Pregnancy & Prenatal

Analysis

By
Dr Waleed Shaaban Dr. Marwa Abdelhaq
DIAGNOSIS OF PREGNANCY
Pregnancy tests detect human chorionic gonadotropin
(hCG) in serum or urine.
Human chorionic gonadotropin is a glycoprotein hormone
produced by syncytiotrophoblastic cells of conceptus and
later by placenta
It circulates in maternal blood and excreted by the
kidneys.
Immunological tests use antibodies directed against β-
subunit of hCG to avoid cross-reactivity with LH, FSH, and
TSH.
 DIAGNOSIS OF PREGNANCY
Indications for measurement of β human chorionic
gonadotropin
1. Early diagnosis of pregnancy
2. Diagnosis and management of gestational trophoblastic
disease
3. As a part of maternal triple test screen: This consists of
measurement of hCG, α-fetoprotein, and estriol in maternal
serum at 14-19 weeks of gestation. The maternal triple screen
identifies pregnant women with increased risk of Down
syndrome and major congenital anomalies like neural tube
defects
 DIAGNOSIS OF PREGNANCY
 Two types of pregnancy tests are
available:
1. Qualitative tests: These are
positive/negative result types that
are done on urine sample.
2. Quantitative tests:
 Give numerical result and are done
on serum or urine.
 Positive serum hCG test: 8 days after
conception or 3 weeks after last
menstrual period (LMP)
 Positive urine hCG test: 21 days after
conception or 5 weeks after LMP.
 DIAGNOSIS OF PREGNANCY

In the first trimester (first 12 weeks) of pregnancy, hCG

levels rapidly rise with a doubling time of about 2 days.
Highest or peak level is reached at 8-10 weeks (about
100,000 mIU/ml).
This is followed by a gradual fall, and from 15-16 weeks
onwards, a steady level of 10,000-20,000 mIU/ml is
maintained for the rest of the pregnancy.
What do the pregnancy test results mean?
 If the test gives positive result, this means pregnancy, no matter how
faint the color is. In very rare cases, a false-positive result may obtained.
 False positive:
1. Errors of test application
2. Use of drugs containing the assay molecule
3. Non-pregnant production of the assay molecule.
4. Tests used past their expiration date.
5. A woman who has been given an hCG injection as part of infertility
treatment will give positive result on pregnancy
6. Some diseases of the liver, cancers, and other medical
conditions may produce elevated hCG and thus cause a false positive
pregnancy test. These include choriocarcinoma and other germ
cell tumors, IgA deficiencies, gestational trophoblastic diseases (GTD),
and gestational trophoblastic neoplasms (GTN).
What do the pregnancy test results mean?
False negative:
1. The test is past its expiration date.
2. The test was run in a wrong way.
3. The test is done too early in pregnancy.
4. The urine is too diluted.
5. Certain medications, such as diuretics or
antihistamines.
 Semen Analysis
 Semen (or seminal fluid) is a fluid that is emitted from the male
genital tract and contains sperms.
 Structures involved in production of semen are:
1. Testes and epididymis: 10%
2. Seminal vesicles: 50%
3. Prostate: 40%
 Indications for semen analysis
1. Investigation of infertility: Semen analysis is the first step in the
investigation of infertility. About 30% cases of infertility are due
to problem with males.
2. To check the effectiveness of vasectomy by confirming absence
of sperm.
 COLLECTION OF SEMEN
Semen specimen is collected in a clean, dry, sterile,
plastic container, and brought to the laboratory within 1
hour of collection.
Tests done on seminal fluid
1. Physical examination: Time to liquefaction, viscosity,
volume, pH, color.
2. Microscopic examination: Sperm count, vitality, motility,
morphology, and proportion of white cells.
3. Immunologic analysis: Anti-sperm antibodies.
4. Bacteriologic analysis: Detection of infection.
 Semen analysis
 Physical Examination
 Examination is carried out after liquefaction of semen that occurs
usually within 20-30 minutes of ejaculation.
1. Visual appearance: Normal semen is viscous and opaque gray-white
in appearance.
2. Viscosity: Immediately following ejaculation, normal semen is
viscous. It becomes liquefied within 30 minutes by the action of
proteolytic enzymes of prostate. If liquefaction does not occur within
60 minutes, it is abnormal. Increased semen viscosity affects sperm
motility, it results from infection of seminal vesicles or prostate.
3. Volume: Normally is > 2 ml. It is measured after the sample has
liquefied. Volume < 2.0 ml is abnormal, and is associated with low
sperm count.
 Semen analysis
4. pH: Normal pH is 7.2 to 8.0 after 1 hour of ejaculation. The
portion of semen contributed by seminal vesicles is basic, while
portion from prostate is acidic.
 Low pH (< 7.0) with absence of sperms (azoospermia) suggests
obstruction of ejaculatory ducts or absence of vas deferens.
Semen analysis
 Microscopic Examination
 The most important test in semen analysis for infertility is microscopic
examination of the semen.
1. Sperm Motility
 The first laboratory assessment of sperm function is sperm motility.
 All motile and non-motile sperms are counted in randomly chosen fields
under 40× objective. Result is expressed as a percentage of motile
spermatozoa observed.
 Percentage of: (a) rapidly progressive spermatozoa (moving fast forward
in a straight line).
(b) slowly progressive spermatozoa (slow linear or curved movement).
(c) non-progressive spermatozoa (movement of tails, but with no forward
progress).
(d) immotile spermatozoa (no movement at all).
Semen analysis
 Sperms of grades (c) and (d) are considered to be poorly motile
(asthenospermia).
 Normally, ≥ 25% of sperms show rapid progressive motility, or ≥ 50% of
sperms show rapid progressive and slow progressive motility.
2. Sperm Viability or Vitality
 The result is expressed as a proportion of viable sperms against non-
viable as a percentage. 75% or more of sperms are normally viable.
3. Sperm Count
 Number of spermatozoa is reported in millions/ml
 Normal sperm count is ≥ 20 million/ml
 Sperm count < 20 million/ml may be associated with infertility
Semen analysis
4. Sperm Morphology
 A smear is prepared by spreading a drop of seminal fluid on a glass
slide, stained, and then percentages of normal and abnormal forms of
spermatozoa are counted.
 Normal morphology: A spermatozoon consists of three main
components: head, neck, and tail.
 Normally, > 30% of spermatozoa should show normal morphology.
 Abnormal morphology
 Large heads Bent tails
 Small heads Multiple tails
 Tapered head Short tails
 Round heads Coiled tails
Abnormal morphological sperm forms: (1) Normal sperm, (2) Large head, (3) Small head, (4)
Tapered head, (5) Pyriform head, (6) Round head, (7) Amorphous head, (8) Vacuoles in head,
(9) Round head without acrosome, (10) Double head, (11) Pin head, (12) Round head without
acrosome and thick midpiece, (13) Coiled tail, and (14) Double tail
Terminology in semen analysis
Oligozoospermia: Sperm count <20 million/ml
Azoospermia: Absence of sperms in seminal fluid
Aspermia: Absence of ejaculate
Asthenozoospermia: Reduced sperm motility
Teratozoospermia: increased proportion of
abnormal forms
Leukocytospermia: >1 million white blood cells/ml
of semen
Necrozoospermia: All sperms are non-motile or
non-viable
Prenatal diagnosis
 Employs a variety of techniques to determine the health and
condition of an unborn fetus
 There are a variety of non-invasive and invasive techniques
available for prenatal diagnosis. Each of them can be applied only
during specific time during the pregnancy.
 The techniques used for prenatal diagnosis include:
1. Ultrasonography
2. Amniocentesis
3. Chorionic villus sampling
4. Maternal serum alpha-fetoprotein
5. Maternal serum beta-HCG Maternal Triple test screen
6. Maternal serum estriol
Prenatal diagnosis
 Ultrasonography
 This is a non-invasive procedure that is harmless to both the fetus
and the mother.
 Ultrasound examination is useful to determine the size and
position of the fetus, the size and position of the placenta, the
amount of amniotic fluid, and the appearance of fetal anatomy,
 There are limitations in which subtle abnormalities may not be
detected until later in pregnancy, or may not be detected at all.
 A good example of this is Down syndrome (trisomy 21) where the
morphologic abnormalities are often not marked.
Prenatal diagnosis
 Amniocentesis
 An invasive procedure in which a needle is passed through the mother's lower
abdomen into the amniotic cavity.
 For prenatal diagnosis, most amniocenteses are performed between 14 and 20
weeks gestation analysis to detect genetic disorders.
 Ultrasound examination always proceeds amniocentesis to determine
gestational age, the position of the fetus and placenta, and determine if enough
amniotic fluid is present.
 Within the amniotic fluid are fetal cells (derived from fetal skin) which can be
grown in culture for chromosome analysis to detect genetic disorders.
 In the third trimester of pregnancy, the amniotic fluid can be analyzed for
determination of fetal lung maturity
 Also can be used for the presence and severity of erythroblastosis fetalis.
Prenatal diagnosis
 Maternal serum alpha-fetoprotein (MSAFP)
 MSAFP test is used to determine the levels of AFP from the fetus.
 Ordinarily, only a small amount of AFP gains access to the
amniotic fluid and crosses the placenta to mother's blood.
 However, when there is a neural tube defect in the fetus, from
failure of part of the embryologic neural tube to close, there is a
escape of more AFP into the amniotic fluid.
 The MSAFP can also be useful in screening for Down syndrome.
The MSAFP tends to be lower when Down syndrome or other
chromosomal abnormalities is present.
Prenatal diagnosis
 Maternal serum beta-hCG
 In the middle to late second trimester, the beta-hCG can be used in
conjunction with the MSAFP to screen for chromosomal
abnormalities (Down syndrome in particular).
 An elevated beta-hCG coupled with a decreased MSAFP suggests
Down syndrome.
 Maternal serum estriol
 Measurement of serum estriol levels will give an idea of general
well-being of the fetus.
 Estriol tends to be lower when Down syndrome is present and
when there is adrenal hypoplasia.
 Tests for fetal lung maturity(FLM)
 Lecithin/Sphingomyelin ratio (L/S ratio)
 This test evaluates a change in the relative amounts of lecithin
(phosphatidyl choline) and sphingomyelin (a phospholipid of unknown
origin) in amniotic fluid samples as gestational age increases.
 Until about 32-33 weeks of gestation, the concentration of these two
substances are quite similar; thereafter the concentration of lecithin
increases significantly compared with the relatively constant
concentration of sphingomyelin.
 In absence of complications, the ratio of these two components
reaches 2.0 at about 35 weeks gestation. Infants delivered after
attaining an L/S ratio of 2.0 or higher rarely develop RDS. This value
of 2.0 has become the commonly accepted standard value indicating
maturity in the fetus of a non-diabetic woman.
Tests for fetal lung maturity(FLM)
 Phosphatidylglycerol (PG) is a minor constituent of surfactant that
generally appears several weeks after the increase in lecithin
concentration.
 Because PG enhances the spread of phospholipids on the alveolar
surface, its presence indicates an advanced state of fetal
pulmonary maturity.
 PG determination can be accomplished by either thin-layer
chromatography or by slide agglutination.
 The test is reported as either "PG detected" or "PG absent." When
PG is detected, the risk of RDS is minimal.
Tests for fetal lung maturity(FLM)
 An advantage of PG determination in assessing fetal maturity is
the fact that blood, meconium, or other contaminants do not
generally affect it. This characteristic allows PG determination by
using vaginal pool samples from patients who have spontaneous
rupture of membranes.
 A disadvantage of using PG for assessing fetal maturity is its
relatively late appearance in pregnancy. Compared with other
tests, an “immature” result (negative PG) is associated with a
greater proportion of infants who will not develop RDS.
Tests for fetal lung maturity(FLM)
 Fluorescence polarization (surfactant/albumin [S/A] ratio)
 Test of choice for rapid and accurate results.
 Sensitivity 99%, specificity 70%; high predictive value for lung
maturity.
 Commercially available test (TDx FLM II) reported in units of mg
surfactant/g albumin.
 S/A values increase during gestation in parallel with lung
maturation.
 ARUP-produced test reported in units of mPol Polarization values
decrease during gestation in parallel with lung maturation
 Blood contamination can affect results.
 Meconium contamination produces an unpredictable effect on
results.
Erythroblastosis Fetalis

Erythroblastosis Fetalis: Amniotic Fluid Absorbance

 Specific tests for diagnosis depend on the type of
erythroblastosis, include:
1. Complete blood count
2. Bilirubin level
3. Blood typing
 Erythroblastosis Fetalis
 Determination of Bilirubin
 In normal pregnancy a small concentration of unconjugated bilirubin is
present in the amniotic fluid, and this concentration falls as term is
approached.
 In erythroblastosis fetalis, the bilirubin concentration increases with time
as the hemoglobin liberated by the hemolytic process is converted to
bilirubin. Due to fetal hepatic immaturity all bilirubin is generally of the
unconjugated type and is bound to albumin.
 When the bilirubin concentration is so high that the binding capacity is
exceeded, the free unconjugated bilirubin can penetrate the brain and
produce kernicterus.
 Hence, the assessment of the presence and severity of erythroblastosis
fetalis relies on the monitoring of bilirubin in the amniotic fluid.
Erythroblastosis Fetalis
 Bilirubin in amniotic fluid is monitored by direct visible scanning
spectrophotometry of a centrifuged specimen.
 Amniotic fluid absorbance values are interpreted by plotting them
semi-logarithmically on a Liley-Prognostication Chart, which
contains weeks of gestation on the linear axis and absorbance on
logarithmic axis.
 The chart is divided into three zones:
1. Zone C: very severely affected babies (zone of severe hemolysis).
2. Zone B: indeterminate zone.
3. Zone A: unaffected babies (no hemolysis).