejaculation Sexual Pain Disorders - Dyspareunia - Vaginismus Sexual Dysfunction Not Otherwise Specified Paraphilias - Exhibitionism - Fetishism - Frotteurism - Pedophilia - Sexual Masochism - Sexual Sadism - Transvestism Fetishism - Voyeurism - Paraphilia Not Otherwise Specified Gender Identity Disorders - Gender Identity Disorder - Gender Identity Disorder Not Otherwise Specified V. Sexual Dysfunction Due to General Medical Conditions: Effects of aging, chronic illness and surgical conditions on sexuality - Menopause - Myocardial infarction - Stroke / brain damage - Chronic renal failure - Ileostomy and colostomy - Arthritis - Chronic alcohol abuse and alcoholism - Obesity - Pulmonary disease (COPD, asthma, emphysema, cystic fibrosis) VI. Substance-Induced Sexual Dysfunction: Effects of medical treatments on sexuality VII. Gay, lesbian, bisexual, and transgender health care issues Southern Comfort Video Clip -------------------- PANEL DISCUSSION -------------------------------------------------------------------

RECOMMENDED TEXBOOK TO READ The Guidebook of Sexual Medicine (Paperback): Waguih William IsHak MD, FAPA. A & W Publishing Group, Beverly Hill, CA, 2008. HIGHLY RECOMMENDED READING Kingsberg SA.Taking a sexual history. Obstet Gynecol Clin North Am. 2006;33(4):535-47. Tsimtsiou Z, Hatzimouratidis K, Nakopoulou E, Kyrana E, Salpigidis G,Hatzichristou D. Predictors of physicians' involvement in addressing sexual health issues. J Sex Med. 2006;3(4):583-8. Burd ID, Nevadunsky N, Bachmann G. Impact of physician gender on sexual history taking in a multispecialty practice. J Sex Med. 2006;3(2):194-200. Young F. How to take a sexual history. J Fam Health Care. 2005;15(5):149-51. Jones R, Barton S. Introduction to history taking and principles of sexual health. Postgrad Med J. 2004;80(946):444-6. (Available online at http://pmj.bmjjournals.com/cgi/content/full/80/946/444) Finger WW, Lund M, Slagle MA. Medications that may contribute to sexual disorders: A guide to assessment and treatment in family practice. Journal of Family Practice 1997;44(1):33-43. American Academy of Pediatrics, Committee on Adolescence. Sexual Orientation and Adolescence. Pediatrics 2004;113:1827-1832. OTHER RECOMMENDED READING The Ultimate Guide to Sex and Disability: For All of Us Who Live with Disabilities, Chronic Pain, and Illness, second Edition. Miriam Kaufman, Cory Silverberg, Fran Odette. Cleis Press, Inc., San Francisco, CA, 2007. Maurice, WL. Sexual Medicine in Primary Care. Mosby, Inc., 1999. Chapter 2 Talking about sexual issues: Interviewing methods Chapter 3 Screening for sexual problems Chapter 7 Talking about sexual issues: Gender and sexual orientation OTHER REFERENCES Gay, Lesbian and Transgender Issues Sanchez NF, Rabatin J, Sanchez JP, Hubbard S, Kalet A. Medical students' ability to care for lesbian, gay, bisexual, and transgendered patients. Fam Med. 2006 Jan;38(1):21-7. Ryan Caitlin and Futterman D. Lesbian and Gay Youth: Care and Counseling. Columbia University Press, NY, 1998. Stevens PE, Morgan S. Health of lesbian, gay, bisexual, and transgender youth. J Pediatr Health Care. 2001;15(1):24-34.

Gay, Lesbian and Transgender Issues (contd) Feldman J, Bockting W.Transgender health. Minn Med. 2003;86(7):25-32. De Cuypere G, Tsjoen G, Beerten R, Selvaggi G, De Sutter P, Hoebeke P, Monstrey S, Vansteenwegen A, Rubens R. Sexual and physical health after sex reassignment surgery. Arch Sex Behav. 2005;34(6):679-90. Male and Female Sexual Dysfunction Montorsi F.Assessment, diagnosis, and investigation of erectile dysfunction. Clin Cornerstone. 2005;7(1):29-35. Berman JR. Physiology of female sexual function and dysfunction. Int J Impot Res. 2005;17 Suppl 1:S44-51. Frumovitz M, Sun CC, Schover LR, Munsell MF, Jhingran A, Wharton JT, Eifel P, Bevers TB, Levenback CF, Gershenson DM, Bodurka DC. Quality of life and sexual functioning in cervical cancer survivors. J Clin Oncol. 2005;23(30):7428-36. Fink HA, Mac Donald R, Rutks IR, Nelson DB, Wilt TJ. Sildenafil for male erectile dysfunction: a systematic review and meta-analysis. Arch Intern Med. 2002;162(12):1349-60. Hatzichristou D, Tsimtsiou Z. Prevention and management of cardiovascular disease and erectile dysfunction: toward a common patient-centered, care model. Am J Cardiol. 2005;96(12 Suppl 2):80-4. Montejo AI, Llorca G, Izquierdo JA, Ledesma A, Bousoo M, Calcedo A, Carrasco JL,Daniel E, de Dios A, de la Gndara J, Derecho J, Franco M, Gmez MJ, Macas JA, Martn T, Prez V, Snchez JM, Snchez S, Vicens E.Sexual dysfunction secondary to SSRIs. A comparative analysis in 308 patients. Actas Luso Esp Neurol Psiquiatr Cienc Afines. 1996 Nov-Dec;24(6):311-21. Sexuality and Aging Naumburg EH. Sexual health across the life cycle: a practical guide for clinicians. Fam Med. 2006;38(1):59-60. Sarrel PM. Sexual dysfunction: treat or refer. Obstet Gynecol. 2005;106(4):834-9. Nusbaum MR, Lenahan P, Sadovsky R. Sexual health in aging men and women: addressing the physiologic and psychological sexual changes that occur with age. Geriatrics. 2005;60(9):18-23. WEB SITES TO KEEP YOU INFORMED American Association for Health Education American Association of Sex Educators, Counselors, and Therapists Association of Reproductive Health Professionals Gay and Lesbian Medical Association National Education Association Health Information Network National Gay and Lesbian Task Force MORE WEB SITES TO KEEP YOU INFORMED www.aahe.org www.aasect.org www.arhp.org www.glma.org www.nea.org www.ngltf.org

National Family Planning and Reproductive Health Association Parents, Families, and Friends of Lesbians and Gays Religious Coalition for Reproductive Choice and Health Sexual Health Sexuality Information and Education Council of the United States The Alan Guttmacher Institute Persad Center (Mental health provider for GLBT in Pittsburgh) Metro Family Practice (Medical provider for GLBT in Pittsburgh)

www.nfprha.org www.pflag.org www.rcrc.org www.SexualHealth.com www.siecus.org www.agi-usa.org www.persadcenter.org www.metrofamilypractice.org

PHARMACOLOGY OF ESTROGENS AND SERMS Don DeFranco, Ph.D. Wednesday, February 25, 2009 3:45 p.m. LEARNING OBJECTIVES 1. Understand the structure and pharmacological properties of various estrogens and estrogen analogs used clinically 2. Understand the properties and uses of nonsteroidal compounds with estrogenic activity 2. Explain the properties and functions of the two estrogen receptors 3. Describe the mechanism of action of synthetic estrogen receptor antagonists and estrogen receptor modulators (SERMs) and explain why different SERMs have distinct effects on target organs 5. Understand possible mechanisms of SERM resistance 6. Understand the mechanism of action and uses of aromatase inhibitors OUTLINE A. B. C. D. E. F. G. Estrogen Structure Estrogen Synthesis Estrogen Receptors (ER and ER) Pharmacology of Estrogens ER Antagonists and SERMs Clinical Uses of Estrogens, ER Antagonists and SERMs Aromatase Inhibitors

RECOMMENDED READINGS 1. Goodman & Gilmans The Pharmacological Basis of Therapeutics, 11th Edition, (Brunton, Lazo & Porter, Eds.), McGraw Hill, 2006. Chapter 57, Estrogens and Progestins, pp 1541-1558; Chapter 51, Antineoplastic Agents, pp 1381-1387. 2. Williams Textbook of Endocrinology, 10th Edition (Larsen et al., Eds.), Saunders, 2003. Chapter 4, Mechanism of Action of Hormones that Act on Nuclear Receptors, pages 35-44. 3. Lippincotts Illustrated Reviews: Pharmacology, Third Edition (Howland & Mycek, Eds.), Lippincott, Williams & Wilkins, 2006. Chapter 26, Estrogens and Androgens, pages 295-299. 4. Goldstein SR, SIddhanti S, Ciaccia AV, Plouffe L Jr (2000) A pharmacological review of selective oestrogen receptor modulators. Human Reproduction Update, Vol. 6, no. 3, pp. 212-224.

A. Estrogen Structure The Phenolic A ring of estrogens is the most critical structural determinant of high affinity binding to the estrogen receptor. However, the 3-hydroxyl group, and 17beta hydroxyl group also make contacts with binding pocket of the estrogen receptor. The rest of the molecule makes more subtle hydrophobic contacts. The molecule is relatively flat from A to D rings, with 17- hydroxyl and 18 -methyl groups pointing down. The most abundant and potent circulating estrogen is 17estradiol (also referred to simply as estradiol). Other naturally occurring estrogens include estrone and estrane, both of which are at least 10-fold less biologically active than estradiol. B. Estrogen Synthesis Like all steroid hormones, estrogens are synthesized from cholesterol. The granulosa cells of the ovary are the main sites of estrogen synthesis in premenopausal women. In graulosa cells, androstenedione produced in the adjacent theca cells from cholesterol is converted by the enzyme aromatase to estradiol. In postmenopausal women, adipose stromal cells become the main source of estrogen synthesis using dehydroepiandrosterone (DHEA) produced by the adrenal gland. In men, most circulating estrogen is derived from aromatization of androstenedione in extragonadal tissue although the testes also produce some estrogens. In both men and women, local production of estrogens by aromatization of androgens can affect physiological (e.g. bone mineral density, behavior) and pathophysiological (e.g. cancer) processes. Estrogen levels are regulated during the menstrual cycle peaking at 600 pg/ml at the time of ovulation and reaching a nadir of 100 pg/ml in the follicular phase. During pregnancy the levels of estrogen can reach 20,000 pg/ml. Following menopause, estrogen levels in women approach that of men of similar ages (i.e. 5-20 pg/ml). IMPORTANT CONCEPT 1: The ovaries are the major sites of estrogen synthesis in premenopausal women while extragonadal synthesis of estrogen via aromatization of androgens are the major source of estrogen synthesis in men and postmenopausal women. C. Estrogen Receptors 1. Nuclear Receptor Superfamily The receptors for estrogen (i.e. ERs) are members of a large superfamily of nuclear receptors that have a common structural organization. These receptors contain a centrally localized, highly related DNA-binding domain (DBD), a less conserved carboxyl-terminal ligand binding domain (LBD) and a divergent amino terminal domain that participates in transcriptional activation. Like all members of

the nuclear receptor superfamily, ERs are transcription factors that can either activate or repress transcription from target genes. 2. ER-DNA Binding/Transcriptional Regulation Estrogen regulation of gene transcription most often requires the specific binding of ERs to select DNA sequences (termed estrogen response elements or EREs) usually contained within the promoter region of target genes. Usually ERs bind to their target DNA sequences as a receptor dimer. However, ERs can also associate with estrogen responsive target genes (sometimes as a monomer) through their association with other DNA-bound transcription factors. Finally a number of other proteins (e.g. transcriptional coactivators) that either directly or indirectly interacts with ER or ER-regulated genes are involved in estrogen regulated gene transcription. In fact, recent genome-wide analysis found that most ER binding sites are in close proximity to sites bound by the Forkhead family of transcription factors. The association of these transcription factors with target genes appears to be essential for ER binding and estrogen response. IMPORTANT CONCEPT 2: Estrogens act in target tissue predominantly to activate nuclear ERs, which affect the transcriptional activity of select target genes in cooperation with other proteins recruited directly or indirectly to the estrogen-regulated gene promoter. 3. Nongenomic Effects of ERs In addition to genomic effects on target gene transcription, ERs can exhibit nongenomic effects and rapidly activate multiple signaling pathways (e.g. mitogen-activator protein kinase or MAPK pathways). In these cases, estrogen effects may be mediated by plasma membrane associated ERs. Recent evidence in animal models suggests that rapid nongenomic effects of plasma membrane associated ERs on nitric oxide generation in epithelial cells may influence vascular function. IMPORTANT CONCEPT 3: Emerging evidence supports the possibility of ERs exerting physiological effects, particularly in the cardiovascular system, via rapid nongenomic action on specific signal transduction pathways. C. Two Receptors for Estrogens- ER and ER Two highly conserved ER genes have been identified and designated ER and ER. Both receptors are widely expressed throughout the body with the exception of the uterus where ER levels are low. 17-estradiol serves as a potent ligand for both ER and ER. Heterodimers can form between ER and ER but the relative amount of ER homodimers (i.e. ER/ER or ER/ ER) versus ER heterodimers (i.e. ER/ER) is determined by the expression levels of each receptor in a given cell.

Much has been learned about the contributions of ER versus ER to estrogen effects in select target tissues through the analysis of mice with targeted gene deletions of each receptor. For example, estrogen effects to stimulate uterine growth is due primarily if not exclusively to the action of ER. In addition a major role for ER in bone metabolism is suggested by the reduced bone mineral density and lack of responsiveness to estrogen of a male patient with an inactivating mutation in the ER gene. IMPORTANT CONCEPT 4: Two receptors for estrogen exist, each of which may be responsible for distinct effects of estrogen in specific tissues and cell types. D. Pharmacology of Estrogens 1. Estrogen Metabolism Estrogen is lipophilic and therefore can distribute rapidly and efficiently to all tissues in the body. However, it is rapidly metabolized in liver and has a plasma half-life that is measured in minutes. The major products of estrogen metabolism in the liver, which are excreted in the urine, include 16-hydroxylated and 17keto forms in addition to a variety of sulfate and glucuronide conjugated forms. Estrogen metabolites (i.e. mainly sulfate and glucuronide forms) also undergo enterohepatic recirculation following their excretion via the bile duct into the intestines, hydrolysis and reabsorption. 2. Effects on Estrogen Metabolism Estrogen metabolism can be affected by other drugs and environmental agents (e.g. cigarette smoke) that alter the activity or expression of metabolizing enzymes in liver. Variations in estrogen metabolism can also occur in response to different stages of the menstrual cycle, menopausal status, genetic polymorphisms and disease status. For example, estrogen excess conditions may result from reduced estrogen metabolism in patients with liver damage. IMPORTANT CONCEPT 5: Hepatic metabolism of naturally occurring estrogens limits the efficacy of hormone action and can be influenced by a variety of genetic, pathophysiological and environmental factors 3. Estrogen Preparations Estradiol is used in various transdermal preparations (e.g. estraderm ), which provide slow, sustained release, limited first-pass clearance in the liver, and therefore more stable blood levels than oral preparations. By limiting portal delivery to the liver, transdermal preparations of estrogen also limit some hepatic side effects (see below). Estradiol is also available as a topical cream (estrasorb) and gel (estrogel). Some specialized estrogen creams are available for topical vaginal administration, but significant absorption does occur with these preparations and may trigger systemic effects.

4. Estrogen Derivatives Substitutions at the C17 position of estrogen dramatically limit its first-pass hepatic metabolism. Therefore C17 derivatives such as ethinyl estrogen are commonly used for oral administration. These compounds have a prolonged duration of action. Mestranol, the 3-methyl ether of ethinyl estrogen is converted to ethinyl estrogen upon demethylation in liver. The elimination-phase half-life of ethinyl estrogen can range from 13-27 hours. C17 substituted estrogens are also lipophilic and stored in adipose tissue, from which they are slowly released. Unlike naturally occurring estrogens, ~90% of which circulate in blood bound to steroid hormone binding globulin (SHBG), most circulating C17 substituted estrogens are not bound to SHBG but loosely associated with albumin. One of the most commonly used estrogen preparations for the treatment of postmenopausal symptoms is premarin (i.e. conjugated equine estrogens), which contains a mixture of estrogen sulfates, including estrone, equilin, and 17dihydroequilin. The conjugated estrogens in these preparations are hydrolyzed by enzymes in the lower gut and absorbed through the intestinal epithelium. IMPORTANT CONCEPT 6: Conjugation of estrogens at their C17 position can limit their hepatic metabolism. Therefore C17 estradiol derivatives are effective estrogeic agents for oral administration. 5. Nonsteroidal Compounds with Estrogenic Activity Some non-steroid compounds also possess estrogenic activity (i.e. activate ERs). Many of these are plant derived products (i.e. phytoestrogens, typically from soy) and available as over-the-counter supplements. These preparations typically contain flavonoids such as genistein and are typically weakly estrogenic. Some synthetic industrial chemicals, or their byproducts, (i.e. xenoestrogens) have estrogenic activity via their direct activation of ERs. Common xenoestrogens (or sometimes referred to as environmental estrogens) include polychlorinated biphenyls (PCBs) and 1,1-bis(p-chlorophenyl)-2,2dichloroethylene (DDE). Long-term exposure to xenoestrogens such as PCBs or Bisphenol A (found in coating of food cans, plastic water bottles) may have some detrimental effects on human health. Diethylstilbesterol (DES) is a particularly potent non-steroidal xenoestrogen, which is not bound to SHBG or rapidly metabolized. It was used in the 1950s and 1960s to prevent miscarriages in women with high-risk pregnancies. However, DES is no longer used due to an increased incidence of rare forms of vaginal cancer and genital track anomalies that were observed in offspring of women that received DES during the first trimester of pregnancy. IMPORTANT CONCEPT 7: Both plant and industrial-derived non-steroidal compounds can exert estrogenic effects through direct binding to ERs.

E. ER Antagonists and Selective Estrogen Receptor Modulators (SERMS) 1. Pure ER Antagonists Most antagonist and partial ER agonist have very related core components that allow for high affinity binding to the ER LBD. However, ER antagonists typically have large chemical moieties that are located above the plane of rings. These modifications are tolerated only at the C7 and C11 carbon positions on the estradiol ring. These additional chemical moieties also must be of sufficient size to block protein folding by steric hindrance. Fulvestrant (faslodex , ICI 182,780) is a pure ER antagonist that is approved for treatment of breast cancer. Faslodex is particularly useful for ER-positive breast tumors that are tamoxifen resistant (see below). The ability of Faslodex to trigger ER degradation distinguishes its antagonist effects from SERMs (see below). Faslodex is administered once/month by intramuscular injection and reaches a maximum within 7 days. IMPORTANT CONCEPT 8: Compounds that antagonize ER action can be useful for the treatment of ER-positive breast cancers. 2. SERMs More recent analysis has revealed cell-type specific agonist effects of compounds that were initially identified as ER antagonists. Compounds that exhibit such selective agonist effects are now called Selective Estrogen Receptor Modulators or SERMs. For example, tamoxifen (nolvadex), which antagonizes estrogen action in breast tissue, is widely used to treat ER-positive breast cancers in both premenopausal and postmenopausal women. While the agonist effects of tamoxifen in bone reduce the risk of osteoporosis associated with pure ER antagonists, its agonist effects in uterine tissue leads to an undesirable increased risk of uterine cancer. Other side effects of tamoxifen include hot flashes, vaginal atrophy, hair loss, vomiting and an increased risk of thromboembolism. Tamoxifen is given orally and takes 3-4 weeks of treatment to reach steady state plasma levels due to its two distinct elimination phases (i.e. half lives of 7-14 hrs and 4-11 days). It is converted in liver (within 4-6 hrs) to its very active metabolite 4-hydroxytamoxifen. Raloxifene (evista) is useful for treatment of osteoporosis due to its ER agonist activity in bone. It does not act as an ER agonist in the uterus or breast and therefore does not increase the risk of cancers in these tissues. Raloxifine is also given orally and has an absolute bioavailability of 2%. Raloxifen has recently been shown to have a protective effect for women at high risk of breast cancer.

Clomiphene (clomid ) is one of the most widely used drugs for the management of infertility. It works primarily as an estrogen antagonist in the hypothalamus leading to enhanced release of GnRH (i.e. due to loss of estrogen negative feedback) and subsequent increases in FSH and LH secretion from the pituitary. In animal studies, clomid exhibits estrogen agonist effects in bone and skeletal muscle. Clomid is rapidly absorbed following oral administration (i.e. peak plasma levels within 6 hours oral administration) and has a long plasma half-life (5-7 days) due to its accumulation in adipose tissue. Since clomid is metabolized in liver, its use is contraindicated in patients with liver dysfunction. IMPORTANT CONCEPT 9: SERMs hold promise for allowing ER action to be blocked in specific cell and tissue types (e.g. breast cancer cells). The agonist properties of SERMs in specific tissues not only limit their side effects but also could provide some benefit, particularly for relief of some postmenopausal symptoms. Recent studies identified 27-hydroxycholesterol (27-HC) as an endogenous SERM that is an ER agonist in liver but an antagonist in the vasculature. 27-HC is the most abundant circulating oxysterol and found at high levels in atherosclerotic plaques. Its ER antagonism in the vasculature may limit the cardioprotective effects of estrogen. 3. Molecular Mechanisms of SERM Action SERMs bind directly to ERs but induce a conformational change in the LBD of the receptors that is distinct from that induced by estrogen binding. Specifically, helix 12 of the LBD adopts a different position in ERs occupied by estrogen versus SERMs. As a result of this different conformation, ER interactions with other cofactors is altered. For example, ER occupied by estrogen binds efficiently to a number of coactivator proteins mainly using contacts supplied by helix 12. However, these coactivator interactions are reduced if the receptor is occupied by a SERM. In fact, SERM-occupied ERs have a relatively higher affinity for corepressor proteins than estrogen-bound receptors. IMPORTANT CONCEPT 10: The cell-and tissue-specific effects of SERMs are due to their distinct effects on conformational changes in the LBD of the ER. F. Clinical Uses of Estrogen, ER Antagonists and SERMs 1. Menopausal Hormone Replacement Therapy: The two major therapeutic uses of estrogen are for combined oral contraception and menopausal hormone replacement therapy (HRT). Estrogen is used to relieve vasomotor symptoms and reduce bone loss and urogenital atrophy in postmenopausal women. Conjugated estrogens have been more commonly used for HRT at doses (i.e. 0.625mg/day) ~ 5-fold lower than those used in oral contraceptives. For women that have not had a hysterectomy, progestins are

included to reduce the risk of endometrial cancer associated with estrogen use alone. In this case, progestins decrease the undesirable effects of estrogen on uterine epithelial cell proliferation through decreasing ER levels, local induction of estrogen metabolizing enzymes, and altering the phenotypic state of the uterine epithelium. Since estrogen acts primarily to prevent bone resorption, it is effective at preventing rather than restoring bone loss. Raloxifine is also approved for osteoporosis prevention in postmenopausal women. Unfortunately, both tamoxifen and raloxifene induce rather than reduce hot flashes in postmenopausal women. The association between HRT and breast cancer risk will be discussed in a separate lecture that will highlight the results of a large, randomized clinical trial (i.e. Womens Health Initiative). IMPORTANT CONCEPT 11: Estrogens alone or in combination with progestins can be used to relieve some undesirable symptoms caused by estrogen depletion in postmenopausal women. 2. Breast Cancer Therapy Tamoxifen is widely used for the treatment of women with ER-positive breast tumors in both premenopausal and postmenopausal women. It is even more effective in women whose tumors are positive for both ER and progesterone receptor. In women who are at high risk of developing breast cancer, a 5-year prophylactic tamoxifen treatment can significantly reduce cancer risk. In recent clinical trials, raloxifene has been found to reduce the incidence of breast cancer in postmenopausal women. Raloxifene has recently received FDA approval for treatment of postmenopausal women with breast cancer. Pure ER antagonists are also being evaluated for their effectiveness as anti-cancer agents. 3. SERM Resistant Breast Cancers Patients receiving adjuvant tamoxifen whose tumors express high levels of both HER2/neu (HER2) and the ER coactivator protein Amplified In Breast Cancer-1 AIB1 often develop tamoxifen resistance. AIB1 (SRC-3) is an ER coactivator that, when overexpressed in cultured cells, can reduce the antagonist activity of tamoxifen-bound ERs. Signaling through the HER-2 receptor pathway activates AIB1 by phosphorylation. Other findings point to N-COR1, an ER corepressor, as a promising independent predictor of tamoxifen resistance in patients with ERpositive breast tumors. A mutation at serine-305 in the hinge region of ER shows hypersensitivity to estrogen levels that may activate the receptor most/all of the time. Activation of an efflux transporter (i.e. MDR multi-drug resistance transporter) responsible for the transport of metabolites can reduce cellular SERM levels. IMPORTANT CONCEPT 12: Many mechanisms can lead to resistance of ERpositive breast cancer cells to SERM treatment.

G. Aromatase Inhibitors Since aromatization of testosterone is the major biosynthetic step leading to estrogen production, aromatase inhibitors (AIs) provide an effective strategy to reduce estrogen levels in peripheral tissues. These inhibitors are not effective at blocking estrogen synthesis in ovaries since the decreased estrogen activates the hypothalamus and pituitary to increase gonadotropin secretion and thereby stimulate steroidogenesis in the ovary. Thus, AIs must be combined with other agents that block ovarian steroidogenesis (e.g. the GnRH analog, triptorelin [trelstar]) for chemotherapy in premenopausal women with breast cancer. Steroidal or type I inhibitors (e.g. formestane and exemestane [aromasin]) are suicide substrates and are converted to reactive intermediates by aromatase. They irreversibly inactivate aromatase. Nonsteroidal or type II inhibitors (e.g. anastrozole [armidex], letrozole [femara], and vorozole) reversibly interact with the heme group of aromatase. Exemestane, anastrozole and letrozole are the most widely used AIs for treatment of early-stage and advanced breast cancer. 1. Anastrozole A 1mg/day oral administration of postmenopausal women with anastrozole is effective in women with early-stage or advanced ER-positive breast cancer. Within 28 days of daily anastrozole treatment, total body aromatization can be reduced by ~97%. In advanced stage ER-positive breast cancer, anastrozole may be more effective than tamoxifen. Co-administration of tamoxifen does not affect the metabolism of anastrozole and may be more effective in the treatment of breast cancer than either agent alone. In addition, anastrozole has reduced incidence of some side effects associated with tamoxifen treatment (e.g. hot flashes, vaginal discharge, endometrial cancer, deep vein thrombosis). However, tamoxifen has a lower incidence of musculoskeletal disorders and fractures than anastrozole. 2. Letrozole Letrozole is also efficiently absorbed following oral administration and has a 99% bioavailability. Steady state levels of letrozole are reached within 2-6 weeks of treatment. It has a relatively long elimination half-life (~40 hrs). A once daily dose of letrozole (2.5 mg) has been found to be effective for the treatment of early stage or advanced, ER-positive breast cancer. In some cases of advanced breast cancer, letrozole may be superior to tamoxifen as first-line treatment. The major documented side effect of letrozole is hot flashes although its effects on bone mineral density and lipid profiles have not been definitively established. 3. Exemestane Exemestane has a similar toxicity profile to letrozole but is more effective at lowering estrogen levels than formestane. It also is favored over formestane since it has lower androgenic activity. Exemestane is absorbed rapidly after oral administration (maximum plasma levels after 2 hrs with 25mg/day dose) and has

a 24 hr half-life. Since many of its metabolites are excreted into urine, its doses must be adjusted in patients with renal disease. Randomized trials showed significant reduction in breast cancer risk when exemestane is combined with tamoxifen. When compared to tamoxifen alone, exemestane had reduced gynecological side effects but an increased risk of fractures. IMPORTANT CONCEPT 13: Aromatase inhibitors function to reduce peripheral estrogen production and are effective for breast cancer treatment in postmenopausal women either as first-line treatment or used in combination with tamoxifen.

Menopause and Aging in the Female Kathleen Ryan, Ph.D. Learning Objectives: The student will understand: 1. 2. 3. the changes in ovarian function during climacteric years the biological and physical consequences of this process issues in therapeutic management of symptoms of menopause

RESOURCES: Ferin M, Jewelewicz R, Warren, M. 1993 The Menstrual Cycle, Physiology, Reproductive Disorders and Infertility Chapter 8 Dubey, R, Imthurn B, Lefteris C, Jackson EK, 2004. Hormone Replacement Therapy and Cardiovascular Disease: What went wrong and where do we go from here. Hypertension 44 Naessen T and Rodriguez- Macias, K, 2005 Menopausal estrogen therapy counteracts norm aging effects on intima thickness, media thickness and intima/media ratio in carotid and fem arteries: an investigation using noninvasive high-frequency ultrasound. Atherosclerosis 12:2 Prior, J.L. (1998) Perimenopause: The complex endocrinology of the menopausal transition Endocrine Reviews 19 (4):397-428.

KEY WORDS AND PHRASES: menopause, hormone replacement, estradiol deficit, follicular depletion
Outline: 1. The reproductive life cycle A. B. C. D. 2. regular menstrual cycle: changes in fertility with aging changes in ovarian function hypothalamic consequences of altered ovarian function systemic consequences of altered ovarian function

Hormone replacement therapy for peri- and post-menopausal women A. B. C. Changes in steroid profiles through life cycle Effect of menopause on hormone-dependent tissues. Risks vs benefits of hormone replacement: perceptions vs reality

Lecture Notes: Menopause The changes in ovarian function which occur at menopause in women As stated previously in this course, the lifetime supply of ova in female mammals is present in the ovaries in utero in primordial follicles, and the number of available follicles peaks before birth. The steady progression of follicles from this pool of primordial follicles into the actively growing pool begins decreasing this pool size even before birth and this decline continues throughout the life of the female. Most of the follicles which are recruited become atretic, either as secondary or early antral follicles which do not receive gonadotropic support or as antral follicles which begin to respond to menstrual cycle hormones but which are slightly out of synchrony with hormonal signals and which succumb to atresia when FSH levels decrease in the mid-follicular phase (see Gosman lecture). The depletion of the pool of primordial follicles may accelerate in the last decade of menstrual cyclicity. However, the climacteric or menopause is the physiologic and psychological adjustment to the gradual waning of ovarian function due to decreasing numbers of stimulable ovarian follicles and the consequent fall in circulating estradiol levels. The external manifestation of menopause is the cessation of menstrual cycles. Menopause as a clinical entity is a consequence of the increase in life-span of women over the past several decades such that now, most women live 20 to 30 years or more beyond their reproductive years. The median age at menopause in the USA is 49.8 years, which is similar to data obtained in many developed countries. Because the follicle pool is large (in excess of 300,000 or more at its peak), and the entry of follicles into the proliferating pool is a gradual phenomenon, the onset of menopausal symptoms is usually gradual, and symptoms are highly variable among individuals. The following description is of a "classic" presentation of menopause, but many variations on this pattern occur in normal women. A period of decreasing ovarian function often occurs over 5 to 10 years before complete cessation of cycles. In perimenopausal women who are still menstruating regularly, estradiol concentrations are usually found to be lower and FSH higher than in younger women, while LH remains in the normal range. Since sensitivity of FSH and LH to estradiol negative feedback does not appear to change in menopause, it is hypothesized that the elevation of FSH reflects decreased follicular inhibin levels as well as a decrease in total amount of estradiol produced by the ovary from the decreased numbers of follicles there. The shorter duration menstrual cycles reported by many women may reflect this relative increase in FSH concentrations. Increased FSH should accelerate follicular advancement of antral follicles to maturation, thereby shortening the follicular phase. As the follicle pool shrinks, and the number of hormone-responsive follicles in the ovary at any one time falls, the period required to secrete enough estradiol to elicit an LH surge increases, thereby lengthening cycles. In time, this desynchrony results in anovulatory cycles interspersed with ovulatory ones. If an appropriate follicle is "in the window" to respond to follicular phase FSH, the estradiol-induced surge occurs normally and cycles can still be fertile (!). As the follicle pool decreases, so does ovarian steroid secretion until, with the total absence of follicles in the post menopausal ovary, ovarian production of estradiol and progesterone ceases. The thecal and interstitial cells of the

post menopausal ovary do continue to produce androgens under the stimulus of LH, but in the absence of mature granulosa cells, this androgen is not converted to estradiol. With the decline in ovarian steroids, the negative feedback inhibition of LH and FSH is removed and the amplitude and frequency of gonadotropin pulses increases. Physiology underlying hot flushes of Menopause The vasomotor symptoms of menopause are primarily caused by the decrease in estradiol levels. Vasomotor instability (hot flushes and profuse sweating) have been shown to occur concomitantly with increased pulsatile secretion of LH. This is a coincidence, the two events are not causally related, but control centers for body temperature regulation and GnRH secretion are in proximate hypothalamic regions and both phenoma most likely represent altered hypothalamic function consequent to estrogen deficit. Hot flashes (or flushes) are the manifestation of an inappropriate onset of temperature regulatory events secondary to excitation of neurons in the anterior hypothalamus which regulate body temperature. These neurons possess estrogen receptors and are in close proximity to many GnRH neurons which are also exhibiting increased activity due to the absence of estradiol and progesterone. The diagram below illustrates events of a hot flash episode. These often begin at night, but can occur at any time of day. Some women only have a few such episodes for a few years; others may have them hourly for years on end. onset Vasodil/sweating (<B.T.) Body Temperature: nl end vasoconst/shiver (>B.T.) nl

______________low______________ Time (~ 30 min)

At the onset of the episode, a person with a normal body temperature suddenly has profound vasodilation of the upper torso, neck and face. This brings core blood to the surface, allowing cooling (by radiant heat loss). The flushing also heats the skin, eliciting localized sweating. In addition to the local stimulation of sweating in the skin, the same stimulus which initiated the flushing also causes neurogenic (copious, independent of core body temperature) sweating. These two physiologic events both contribute to dissipation of heat, causing a drop in body core temperature. This new body temperature will be maintained for a time (15-30 min, maybe less) but the episode of hypothalamic excitation will end, and the body temperature regulatory center will return to normal signaling now that body temperature is low. This will cause vasoconstriction and shivering to restore body temperature to normal. Some women exhibit hot flushes as frequently as once per hour/24h for many months or years. This is distracting and annoying, but several studies have now shown that hot flushes which occur at night, even without waking the sleeping subject, still interrupt REM sleep. In persons with frequent hot flashes over years, this can result in chronic sleep deprivation with resultant psychiatric sequelae.

These episodes are completely preventable with administration of estradiol in hormone replacement. However, in large part due to results of the WHI the pendulum is swinging away from use of HRT to prevent menopausal symptoms. Unfortunately, most of the new, non estrogen compounds being used to prevent osteoporosis (evista, etc) do not prevent hot flashes. Hormone Replacement Therapy (HRT) Given recent publicity, and study results, why are we still talking about it?? In the current furor over HRT, it is easy to indulge in armchair quarterbacking and forget why the data emerging from these studies is so surprising. What follows here, is a very brief review and reminder of what estradiol is known to do in several important physiological systems. Furthermore, it is still the case that the cardiovascular protective effect of being female is well documented, it begins with the initiation of ovarian activity at puberty, and declines with the cessation of ovarian function at menopause. Furthermore, no studies have suggested that some other (mysterious) ovarian product, other than estradiol and perhaps or with progesterone, may be responsible for the protective effect of active ovaries. Bone: Osteoporosis begins and is most aggressive in the first few months after cessation of ovarian cycles, due to increased activity of osteoclasts and decreased function of osteoblasts. There are marked gender differences in incidence and severity of hypertension, cardiovascular disease and stroke. How is estradiol (E) doing this (if it is)? A primary observation is that BP fluctuates markedly during the menstrual cycle: falling through the follicular phase, especially during the last few days when E is highest. BP is lowest during the luteal phase, when both E and Progesterone are high. Vascular tone: Estradiol (E) causes vasodilation by both E receptor-dependent and ER independent mechanisms. Long term E treatment counters the vasoconstrictor effect of several agents and this vasodilator effect of E is reduced by certain synthetic progestins. Estradiol stimulates NO production by increasing (endothelial) intracellular free Calcium. E increases coronary vasodilation by opening Calcium activated potassium channels in the endothelium. Also it inhibits several voltage-dependent currents in coronary artery vascular smooth muscle. It may do this through more than one pathway. Further vasodilator effects of Estradiol are: increased cAMP (vasodilator 2nd messenger), increased adenosine production in vascular smooth muscle, increased prostacyclin by increasing prostacyclin synthase and cyclooxygenase, decreased synthesis of Angiotensin II, endothelin-1 and catecholamines (vasoconstrictors)

Summary: Estradiol is a potent vasodilator that decreases vascular resistance by multiple mechanisms. Protective action to prevent Heart Disease and hypertension. E counters the accelerated growth of vascular smooth muscle cells in response to disease or injury. (Review from C-V notes). Short form: Invasion of intima and stimulation of vascular smooth muscle growth are pathological remodeling. Estradiol interferes with this process: a. Blocks inflammation and decreases expression of adhesion molecules at injury site. b. Attenuates or inhibits recruitment of macrophages, lymphocytes and thrombocytes. c. Prevents accumulation of LDL by blocking macrophage invasion of subendothelial spaces. d. Inhibits mitogenic effects at injury site that trigger overgrowth of vascular smooth muscle. Summary: Estradiol profoundly reduces the vascular response to injury.

Estradiol impacts synthesis of circulating vasodilator/vasoconstrictors a. increased Bradykinin synthesis and release (<BP) b. decreased angiotensin converting enzyme (ACE) thus decreased Renin release. c. decreased circulating levels of homocysteine (involved in endothelial cell damage). d. decreased stimulation and synthesis of Endothelin I, also blocks effects of this substance to stimulate muscle mitogensis. Summary: Estradiol reduces the potential for vasoconstrictor injury. These few items are a brief indicator of some factors that comprise the systemic protective effect of Estradiol. There are numerous others that have been validated in well done studies showing protective effects on heart, vasculature, kidneys, autonomic nervous system, stress response, etc All these effects are thought to contribute to the gender differences in health and disease. Some are reduced or opposed by the coincident exposure to progesterone, some are augmented and some unchanged. So why did the most recent HRT trials come out opposite to our understanding of how estradiol works and in apparent contradiction to the physiology we think we understand? One possibility is that all the years of careful physiology and study are wrong and either estradiol is not the protective agent from the ovary, or that the ovarian protective effect is an illusion. At this juncture, this seems unlikely but never say never. If the Biology is correct, then perhaps there is something in the design of the studies that is leading to this disconnect between our expectations and the data. One possibility is

proposed by Dr. Ed Jackson of Pitts pharmacology department. Data from his laboratory suggest that at least some of the effects of estradiol are mediated by metabolites of estradiol specifically methoxyestradiols and catechoestradiols. His studies are examining this issue intensively, and, at present, there are strong data suggesting that several of the protective effects of estradiol, e.g. anti-mitogenic and antihypertensive actions, are the result of these metabolites. Interestingly, and in support of his work, the agents used in the recent large clinical trials of HRT (mostly prempro and premarin) are not metabolized by this pathway, and produce neither methoxyestradiols nor catecholestradiols. The compounds used in WHI were equine hormones with (prempro) and without (premarin) synthetic progesterone added. They were used in the study because they were the most commonly used preparation that was prescribed for post menopausal hormone replacement. A small study in Europe which used Estradiol-17B implants for hormone replacement for over 20 years beginning immediately after ovariectomy in young women shows a strong protective effect on intimal thickness.

This issue is not yet resolved, and it will take many more studies to make progress. The resolution of this or the next iteration of this problem, will still be there when you begin medical practice. Women will still be seeking reliable answers, and you will be one of the people they approach Should I take HRT or not, doctor?? What to say? What to do?

You may find the figure above to be a useful summary of the major hormonal events occurring during the menstrual cycle. The changes at menopause begin here. This figure was used in lecture to explain the changes that occur during the cessation of ovarian cycles.

Pathology of the Breast

Suggested Reading: Robbins & Cotran Pathologic Basis of Disease, 7th edition, Chapter 23, pp. 1119-1154

Sara Monaco, M.D. UPMC Department of Pathology

Overview of Breast Development 5th week- thickening of the epidermis, milk line formation Mammary ridges form from axilla to groin region Involution of mammary ridges, except in chest region o Note: persistence yields supernumerary breast (polythelia) 15th week-downward growth into stroma Last 2 months of gestation- canalization of epithelial cords with formation of branching and lobuloalveolar structures Overview of Categorization of Breast Lesions Non-proliferative cysts, papillary apocrine change, mild hyperplasia Proliferative without atypia moderate/florid ductal hyperplasia, intraductal papilloma, sclerosing adenosis, complex sclerosing lesions, complex fibroadenomas Atypical Hyperplasia (Proliferative with atypia) ductal/lobular (ADH/ALH) Malignant Breast Lesions epithelial derived tumors (DCIS/IDC, LCIS/ILC), Mesenchymal neoplasm (sarcoma), Phyllodes Tumor, Angiosarcoma, Others Lesion Nonproliferative Breast Lesions Proliferative Disease without Atypia Proliferative Disease with Atypia Carcinoma in-situ -most invasive cancers are IDC for both RR of Inv Ca 1.0 1.5-2.0 4.0-5.0 8.0-10.0 Breast at risk Neither Both Both Both for LCIS Ipsilateral for DCIS

Inflammatory Breast Lesions 1. Acute Mastitis Seen in early weeks of nursing Consequence of bacterial infection via the nipple Usually Staph/Streptococcal infection resulting in abscess(es)Staph aureus is most common cause Micro: Infiltrate of neutrophils, +/- necrosis; localized area of inflammation (Staph) or diffuse involvement (Strep, less common) 2. Traumatic Fat Necrosis Secondary to surgical or non-surgical trauma

Updated 12/2008

Can present clinically as a painless, palpable mass Micro: Ill-defined focus of hemorrhage and necrotic adipocytes replaced by histiocytes and eventually scar tissue

3. Lymphocytic Mastopathy (Sclerosing Lymphocytic Lobulitis) Presents with single or multiple firm palpable masses More common in women with type 1 (insulin-dependent) diabetes or autoimmune thyroid disease Micro: collagenized stroma surrounding atrophic ducts and lobules with a thickened basement membrane and prominent lymphocytic infiltrate Benign Breast Lesions 1. Fibrocystic Changes (FCC) Miscellaneous changes in the breast involving ducts, lobules, and stroma; sex hormone responsive Clinical incidence: approximately 40-50% of patients (present with lump) Pathological incidence: greater, approximately 60-80% (20% seen grossly) Terminology- fibrocystic change favored over fibrocystic disease Nonproliferative FCC (no increased risk of breast ca) Proliferative FCC (increased risk of breast ca; 1.5-2 X) Gross: blue domed cysts Micro: Cysts with frequent apocrine metaplasia, fibrosis, and inflammation associated with cyst rupture 2. Fibroadenoma (FA) Most common benign tumor of the female breast Usually in young women (20-30 yo) Benign fibroepithelial tumor, can be multiple & bilateral May increase in size during menstruation, lactation, infarction May regress with hyalinization and calcification in older women Rarely associated with carcinoma Gross: well-circumscribed, mobile nodules (shelled out) with gray bulging cut surface Micro: homogeneous fibroblastic intralobular stroma with compressed/distorted epithelium (pericanalicular/non-compressed and intracanalicular/compressed patterns) Complex Fibroadenoma- cysts, sclerosing adenosis, calcification, papillary apocrine change (any one of these) slight increased risk of breast ca

3. Pseudoangiomatous stromal hyperplasia (PASH) Benign, can cause a mass-like lesion in breast Micro: proliferation of interlobular stroma

Updated 12/2008

4. Gynecomastia Small subareolar swelling, bilateral Causes: unknown, relative estrogen excess (estrogen therapy), drugs (hormones, digitalis, spironolactone), metabolic or endocrine disorders (cirrhosis, starvation, malnutrition, hyperparathyroidism), neoplasms (estrogen secreting tumors), Klinefelters syndrome Physiological gynecomastia is most common in puberty and extreme old age No clear cut association with the development of carcinoma Micro: intraductal epithelial hyperplasia and increased periductal stromal cellularity and edema 5. Proliferative Breast Disease without atypia (risk of breast ca 1.5-2 X) a. Florid Ductal Epithelial Hyperplasia i. Epithelial hyperplasia is >2 cell layers moderate-to-florid with >4 cell layers ii. Micro: hyperplastic epithelium fills & expands the ducts/lobules with irregular lumens b. Sclerosing Adenosis i. Microscopic entity with at least 2X the number of acini, and associated with calcification ii. Rarely, in-situ carcinoma can arise (LCIS>DCIS) iii. Mimic of invasive carcinoma, can require myoepithelial cell stains to differentiate iv. Micro: lobular arrangement of distorted small acini with myoepithelial cells present, stromal fibrosis, calcifications c. Complex Sclerosing Lesions i. Includes radial scars and other complex lesions with areas of sclerosing adenosis, papilloma, and hyperplasia d. Papillomas i. Benign papillary proliferation within a duct ii. Identified peripherally (small duct papilloma) or centrally (large duct papilloma; associated with bloody nipple discharge) iii. Mild increased risk (1.5-2X) of development of invasive cancer in patients with multiple peripheral intraductal papillomas iv. Micro: Fibrovascular cores lined by epithelial and myoepithelial cells 6. Proliferative Breast Disease with Atypia (risk of breast ca 4-5 X) a. Atypical Ductal Hyperplasia i. Incidence: approximately 5% ii. Approximately 10% of patients will develop cancer iii. Risk for cancer is bilateral and is greatest in first decade following diagnosis iv. Patients with ADH and a family history have same risk as in-situ cancer group for the development of invasive ca: 8-10X v. Prognosis of ADH-associated cancer is the same as cancer lacking ADH

Updated 12/2008

Malignant Breast Conditions Overview Breast carcinoma is the most common malignant tumor affecting women o 180,000-240,000 new cases annually, and >40,000 deaths o Approximately 12% of women born today will be diagnosed with cancer of the breast during their life (approx 1 in 8 women) o Increase in incidence in the 1980s coincides with increase in mammography screening o Second most common cause of cancer death in women (#1 is lung) o Rare in women <25 years old, peak incidence in 4th-6th decade Most cancers (90%) arise from the ductal epithelium 10% arise from lobular epithelium About 10-30% of breast carcinomas are identified at in situ stage In situ carcinomas do not extend below the basement membrane and do not metastasize (unlike invasive carcinomas) Breast Carcinoma in men: rare, associated with BRCA2 mutations Metastatic tumors can arise: contralateral breast carcinoma, skin, lung Clinical presentations: breast pain, mass (solid mass or cyst), nipple discharge or inversion, mammographic lesion, pseudoinflammatory presentation (cancer involving lymphatics), peau dorange Triple test: clinical, radiology, pathology Treatment: surgery, radiation, chemotherapy Risk Factors for Breast Carcinoma 1. Older age 2. Early menarche or late menopause 3. First pregnancy at older age (>35 yo), nulliparity 4. Increased number of affected 1st degree relatives 5. Other

Prognostic Factors of Breast Cancer 1. TNM staging (in situ vs invasive tumor, size of primary tumor, lymph node involvement and extent, metastases) 2. Histologic type (better prognosis for tubular, mucinous/colloid, medullary) 3. Grade (Nottingham grade incorporates tubule formation, nuclear grade, mitotic activity) 4. Lymphovascular space invasion (LVSI) 5. ER/PR/Her2 status 6. Race (African American women present at more advanced stages and have increased mortality compared to Caucasian women)

* Axillary LN status is the most important prognostic factor in absence of distant metastases.

Updated 12/2008

1. Ductal Carcinoma In Situ (DCIS) Neoplastic transformation of ductal epithelium within ducts or lobules (intraductal), confined by the basement membrane Various histologic patterns: comedo, solid, cribriform, clinging and papillary type May be detected by microcalcifications (irregular, clustered or linear and branching) most common malignancy associated with calcifications May represent up to 25% of breast carcinoma Less likely than LCIS to be bilateral (10-20%) High grade and large size of the in situ ca predicts multifocality and propensity for invasion Relative risk for invasive carcinoma: 8-10X, primarily ipsilateral Micro: cohesive cells fill and distort ducts and lobules, round regular lumens, myoepithelial cells are present at periphery; Microinvasion if focus of invasion <1mm in diameter 2. Lobular Carcinoma In Situ (LCIS) Neoplastic transformation of epithelial cells lining terminal ducts and acini Typically multifocal and bilateralmulticentric in 70% cases, bilateral in 30-40% cases Usually incidental findings, NOT associated with calcifications Relative risk for invasive carcinoma: 8-10X Bilateral risk for the development of invasive cancer, subsequent cancer may be ductal or lobular 75% of invasive cancers are ductal type Micro: dyscohesive cells with bland round nuclei with occasional signet-ring morphology fill and expand the lobule 3. Invasive Ductal Carcinoma (IDC) Infiltrative malignant epithelial process resembling cells lining ducts-most common breast carcinoma Micro: tumor cells invading beyond basement memebrane in cords/nests, absence of myoepithelial layer Classified according to histologic appearance as either: o Carcinoma not otherwise specified- majority o Special good prognosis subtypes: medullary, colloid/mucinous, tubular 4. Invasive Lobular Carcinoma (ILC) Infiltrative carcinoma resembling cells lining the lobules Micro: Infiltrates as single cells in classical Indian file pattern and targetoid bulls eye pattern; composed of relatively small cells with scant cytoplasm, minimal desmoplasia, +/- signet-ring morphology Represents approximately 5-10% of breast cancer with a higher than usual incidence of bilaterality (approximately 20%) 5. Phyllodes Tumor Fibroepithelial neoplasm of variable malignant potential Most are benign and usually NOT cystic (avoid cystosarcoma phyllodes)

Updated 12/2008

Neoplastic component is the stroma Degenerates into a frank sarcoma with metastasis to lung and distant organs (hematogenous spread; only stromal component metastasizes; seen in about 30%) thus, axillary LN dissection is usually NOT indicated The majority can be cured by complete excision Peak incidence at 50 years of age, more common in Hispanics Micro: proliferating stromal nodules that create a leaf-like appearance, increased cellularity, mitotic figures, stromal overgrowth & heterogeneity Classification o benign- locally recurrent o malignant- low grade/high grade, potentially metastasizing (lung, bone) Angiosarcoma Primary in young individuals Occurs following radiotherapy in the overlying skin or spontaneously Stewart-Treves syndrome: angiosarcoma arising in the skin of patients with lymphedema after mastectomy Latency: arises 5-10 years after treatment


7. Lymphomas Primary- NHL (commonly DLBCL), MALT, Burkitt Lymphoma Secondary- part of generalized process 8. Pagets Disease of the Breast In situ carcinoma of lactiferous ducts with extension to epidermis Involves the nipple and areola presents with discharge, crusting or excoriation Over 95% are associated with an underlying carcinoma DCIS +/- invasion DDx: clear cell change in epidermis (Toker cells), Bowens disease, Melanoma

10. Male Breast Carcinoma Rare, ratio of male:female breast cancer is 1:125 Presents at an advanced stage Identified in peri-nipple/areolar region Associated with BRCA2 mutations Hereditary Breast Carcinoma 13% of women have reported family history of breast carcinoma in a 1st degree relative Lifetime risk of breast carcinoma is 60-85% Increased risk of ovarian carcinoma (greater for BRCA1 carriers, 20-40%) and other tumors (colon, prostate, pancreas) BRCA1: chr. 17q, increased incidence of poorly differentiated, triple negative, basal-like phenotype, & medullary carcinomas BRCA2: chr 13q, associated with male breast carcinoma Genetics: 1 mutated allele inherited, inactivation of 2nd allele (2nd hit) is acquired mutation

Updated 12/2008

Pathology of the Ovary

Suggested Reading: Robbins & Cotran Pathologic Basis of Disease, 7th edition, Chapter 22, pp. 1092-1104.

Sara Monaco, M.D. UPMC Department of Pathology

Non-neoplastic Ovarian Lesions 1. Cysts Cystic follicles are common (called Follicular Cyst if >2cm) & multiple Most common in non-pregnant reproductive age women Micro: clear serous fluid contents with thin smooth wall comprised of granulosa (inner; may be denuded) and theca (outer) cells 2. Surface Epithelial Inclusion Cysts Due in invagination of overlying surface epithelium Usually small and an incidental finding Micro: benign ciliated or nonciliated cuboidal/flattened lining

3. Hemorrhagic Cysts Includes primarily corpus luteal cysts and endometriotic cysts Corpus Luteal Cyst o Gross: cyst wall has bright yellow lining, bloody cyst contents o Micro: thickened wall contains granulosa cells with luteinization Endometriosis/Endometriotic Cysts o Clinical presentation: severe dysmenorrhea, infertility in 30-40% o Gross: chocolate cysts, may have fibrous adhesions o Micro: 3 components include endometrial stroma, endometrial glands, hemosiderin-laden macrophages 4. Polycystic Ovarian Disease (PCOD; Stein-Leventhal Syndrome) Incidence: about 5 % of reproductive age women Clinical presentation: anovulation/oligomenorrhea, obesity, hirsutism, decreased glucose tolerance (diabetes), virilism Gross & Micro: enlarged ovaries with thickened cortex and multiple small follicle cysts 5. Stromal Hyperthecosis (SH) Clinical presentation: similar to PCOD, but with more gradual onset & usually in postmenopausal women Gross & Micro: enlarged ovaries with hypercellular stroma containing luteinized stromal cells HAIR-AN syndrome: rare, multisystem disorder of hyperandrogenism (HA), insulin resistance (IR) and acanthosis nigricans (AN); described in a subset of women with SH or PCOD

Updated 12/2008

6. Pelvic Inflammatory Disease (PID) Ovarian involvement usually occurs secondary to salpingitis with a tubo-ovarian abscess, and sometimes peritonitis Can lead to tubo-ovarian fibrous adhesions IUD related PID can be due to actinomycosis 7. Ovarian torsion and infarction Usually a complication of an associated ovarian lesion Clinical presentation: similar to appendicitis with abdominal pain Gross & Micro: swollen, hemorrhagic ovary with possible infarction due to loss of blood supply

Updated 12/2008

Malignant Ovarian Lesions Overview Approximately 20,000 new cases annually, and 15,000 deaths large number end in death because hard to detect, so often widespread with high stage Approximately 1.5% of women born today will be diagnosed with cancer of the ovary during their life (approx 1 in 72 women) Third most common tumor of the female genital tract (after cervix and endometrium) Fifth most common cancer in women 80% are benign, usually in younger women (<40 years old) Malignant tumors are more common in older women Older women surface epithelial tumors predominate Younger women germ cell tumors predominate Risk factors: nulliparity, family history, BRCA mutations (BRCA1>BRCA2) Benign tumors tend to be cystic, non-complex, simple lining (cystadenoma) Borderline tumors tend to have increasing complexity, non-invasive, may require less surgery Malignant tumors are invasive (cystadenocarcinoma)

Tumor Origin
Surface Epithelium Serous (40%), 60%B9/10%BT/30%M Mucinous (10%), 80%B9/15-20%BT/<5%M Endometrioid (20%) Clear cell (<10%) Brenner (<10%) Germ Cells Teratoma (>95%) Dysgerminoma Yolk sac/Endodermal Sinus Tumor Choriocarcinoma Embryonal Sex Cord-Stroma Granulosa-theca Fibroma-thecoma Sertoli-Leydig Metastases Mullerian origin tumors (FT, opposite ovary) GI tract (Krukenberg tumor-signet ring tumor of stomach metastatic to ovaries) Appendix Breast

Overall Frequency

Proportion of Malignant tumors








Updated 12/2008


Surface Epithelial Tumors Includes the majority of the primary ovarian neoplasms Can be benign, borderline, or malignant May have a variety of components, including: cysts, fibrous stroma, and a mixture Serous Tumors (ST) o Most are benign o More likely to be bilateral & unilocular than mucinous tumors o Bilaterality is more common in malignant lesions (65% bilateral) o Peritoneal spread can occur as invasive or noninvasive implants 30% of SBTs are associated with implants & 90% of these are non-invasive o Classification: Cystadenoma, Borderline ST, Cystadenocarcinoma o Gross: usually a unilocular cyst; must examine lesion for solid areas, which may be a papillary-type projection, thickening or mass within the lesion, or necrosis, because all of these findings may be associated with malignancy o Micro: cystic lesions with variable amounts of epithelial thickening or papillary projections; benign lesions have non-complex columnar epithelium +/- cilia; malignant lesions are more complex; frequently find Psammoma bodies Mucinous Tumors (MT) o Most are benign o Usually Multilocular & Unilateral (MUcinous)- opposite of serous tumors o If bilateral, must exclude a metastatic origin (ex. Appendix, GI tract) o Classification: Cystadenoma, Borderline MT, Cystadenocarcinoma o Gross: usually multilocular with thick mucin; usually large o Micro: tall columnar mucinous epithelium o Pseudomyxoma Peritonei: mucinous tumor associated with mucinous ascites (jelly belly), implants, adhesions, and other complications that can result in death Endometrioid Tumors o Most are malignant o 15-30% associated with endometrial carcinoma; probably synchronous o 15% coexist with endometriosis Clear Cell Tumors o Most are malignant o Associated with endometriosis o Aggressive Brenner Tumor o Most are benign & usually unilateral o Micro: nests of cells resemble urothelial lining of the bladder with associated fibrous stroma Malignant Mixed Mullerian Tumor (MMMT) o Uncommon, <1% of ovarian tumors

Updated 12/2008

o o

Usually in older women (6th-8th decade) Gross/Micro: large, hemorrhagic & necrotic; epithelial (usually serous or endometrioid) and sarcomatous (homologous vs heterologous) elements

2. Germ Cell Tumors (GCT) Majority (>95%) are benign mature cystic teratomas In first 2 decades, GCTs account for 60% of ovarian tumors & 1/3 are malignant (accounts for 2/3 of ovarian cancer in this group) Embryonal carcinoma is rarer in the ovary than in the testis Teratomas o 3 subtypes: mature/benign, immature/malignant, monodermal/specialized o Mature/Benign Teratomas: Often cystic (dermoid cysts) Usually occur in young women, bilateral in 10-15% Usually benign, only 1% have malignant transformation (Squamous Cell Carcinoma is most common) Gross/Micro: unilocular with hair, teeth, and sebaceous material; components of all 3 germ layers (ectoderm, mesoderm, endoderm); squamous epithelium with hair and adnexal structures (ectodermal components) predominate usually o Immature/Malignant Teratomas: Often solid Usually in prepubertal girls Rapid growth, can penetrate capsule and metastasize Micro: Contains immature tissue which may show some differentiation, grading is based on amount of immature neuroepithelium o Monodermal/Specialized Teratomas: Includes struma ovarii (mature thyroid tissue), carcinoid (+/- carcinoid syndrome), strumal carcinoid (mixture) Dysgerminoma o Counterpart in the male is seminoma o Rare overall, but comprises 50% of malignant germ cell tumors o 75% occur in 2nd-3rd decade o Usually unilateral o Very radiosensitive, favorable prognosis o About 30% are aggressive, control is usually possible with radiotherapy o Gross/Micro: solid, white-gray soft tumors; sheets of cells with clear cytoplasm, round nuclei, prominent nucleoli, lymphocytes and granulomas in stroma Yolk Sac Tumor (Endodermal Sinus Tumor) o Rare, 2nd most common malignant germ cell tumor (after dysgerminoma) o Associated with elevated AFP o Aggressive, less favorable prognosis o Gross/Micro: variety of patterns, Schiller-Duval bodies (glomeruloid structures), hyaline globules that are + AFP

Updated 12/2008

Choriocarcinoma o Usually exists in combination with other germ cell tumors (Mixed types) o Usually aggressive with widespread hematogenous metastases more aggressive and less responsive to treatment than placental type o Associated with elevated HCG 3. Sex Cord-Stromal Tumors Derived from ovarian stroma, which arises from the sex cords of the embryonic gonad Clinically present with adnexal mass, sex steroid hormone production (with associated manifestations), and are potentially aggressive (about 5-20%) Granulosa Cell Tumors o 2 types: Adult GCT, Juvenile GCT (dilated, irregular cysts; lack of Call-Exner bodies, young patient with precocious puberty) o Most (over 60%) occur in post-menopausal women o Usually unilateral o Clinically associated with increased estrogen (precocious puberty in young girls, endometrial hyperplasia/carcinoma in older women), less likely causes increased androgens, & small risk of malignancy 10-15% of women with a steroid-producing tumor eventually develop an endometrial carcinoma o Malignancy occurs in 5-25%, indolent with late recurrences that can be resected o Gross/micro: solid or cystic, polygonal cells with coffee-bean nuclei containing grooves and Call-Exner bodies (rosette with pink lumen); inhibin + Fibroma-Thecoma o Usually a mixture of fibroblasts (fibroma) & spindle cells with lipid (thecoma) o Usually unilateral o Association with Meigs Syndrome and Basal Cell Nevus Syndrome o Meigs Syndrome: ovarian fibroma, pleural effusion (R>L), and ascites o Gross/Micro: well-circumscribed, solid, gray-white, firm mass with plump differentiated spindle cells Sertoli-Leydig Cell Tumors (Androblastomas) o Usually unilateral o Clinically associated with androgenic manifestations (masculinization, hirsutism, etc) o Peak incidence in 2nd-3rd decade o Gross/Micro: solid, may be golden brown/yellow; tubules and stroma; Leydig cells may be absent or hard to find; can have a dedifferentiated form that mimics a spindle cell neoplasm

Updated 12/2008