The Guide to Investigation of Mouse Pregnancy

I

Atlas

Outline

1 The Cycling and Pregnant Mouse

2 Implant Site Dissections

3 Microscopic Anatomy of the Pregnant Mouse Uterus Throughout Gestation

4 Anatomy of the Mouse Placenta Throughout Gestation

5 Whole-Mount Immunohistochemistry of Early Implantation Sites

6 Magnetic Resonance Imaging of the Mouse Placenta

1

The Cycling and Pregnant Mouse

Gross Anatomy

Stephen C. Pang, Judith Janzen-Pang, M. Yat Tse and B. Anne Croy,    Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada

Patricia D.A. Lima,    Institute of Biology, University of Campinas, Campinas, São Paulo, Brazil

Chapter Summary

Studies of pregnancies in most strains of mice are optimized by attentive observation of external features of the estrous cycle to ensure pairing with a male during the female’s brief interval of receptivity to mating. Also important are recognition of the copulation plug, indicative of mating, and observation of the external appearance of the pregnant female that becomes distinctive from midgestation. Monitoring of external appearance can identify young females who became pregnant following undetected mating. This permits early decisions for appropriate usage of such animals. Premating obesity will complicate visual assessment of pregnancy and necessitate palpation of fetuses in the female’s abdomen to confirm that she is at mid- to late pregnancy. Palpation should not be undertaken before suspected gestation day 12 of pregnancy since it may cause implantation site damage. External features as well as postmortem dissections of the gravid mouse and its uterus are illustrated to show changes that progress over pregnancy.

Keywords

Copulation plug; Estrous cycle; Gestation; Placenta; Pregnancy; Uterus; Vagina; Vaginal smear

Abbreviations

gd Gestation day

h Hour

kg Kilogram

mg Milligram

Introduction

Research investigations requiring timed pregnant mice are much easier to plan and to schedule if the investigator can be certain of having one or more experimental matings available on a specific experimental day. Learning to recognize estrus in the female mouse is essential for the reliable availability of experimental pregnancies. For most general purposes, young, 8–12-week-old male and female mice at the peak of their fertility are used to optimize breeding for timed experiments.

Research costs will be lowered if only females entering estrus are placed with the male. These pairings only need to be 1:1 or may be up to 1:3 male:female if estrous selection is practiced. Large harems of females are not needed for each male. Additionally, when using estrous selection and recording the identity number of the copulating male on the card of the mated female, it rapidly becomes apparent whether or not the stud male mouse is fertile.

The time of observation of the copulation plug, which forms from reproductive tract secretions of the male mouse, is widely called either gestation day (gd) 0.5 or 1.5. It is critical that investigators define in all of their communications which gd-counting system they are using because large changes occur over 24 h in mouse implantation sites. In our laboratory, with the dark period from 7 pm to 7 am, the morning of the copulation plug is called gd 0.5. Mice are nocturnal animals, meaning that they are more active during darkness and that is when mating usually occurs. Copulation plugs are estimated to occur at the midpoint of the dark interval (i.e. 1 am in our example colony), making the morning of plug detection within the first 12 h postmating, thus gd 0.5. Overuse of stud males should be avoided with an optimal 24–48 h of rest from breeding after he has mated a female. Stud males should be housed individually to prevent fighting, and we always prefer to add the female to the male’s home cage, feeling that this is less stressful for the stud and will let him exert dominance.

Selection of estrous mice is done by vaginal examination. The features are more clearly seen in some mouse strains than in others. They are particularly prominent in albino (white, pink-eyed) mice, as shown in Plate 1. Vaginal smears can also be used to monitor the estrous cycle. For mouse strains and species that do not reliably form a copulation plug, vaginal smears are used the morning after pairing of the male and female. These smears are assessed for the presence of spermatozoa. If spermatozoa are present, copulation occurred and a pregnancy should ensue.

Plate 1 Appearance of the mouse vagina over the estrous cycle and the associated vaginal smear cytology.

Stages of the estrous cycle can be evaluated by visual observation (A–D) or by vaginal cytology (E–H). These images were collected serially over 4 days from a single CD1 mouse. Changes in the vaginal cytology correlate with the visual changes in the vaginal mucosa and vulva. Usual cycle length in the mouse is 4–5 days.

(A and E) In diestrus, the vaginal opening is small, and labia of the vulva are not swollen. Diestrous cytology is characterized by smaller polymorphonuclear leukocytes and a few larger epithelial cells.

(B and F) In proestrus, the vaginal opening (labia and dorsal commissure of the vulva) is swollen and moist. The vaginal mucosa is darker in color than at full estrus. Proestrous cytology is characterized by a predominance of epithelial cells that are nucleated. A few cornified epithelial cells may be present.

(C and G) In estrus, when the female mouse is receptive to breeding by the male mouse, the vaginal opening (lateral labia and dorsal commissure of the vulva) is swollen, with the dorsal region showing distinct ridges. The vaginal mucosa is paler and dryer than at proestrus. If whitish soft debris is present on the vaginal mucosal surface, estrus has passed and the female is not receptive to breeding. Estrous cytology is characterized by the predominance of cornified epithelial cells.

(D and H) In metestrus, the vaginal opening is small to closed, and the labia of the vulva are not swollen. Vaginal cytology during metestrus, the shortest stage of the estrous cycle, is characterized by the presence of a few epithelial cells and increasing numbers of polymorphonuclear leukocytes. Bars: E–H: 50 μm.

Mating is usually but not always followed by pregnancy. When pregnancies do not occur, the investigator should ensure that the females are cycling by vaginal cytology and by pairing of females with males of proven fertility (i.e. recently sired a confirmed pregnancy). If copulation plugs still do not result in pregnancy, flushing of the contents from the lumen of the uterus on gd 3.5¹ is helpful in identifying the problem. At this time point in normal mouse pregnancy, blastocysts enclosed in the zona pellucida are present in the uterine lumen (Chapter 3 of ATLAS by Edwards/Yamada, Plate 2, Fig A,B,C.). In infertile mice, unfertilized ova, retarded embryos at the morula stage, normal blastocysts, or a mixture of these may be flushed. If normal blastocysts are present in infertile females, male fertility would be proven and implantation failure would be expected. Unfertilized ova would suggest sterility of the stud male, and retarded embryos would be more suggestive of developmental rather than parental gamete problems. It is important to have continual awareness about the success of matings in your study populations.

Anatomic Changes in the Appearance of the Cycling and Pregnant Uterus

Mouse uterine horns are long tubular structures that each terminate cranially at the uterine tube (oviduct), which is much narrower and is associated with an ovary. At ovulation, hemorrhage into the vacated ovarian follicle occurs, and for several days these red ovulation sites are clearly visible to the naked eye on the surface of the ovary. It is strongly recommended to count and record the corpora hemorrhagica or their descendent, yellower corpora lutea in a mated mouse upon dissection of the reproductive tract. Normally, litter size should equal the number of corpora lutea. Caudally, the uterine horns end at the cervix, a firm white structure in the pelvic cavity. The mouse is a species with a duplex cervix. That is, each uterine horn has its own cervical canal, and there is no uterine body. This anatomic feature prevents movement of conceptuses between mouse uterine horns. Thus, the number of ovulations from the ipsilateral ovary (same side) controls the number of implantation sites possible in a specific uterine horn. The duplex cervix also permits the design of pregnancies in which an experimental and control condition can be studied in a single female. Comparisons between embryo transfer conceptuses of two different genotypes or between pseudopregnancy and pregnancy are examples of how the duplex cervix is useful in research.

The appearance of the uterus changes considerably over the estrous cycle. At most stages, the horns are long, flaccid, relatively thick walled, and beige-pink in color. At estrus, however, the horns are thin walled, nearly transparent, and less colored, and they appear to be fluid filled and turgid. The latter appearance should not be of concern but should be recognized as estrus, particularly when seen during the surgery or dissection of a mouse the investigator expected to be pregnant.

Dissection of the uterus from a recently euthanized mouse releases the uterus from its stabilizing membranes and lowers its temperature to ambient or that of chilled medium if it is used. Postmortem contraction of the uterine smooth muscle walls occurs rapidly and creates transverse ridges across the uterus that mimic the appearance of implantation sites. No implantation sites are present prior to gd 4.5 (from copulation counted as gd 0.5). Early implantation sites are not reliably recognized by unaided study until gd 6.5. Intravenous dye injection prior to euthanasia can be a useful aid in identification of very early implantation sites as vascular leakage occurs at all implantation sites and they become stained.

Mouse embryos implant on the antimesometrial side of the uterus, where they induce primary decidualization. This is followed by a wave of differentiation of secondary decidua that begins antimesometrially and sweeps laterally and then mesometrially, where it becomes the decidua basalis. At gd 4.5 to 7.5, most of the tissue in the mouse implantation site is maternal decidua. The antimesometrial decidua begins to regress at about gd 8.5 and eventually becomes a thin layer, while the decidua basalis persists. The shift in embryo shape from cylindrical to globular between gd 7.5 and 8.5 marks the transition to conceptus tissue dominance within normal implantation sites. The mature placenta is found after the fetal allantois (the tissue progenitor of the umbilical cord) fuses with the chorion at gd 9.0. The chorion is a bilayered, extraembryonic tissue of mesoderm and ectoderm. The latter layer arises from the trophoectoderm of the blastocyst and gives rise to trophoblast cells of the placenta. After fusion, fingers of allantoic tissue that contain new fetal blood vessels grow out into the placental primordial tissue known as the ectoplacental cone and establish the layered architecture of the mature mouse placenta. Placental circulation begins between gd 9 and 10 and is followed by increases in placental weight and volume. These are estimated to continue to gd 16.5, while exponential fetal growth occurs between midpregnancy and term.² Thus, fetal weight or size ratios to placental weight or size ratios become increasingly larger over the later stages of mouse pregnancy.

By gd 10.5, females carrying large litters (>6) begin to show abdominal distension. This is delayed in females with smaller litters. As fetuses continue growing, abdominal distension becomes more noticeable. Postmortem study will reveal displaced abdominal organs, a thinned abdominal wall, and prominent epigastric blood vessels that supply the mammary glands. Details of the placentas and the fetuses become apparent through the nondissected uterine wall. From about gd 12.5, growth of the conceptus in length is very noticeable, and it begins to crowd the fetuses along the uterine horns and distort them. In the last few days of gestation, all abdominal and pelvic contents are displaced by the greatly expanded gravid uterine horns.

Also noticeable late in pregnancy in live mice is a lack of activity and the greater prominence of nipples.

Death of a Conceptus Does not Mean Pregnancy Failure

It is not uncommon to find an implantation site within a litter of a uterus dissected in mid- to late pregnancy that is slightly smaller and paler or is quite dark red compared with littermate sites. These anomalous sites contain failing and dead conceptuses, respectively, and should be recorded as incidental findings. If anomalous sites occur regularly or if many implantation sites of the litter are involved, a problem is present. Problems can be husbandry related, infectious, genetic, or experimentally induced. The cause needs to be ascertained. It is remarkable that in mice, loss of one or a few implantation sites can occur in females that go on to give birth to and rear litters. Postmortem or imaging studies are the only ways in which spontaneous loss of implantation sites are detected. Vigorous breeding colonies are derived by reserving as breeding stock those females and their offspring that always give the largest litters of healthy pups.

Postpartum Uterine Involution

Involution, or the return of the uterus toward its virgin size and vascularity, occurs rapidly in mice. A postpartum ovulation occurs in mice, and if a male is present, the female may be bred within 24–48 h after delivery of her litter even if she is nursing newborn pups. The involuting uterus will have adequately returned to a fertile state within 3.5–4.5 days after the postpartum mating to allow blastocyst implantation and successful pregnancy. Mouse pups are usually weaned at 19–21 days of age to prevent delivery of the new litter into a cage with active juvenile mice.

Because the mouse estrous cycle is 4–5 days, extended intervals between litters are not anticipated. By 6 weeks postpartum, a female who has continuously cohabited with a male should have had her second litter if the pair remains fertile. If there is no litter, the male will have failed to copulate on at least five or perhaps six estrous cycles or the female has become infertile. Such pairs should be immediately replaced.

Illustrative Plates

Atlas Plates 1–13 illustrate many of the features described in this chapter using random-bred albino mice (NIH Swiss and CD1). The choice of NIH Swiss was due to the use of this strain by Kaufmann in The Atlas of Mouse Development³ and the availability of a large bank of archived, paraffin-embedded implantation sites from this strain in the laboratory of Dr Yamada, which were used for the preparation of histological images for this Atlas (Chapter 3). However, significant, unexplained fetal losses occurred in pregnancies of NIH Swiss mice purchased in early 2011 for preparation of the gross anatomical images for this Atlas. We switched to CD1 mice, a related strain, to complete the gross anatomy illustrations of the normal, healthy, gravid mouse uterus.

Plate 2 The copulation plug.

Following mating, secretions from the reproductive tract of the male laboratory mouse harden in the female’s reproductive tract. This is called the copulation plug. It is indicative of mating but not of conception or pregnancy. For example, a vasectomized mouse that is reproductively sterile produces a copulation plug. Matings with vasectomized males are commonly used to prepare females as embryo transfer recipients.

Copulation plugs are transient in the vagina and vary in position. Plugs fall out naturally during the day of mating and are most reliably detected within the first 12 h after copulation. Laboratories usually estimate the time of copulation as the middle time point of the dark part of the husbandry room’s daily illumination cycle. For example, if the room is dark from 7 pm to 7 am, one would estimate that matings occurred at 1 am.

The position of the plug can range from being very deep in the vagina to very superficial. The color of the plug can vary from somewhat translucent to opaque and from white to yellow. Copulation plugs are hard and have a rough pebbled or ground glass–like external surface. They should not be confused with the soft, sloughed, cornified epithelium that is present in the vagina at specific stages of the estrous cycle.

To visualize the plug well, it is recommended that the mouse be picked up by the tail and examined in a vertical position, rather than examined in her natural, horizontal standing position. To assess the texture of the plug and to ensure that it is not placed deeply against the cervix, use of a round-edged microspeculum is always recommended. It is important to clean the speculum between mice to prevent the spread of microorganisms. Sterile saline or water is recommended for this since contact with chemical disinfectants, including alcohol, is highly irritating to the delicate vaginal mucosa.

Plugs are easier to observe in some strains than in others. Illustrated in this plate is a CD1 female that we estimate was mated by a CD1 male 10 h before she was photographed.

(A) An unassisted view of the vagina showing the copulation plug. The plugged vagina lies between the rectum dorsally and the vulva ventrally.

(B) Visualization of the plug is improved if gentle downward pressure is applied to the sacral region of the pelvic girdle. This projects the vagina and vulva caudally and dorsally to improve visualization of their contents.

(C) Use of a speculum is important. Microweighing spatulas with smooth round ends are highly suitable and need to be wiped between animals with a sterile solution that is nonirritating to the vaginal mucosa. The depth that the speculum can be inserted into the vagina is one component of the mating assessment. If it inserts deeply to the firm cervix, no mating occurred. If it inserts deeply to a rough, gritty-textured surface, there is a deep plug. If only shallow insertion is possible, a plug is present. The speculum is also used to assess the surface of the suspected copulation plug for roughness and hardness. These features distinguish the plug from the softer, postestrous, white, cornified, shed epithelium. Finally, the speculum can be used to retract the edge of the vulva, to improve visualization of the plug.

After about 2 weeks, it is important to observe females who were paired with males but showed no copulation plug for external signs of pregnancy. This is often called a missed plug. Pregnant missed plugs should be very infrequent in well-managed colonies. Mating in some mouse species, such as Mus caroli, does not produce copulation plugs. For these matings, a vaginal smear is conducted (see Plate 1) within 12 h of the estimated mating time. The smear is examined for the presence of sperm. Sperm can be recognized in unstained wet mounts. A microscope in the breeding room is most helpful in the management of timed matings for these types of mice.

Plate 3 Physical changes in body appearance during a mouse pregnancy—dorsal view.

(A) Normal, unmated, young adult female mouse.

(B) Gestational day 7.5. This stage of gestation is not recognized grossly. The imaged mouse was carrying a litter of 15 conceptuses.

(C) Gestational day 15.5. This stage of gestation is easily recognized by an increase in abdominal size. The conceptuses can also be detected by abdominal palpation. The imaged mouse was carrying a litter of eight conceptuses.

(D) Term pregnancy at gestational day 19.5. In late pregnancy, the gravid uterus occupies most of the abdomen, and the conceptuses create irregular distortions of the abdominal wall. The imaged mouse was carrying a litter of nine conceptuses. Studies using continuous radiotelemetric recording have shown that physical activity levels decrease in pregnant mice between gestational days 12.5 and term.⁴ The same animals are depicted in Plates 3 and 4.

Plate 4 Physical changes in body appearance during a mouse pregnancy—ventral view.

(A) Normal, unmated, young female mouse. Three pairs of thoracic (more cranial) and two pairs of inguinal (more caudal) mammary gland teats (nipples) are distinctly visible in the virgin mouse.

(B) Gestational day 7.5. This stage of gestation is not recognized grossly. The imaged mouse was carrying a litter of 15 conceptuses.

(C) Gestational day 15.5. This stage of gestation is easily recognized by an increase in abdominal size. Pregnancy can also be detected by abdominal palpation of the implantation sites. The imaged mouse was carrying a litter of eight implantation sites.

(D) Term pregnancy at gestational day 19.5. In late pregnancy, the gravid uterus occupies most of the abdomen, and each implantation site is large. The imaged mouse was carrying a litter of nine. The same animals are depicted in Plates 3 and 4.

Plate 5 The pregnant mouse abdomen, gestational days 5.5–18.5—ventral view: abdominal wall intact (upper row: A–D) and abdominal wall opened (lower row: E–H).

The same mice are imaged in each row. In the upper row (A–D), the abdominal wall is intact. In the lower row (E–H), the abdominal wall has been opened. In all images, the same anatomical landmarks are present. In (A–D), the central linea alba (LA) runs axially in the ventral abdominal wall. Right and left superior epigastric blood vessels (SE) run parallel to the linea alba. These vessels enlarge over pregnancy to support mammary gland development. In (A–H), the xyphoid process (sternum), costal arch (ribs), and liver mark the thoracic–abdominal transition.

(A and E) Gestational day 5.5. Intestinal tract and inguinal fat pads (IF) cover the uterus. Up to this day of gestation, it is difficult to determine that a mouse is pregnant by gross assessment of the abdomen or uterus. Implantation sites become distinctly visible in the mouse uterus at gestational day 6.5. The imaged mouse was carrying a litter of nine conceptuses. Dye injection prior to euthanasia is commonly used at this stage of pregnancy to aid in the identification and localization of implantation sites (picture (E), Plate 8). The implantation sites are revealed as regions of dye leakage, reflecting increased capillary permeability where the blastocysts have attached.¹

(B and F) Gestational day 7.5. At this stage of pregnancy, inguinal fat pads and intestine cover the uterus. The imaged mouse was carrying a litter of 15 implantation sites.

(C and G) Gestational day 14.5. By this stage of pregnancy, the gravid uterus has distended the abdomen and displaced the inguinal fat pads and intestinal tract. The uterus is clearly visible before the abdominal wall is incised. The imaged mouse was carrying a litter of 12 implantation sites.

(D and H) Gestational day 18.5. In late pregnancy, one day before expected birth, the gravid uterus occupies most of the abdominal cavity, displacing all other organs. The abdominal wall is greatly distended (D), and its tension restrains the uterus. The uterus spills out of the abdomen upon its incision. The imaged mouse was carrying a litter of six conceptuses.

Plate 6 Ventral abdomen of the mouse during pregnancy, with the abdominal wall removed and the gravid uterus reflected caudally to show typical changes in appearance of the uterine vessels over pregnancy.

(A) Gestational day 5.5. The uterine arteries arise from two vessels: cranially from the ovarian arteries and caudally from the cranial vesical arteries, which arise from the internal iliac arteries. The uterine arteries course through the mesometrium (uterine suspensory ligament) parallel to and along the full length of each uterine horn. Arcuate arteries branch from the uterine arteries in a relatively uniform pattern to supply the uterus. These arteries give rise to radial arteries that penetrate through the uterine wall. To enable uterine reflection at this early gestational stage, the attachments at the ovarian ends were cut. The ovaries (red) are present in the fat pads at the end of the reflected uterine horns. Uterine contraction due to death and drop in body temperature can mimic implantation sites. Implantation sites cannot always be accurately counted by gross examination at this gestational stage.

(B) Gestational day 7.5. In this pregnancy, containing 15 conceptuses, enlargement of the uterine vascular supply has become obvious.

(C) Gestational day 14.5. In this pregnancy, containing 12 conceptuses, the positions of the arcuate arteries relative to the decidua (white and proximal to arteries) and the placenta (pink over the deciduas), and the fetal positions (bulges antimesometrially), are apparent.

(D) Gestational day 18.5. In late pregnancy, the vessels to the gravid uterus are dilated and turgid with blood. In most cases, each artery is accompanied by the corresponding vein. In these photographs, only veins are visible. Each uterine artery arises from two sites. One end arises from the left or right ovarian artery, which branches from the abdominal aorta toward the upper part of the image shown. The other end branches from the cranial vesical artery, which arises from the internal iliac artery near the middle of the image shown. Blood flowing from this end travels caudally and then cranially as the vessels curve to begin their supply of the uterus and of the placentas that are nearest to the cervix (Chapter 31). The blood-filled placentas are clearly visible in their in situ positions between the maternal blood vessels and each fetus. Residual decidua basalis is seen over each placenta as a small whiter area positioned centrally on the mesometrial facing side of the placenta. The arcuate arteries are not uniform in width. Wider arcuate arteries supply the implantation sites. Narrower arcuate arteries supply the uterine wall between implantation sites. Fetuses are now much larger than their placentas. The imaged mouse was carrying a litter of six.

Plate 7 Birth at gestational day 19.5.

The same mouse is imaged in each panel of this plate. It was only during the dissection that the position of the first fetus in the dilated cervix (birth canal) was noted, indicating that parturition had commenced in this female.

(A) Ventral abdomen of a peripartum mouse with the abdominal wall opened; organs in situ. The gravid uterus with nine conceptuses obscures most of the other abdominal organs. A small piece of intestinal tract is visible between the two uterine horns. The liver is pushed cranially relative to a nonpregnant abdomen. The urinary bladder is visible. Upon dissection, fetus #1 (arrow) was observed to be vertically aligned along the midline and present in the dilated cervix (birth canal).

(B) Ventral abdomen of the peripartum mouse with the uterus withdrawn cranially. Lateral to the urinary bladder, the trimmed edges of the abdominal wall are visible. The head of fetus #1 (arrow) has entered the dilated cervix. The bony pelvic girdle is intact in this image.

(C) Ventral abdomen of the peripartum mouse with the pelvic floor removed and the uterus retracted caudally to show the birth canal. The uterine vessels are prominent as are fetal details such as tails and digits. The relative size of each fetus to its placenta should be noted as should the white remnant of the decidua basalis over the red placenta. The head of fetus #1 (arrow) has entered the dilated cervix.

(D) The dissected peripartum uterus shows the fur of the perineum surrounding the vulva and vagina. The head of fetus #1 (arrow) is clearly in the uterine body and cervix, while the more caudal parts of the fetus remain in the uterine horn.

Plate 8 Appearance of the cycling and early postimplantation uterus (gestational days 4.5–6.5).

A series of uterine horns is shown to illustrate progressive changes in the appearance of the uterus over the estrous cycle (A and B) and on gestational days 4.5–6.5 following implantation (C–F). (E) A uterus from a mouse at gestational day 5.5 who received an intravenous injection of Chicago Sky Blue 6B dye (Sigma Chemical Co., Oakville, ON, Canada) solution prior to euthanasia to illustrate the enhanced visualization of implantation sites provided by this technique (compare D to E). The ovary is placed uppermost in each image, the cervical region is toward the bottom, and the mesentery showing a uterine vessel and its branches is placed to the left of each horn. Red corpora lutea are present in the ovaries early postimplantation (C, D, and F).

(A) The diestrous uterine horn is long and narrow. The stage of estrus of this donor mouse was confirmed by use of a vaginal smear (see Plate 1).

(B) The estrous uterine horn is noticeably swollen. Its wall is thin and pale in color. The uterus can be quite turgid and give the impression of being rigid. It is usually shorter in length than a nonestrous horn. The stage of estrus of this donor mouse was confirmed by use of a vaginal smear (see Plate 1).

(C) Implantation sites are not visible to the naked eye at gestational day 4.5.

(D) Although implantation site swelling may be apparent at gestational day 5.5, postmortem contraction of the uterus at room temperature or if placed into a chilled medium can give a very similar appearance.

(E) Because it is difficult to visualize early implantation sites, injection of dye just prior to euthanasia can be used to aid in implantation site localization. As implantation is associated with neoangiogenesis and vascular leakage, dye spots will indicate the implantation sites. The donor of this gestational day 5.5 uterus received an external jugular vein injection of 0.2 ml of 10% (wt/vol) Chicago Sky Blue 6B solution in saline 2 min before euthanasia. Five darker blue sites can be identified between the ovary and cervix. Note the branching pattern of the uterine arteries in the mesentery.

(F) Individual implantation sites are clearly visible by gestational day 6.5. This is the earliest stage that can be dissected, as described for gestational day 7.5 in Plate 1 in Chapter 2.

Plate 9 Appearance of the gravid uterus from gestational days 7.5–12.5.

A series of uterine horns is shown to illustrate progressive changes in the appearance of the uterus between gestational days 7.5 and 12.5. (A–C) The stages during which development of the placenta occurs. (D–F) The earliest stages with an anatomically complete and functioning placenta. The ovary is placed uppermost in each image, the cervical region is toward the bottom, and the mesentery containing the uterine vessels is placed to the left of each horn.

(A) Gestational day 7.5 implantation sites are oval in shape and contain a cylindrically shaped, primitive streak embryo that is supported by invading trophoblasts and other membranes. Mesometrial trophoblasts, which are the primordia of the placenta, form the ectoplacental cone. Ectoplacental cone invasion results in a hemorrhagic center within each implantation site. This embryonic crypt is not usually seen through the uterine wall, but hyperemia of the mesometrial side of the uterus is usually detectable.

(B) Gestational day 8.5 implantation sites have a distinct round appearance and contain an embryo that has become globular in shape as the somites differentiate, the allantois grows toward the placental primordium, and the embryo rotates. The uterine arteries are prominent and their branches to implantation sites are wider than their branches to uterine tissue between implantation sites. The mesometrial sides of the implantation sites are paler than the antimesometrial sides. This color gradation aids in orientation during dissection. The most hyperemic area is central and is thought to represent the limit of trophoblast invasion. The decidua basalis gives the paler color since it persists while the antimesometrial decidua regresses.

(C) Gestational day 9.5 implantation sites show continued enlargement, with little or no space now remaining between adjacent sites in horns holding large litters. Prominent pale regions can be recognized through the uterine wall where maternal vessels enter and egress. This represents the initial development of a transient leukocyte-enriched area that develops between the circular and longitudinal smooth muscle layers at each implantation site.

(D) Gestational day 10.5 implantation sites are larger. The placenta now has a functional blood supply.

(E) Gestational day 11.5 implantation sites show continuing growth of both the placenta and the fetus. The placental position is now clearly apparent from external examination of the uterus.

(F) Gestational day 12.5 implantation sites show increasing size of the fetus and placenta. The placenta appears darker red in color as gestation continues.

Plate 10 Appearance of the gravid uterus from gestational days 13.5–18.5.

A series of uterine horns is shown to illustrate progressive changes in the appearance of the uterus between gestational days 13.5 and 18.5 (A–F). The placenta is mature in all of these images. Fetal growth continues rapidly while increases in the overall size of the placenta are limited. Thus, the placenta-to-fetus ratio decreases during the second half of mouse pregnancy. Over these gestational days, the placental labyrinthine region enlarges while the decidua basalis regresses. The uterine wall becomes very thin due to the increasing size of each implantation site. Numerous fetal details, such as tails and limbs, are visible without incision of the uterine wall. During dissections to remove the uterine wall, it is very easy to nick the amniotic cavity, and it drains quickly due to the contractile force of the uterine wall. The ovary is placed uppermost in each image, the cervical region is toward the bottom, and the mesentery containing the uterine vessels is placed to the left of each horn.

(A) A gestational day 13.5 uterine horn containing eight implantation sites.

(B) A gestational day 14.5 uterine horn containing five viable implantation sites. A sixth implantation site proximal to the cervix is in an advanced state of resorption. Sporadic failure of implantation sites is seen occasionally in most strains. In normal mice, this usually does not compromise the rest of the litter. The gain in fetal length is now obvious along an axis parallel to the length of the uterine horn.

(C) A gestational day 15.5 uterine horn containing seven implantation sites. Crowding that rotates some fetuses relative to others is seen.

(D) A gestational day 16.5 uterine horn containing eight implantation sites.

(E) A gestational day 17.5 uterine horn containing seven implantation sites. Crowding due to fetal growth becomes more evident. Vessels overlying the fetuses are not maternally derived and are not in the uterine wall. Rather, they are conceptus derived and are in the fetal membranes. The gaining vascularity of the placental labyrinth is evident from the darker red color of the placenta seen through the uterine wall.

(F) A gestational day 18.5 uterine horn containing three implantation sites.

Plate 11 The postpartum uterus.

If a fertile male mouse is present in the cage of a pregnant female and she delivers, she may be mated during the postpartum estrus that occurs within 24 h of giving birth. In the upper row (A), a mouse uterus estimated to be at 10 h postpartum is depicted. In the lower row (B), a mouse uterus estimated to be at 48 h postpartum is depicted. The latter animal was not housed with a male.

(A) The dorsal view of the involuting uterus 10 h following delivery of a litter of 11 viable pups. Prominent longitudinal muscle banding is seen along the uterine horns. The attachment sites of each placenta remain distinct as a series of small, dark red mesometrial marks. The uterus remains swollen.

(A-i) and (A-ii) The lateral views of the horns of the same uterus shown in (A) and their vasculature.

(B) The dorsal view of the involuting uterus 48 h following delivery of a litter of 11 viable pups. The uterine horns have changed considerably during the 48 h following parturition, except at the sites of former placental attachment. These remain distinct but much less prominent than in (A). The placental attachment sites have turned gray-greenish, indicating normal uterine tissue regeneration.

(B-i) and (B-ii). The lateral views of the horns of the same uterus shown in (B) and their vasculature. Note that the ovaries in each image show several bright red corpora hemorrhagica indicative of the typical postpartum ovulation seen in mice.

Plate 12 Appearance of implantation sites early in the process of fetal growth retardation and implantation site failure.

Visual gross assessment of the pregnant uterus of a normal mouse will reveal whether there is heterogeneity in size or color between implantation sites. Sites that are smaller and/or paler are likely abnormal and have a very high probability of resorption before the remainder of the litter is born. This is difficult to establish up to about gestational day 7.5. From gestational day 8.5 to term, it is an easier and important observation to make. One or more implantation sites in either or both horns can be involved. It is only by euthanasia and study of the gestational uterus or ultrasound that postimplantation pregnancy failure can be advanced as an explanation for small litters at birth or long intervals between litters in breeding pairs. Collection of uteri at the stages shown in this Plate should permit recovery of sufficient conceptus material for genotyping or for informative histology. This is particularly important for the development of new, modified mouse strains.

(A) Uterine horn at gestational day 9.5 showing two resorbing (arrow) and four viable implantation sites.

(B) Uterine horn at gestational day 10.5 showing one resorbing (arrow) and four viable implantation sites.

(C) Uterine horn at gestational day 10.5 showing three resorbing (arrow) and four viable implantation sites.

(D) Uterine horn at gestational day 11.5 showing four resorbing (arrow) and two viable implantation sites.

(E) Uterine horn at gestational day 11.5 showing four resorbing (arrow) and three viable implantation sites.

(F) Uterine horn at gestational day 11.5 showing two resorbing (arrow) and two viable implantation sites.

Plate 13 Appearance of implantation sites late in the process of fetal death and implantation site resorption.

Visual gross assessment of the pregnant uterus of a normal mouse will reveal whether large or small hemorrhagic-to-black implantation sites are among a litter. This observation is easiest to make after gestational day 7.5, when the antimesometrial decidua begins to thin and the growth of fetuses causes stretching and thinning of the uterine wall. Sites that are hemorrhagic-to-black indicate the death of the fetus and the process of implantation site resorption. If large fetuses of healthy color remain in the uterus, they will likely be carried to term despite the death of one or more members of the litter. While euthanasia and study of the gestational uterus permit investigators to score resorbed versus viable implantation sites at a specific date of study, meaningful histological or genetic information is difficult to obtain from sites of advanced failure.

(A) Uterine horn at gestational day 11.5 showing three resorbing (arrow) and three viable implantation sites.

(B) Uterine horn at gestational day 12.5 showing four resorbing (arrow) and three viable implantation sites.

(C) Uterine horn at gestational day 12.5 showing three resorbing (arrow) and three viable implantation sites.

(D) Uterine horn at gestational day 15.5 showing one resorbing (arrow) and two viable implantation sites.

(E) Uterine horn at gestational day 13.5 showing two resorbing (arrow) and one viable implantation sites.

(F) Uterine horn at gestational day 17.5 showing two resorbing (arrow) and three viable implantation sites.

Pregnant females were anesthetized using sodium pentobarbital (40 mg kg−1) for photography prior to euthanasia by cervical dislocation for dissection. Use of these mice for preparation of this Atlas was approved by the Animal Care Committee, Queen’s University.

References

1. Nagy A, Gertsenstein M, Vintersten K, Behringer R. Manipulating the mouse embryo A laboratory manual. 3rd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2003.

2. Coan PM, Ferguson-Smith AC, Burton GJ. Developmental dynamics of the definitive mouse placenta assessed by stereology. Biol Reprod. 2004;70(6):1806–1813.

3. Kaufmann MH. The atlas of mouse development. Revised edition London, UK: Elsevier Limited; 1992.

4. Burke SD, Barrette VF, Bianco J, et al. Spiral arterial remodeling is not essential for normal blood pressure regulation in pregnant mice. Hypertension. 2010;55(3):729–737.

2

Implant Site Dissections

Stephen C. Pang, Judith Janzen-Pang, M. Yat Tse and Anne Croy,    Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada

Denise Tse,    Denise Tse Graphic Design, Kingston, ON, Canada

Chapter Summary

Between implantation of the blastocyst and birth of the neonate, change is continuous in normal implantation sites. This dynamic period of change has been very well characterized by embryologists as the developmental continuum of the embryo and fetus, but less thorough attention has been given to mapping the patterns of change in the uterus and in the extraembryonic membranes, including the placenta. Publications addressing uterine and placental changes are often limited to specific days of study or focus on a single feature of the implant site such as its vascular development, immune cell populations, or the relative ratios of one component of the placenta to another. While there can be variability between strains, overall patterns of developmental change characterize the mouse uterus and the extraembryonic membranes of conceptuses over pregnancy. These changes are briefly discussed and then illustrated at increments of 24 h using photomicrographs of dissected maternal and conceptus-derived implantation site tissues from random-bred mice between gestation day 6.5 and parturition on gestation day 19.5.

Keywords

Decidua; Embryonic crypt; Embryo; Fetus; Mesometrial lymphoid aggregate of pregnancy (MLAp); Myometrium; Placenta; Pregnancy; Umbilical cord; Uterus

Abbreviations

MLAp Mesometrial lymphoid aggregate of pregnancy. This term continues to be debated particularly by reproductive immunologists. It refers to a non-encapsulated, immune cell rich, transient tissue that develops in the muscular layers of the mouse uterine wall about mid pregnancy. One MLAp develops at each implant site at the point of contact of the decidual capsule with the uterine wall

uNK cell uterine Natural Killer lymphocyte, a specialized lineage differentiation dependent upon uterine stromal decidualization

Introduction

The earliest gross appearance of mouse implant sites could be called round, but by gestation day (gd) 6.5 (counting from the copulation plug as gd 0.5), when the implant sites visibly stretch across the uterine wall, oval to pear shaped would be more appropriate. The tissue that makes the early implant site visible to the naked eye is primarily maternal decidua. The blastocyst, which implants on the antimesometrial side of the uterus, initiates antimesometrial primary decidua formation. This rapidly induces secondary decidua on the antimesometrial side and the progressive development of lateral and mesometrial secondary decidual regions. The secondary decidua produces the observable uterine swellings and encloses a relatively tiny conceptus¹. Decidua forms only at implant sites; regions of nondecidualized endometrium are present between each decidualized region. This gives the early pregnant uterus an appearance of beads threaded onto a string.

From gd 6.5 to 9.5, elongation of the implant site occurs primarily in a direction transverse to the length of the uterine horns. The conceptus is changing shape from a narrow cylinder to a globe. Thus, radial expansion of the implant sites is seen and space between implant sites diminishes. It is important to recognize that even within litters, the development of each implant is not synchronous.¹ Simultaneously with decidualization, other structural changes occur in the myometrium and uterine mucosa. The most noticeable of these are development of the mesometrial lymphoid aggregate of pregnancy (MLAp) and increased prominence of blood vessels. The MLAp is a hard white structure seen in the uterine wall on the mesometrial side of each implant site from gd 8.5. The MLAp develops between the two layers of the myometrium. This site that was previously known as the metrial gland² is recognized histologically to contain large numbers of immature-stage lymphocytes called uterine natural killer (uNK) cells, which are unique to decidual tissue. Dissection of preplacental-stage implant sites is a skill that requires practice for proficiency and patience for success. These gd 6.5–9.5 implantation site dissections are usually conducted with the aid of a dissection microscope.

Following the preplacental stages, when gastrulation, onset of organogenesis, turning of the embryo, and fusion of the allantois to the chorion are occurring, the major direction of growth of the implant site is shifted by 90°. This is observable from gd 10.5 as implantation site enlargement parallel to the long axis of the uterine horns. Dominance of this direction continues until birth. Between gd 10.5 and term, implant sites are easier to dissect than at preplacental stages because they are less fragile and their structural elements are distinctly visible. In addition, the major changes that now occur are those of growth rather than major changes in structure. Additionally, blood-filled fetal vessels become clearly visible red structures that provide dissection landmarks. Fetal growth that stretches and thins the uterine wall in combination with regression of the decidua makes many features of interest for dissection visible before incision of the uterus. The size of the fetus and amniotic cavity become dominant over the size of the placenta between midpregnancy and birth. The initial steps in dissection of implantation sites containing fetuses with placentae are usually conducted without the aid of a dissection microscope. The dissection microscope remains important for isolation of subcomponents of the placenta, insertion of cannulas for perfusion or casting compounds, and in vivo studies of the circulation, as described in Chapters 20, 30 and 62.

Dissection of Gestation Day 6.5–9.5 Uteri and Implant Sites

Prior to gd 6.5, dissection of implant sites is not routinely practiced due to their small size and fragility and due to difficulties in their visual identification. As described in Chapter 51, uterine horns that are separated at the cervix and then cut transversely at their midpoint can be cut into two lateral halves to successfully reveal gd 4.5 implant sites. Successful access to the implantation site is rather hit or miss, subject to rotational deviations during the dissection and without landmarks to the implant site since prior intravenous dye injection to reveal vascular leakage at implant sites would cause unacceptable autofluorescence in the whole-mount assay. Fixation of the intact uterine horn followed by histological processing and sectioning is a more routine approach than implant site dissection for studies of gd 4.5–5.5 uteri. At gd 5.5, using a dissection microscope, we were more successful in opening the embryonic crypts with a cut longitudinal to the uterine horn, but the sites were too fragile to make any attempt at further dissection such as removal of the decidua. These obstacles are not present from gd 6.5.

Gestation day 6.5 implant site dissections are very similar to those at gd 7.5, but the latter are easier due to their larger size. To gain skill in recovery of the ectoplacental cone or in removal of the extraembryonic region from these primitive streak staged embryos, it is strongly recommended that investigators begin with dissections of gd 7.5. Once proficiency and confidence have been achieved, the gd 6.5 conceptuses will be much less challenging. Care, gentleness, and patience are needed to not crush materials. Very sharp forceps (#5 watchmakers’ forceps are commonly used) are also essential. They should not have been damaged in ways that make one arm shorter than the other or that make one point slightly curled. They should not have been sharpened to the extent that the points are wide, and they should not be fatigued so that the points cross. Striking forceps tips against the dissection microscope itself is sufficient to require a pause for dissection instrument repair. We also recommend that a large volume of saline and numerous dissection dishes be available. This permits movement of specimens into clean medium during the dissection process to keep a very clear field of vision. In sum, when you keep your instruments in excellent condition and your field of vision unobstructed, dissections of early mouse implantation sites will not be difficult.

There are two different ways that gd 7.5 implant sites can be approached (Plates 1, 2, and 3). One way is to cut between each implant site and then dissect each site individually (Plate 3). This is the best way to learn the dissection, but it is more time consuming than opening the full length of the horn (Plates 1(A), 1(B), and 2(A), 2(B)) and then recovering the decidual capsules that surround each conceptus. For either approach, the dissector requires two pairs of very sharp #5 watchmaker’s forceps. One is used for holding tissue (for right-handed individuals, this is the forceps held in the left hand), while the second pair of forceps is rotated at 90° to the holding position and used like a pair of scissors. Very, very fine scissors (such as Vannas design) may be substituted for the first cut (see Plate 3(G)) made by the latter forceps, but they are unnecessary and must be replaced by forceps held in the right hand after the first incision is made.

Plate 1 Appearance of dissected implantation sites at gestation day (gd) 6.5.

A series of photographs illustrate the appearance of implantation sites at gd 6.5. Three consecutive implantation sites are enclosed within a segment of the uterine horn. The line of mesenteric attachment is uppermost on the right implantation site and spirals across the image to the midpoint of the left implantation site (A). One implantation site from a transected uterine horn is isolated for further dissection; the position of the developing implantation site is visible as a granulated red area seen through the uterine wall (B). As the uterine horn is cut and folded back, three consecutive decidual capsules are seen, with the attached uterine wall forming a ring-like structure uppermost in the image (C). The embryonic crypt is visible in this photograph as a central red streak when the decidual capsule is bisected. The primitive streak embryo is transparent and encircled by the most antimesometrial red tissue (D). Note the thick, dense, antimesometrial decidua in comparison to the less dense, still-developing mesometrial decidua basalis.

Plate 2 Appearance of dissected implantation sites at gestation day (gd) 7.5.

A series of photographs illustrate the appearance of implantation sites at gd 7.5. Three consecutive implantation sites are encased within a segment of the uterine horn (A). As the uterine horn is cut open at its antimesometrial edge and folded back, two adjacent implantation sites (decidual capsules) are seen, with the uterine tissue uppermost in the image. The uterine wall can be further retraced to permit the removal of intact decidual capsules (B). Alternatively, most of the uterine tissue can be trimmed away and the remaining tissue transected between implantation sites. This provides isolated implantation sites with mesometrial tissue (at the top) and a decidual capsule (at the bottom), as illustrated in (C). The developing embryo is seen in (D) as a central red streak when the decidual capsule and uterine wall remnant are cut parasagittally (slightly off-center). The transparent embryo is in the decidua to the left side of the image, and the hemorrhagic edge of the embryonic crypt is in the decidua to the right side of the image.

Plate 3 Alternate methods for dissection of the gestational day (gd) 7.5 mouse implantation site.

As illustrated in (A) and (B), an individual implantation site can be isolated by making two vertical cuts across the short axis of the uterine horn. An implantation site isolated by this method is illustrated in (C) and (D). The dashed lines along the antimesometrial aspect of the uterine horns in (A) and (C) indicate the line of incision along the long axis of the uterine horn to expose decidual capsules. Retraction of the cut edges over the capsule permits visualization of the decidual capsule attachment site to the myometrium. Blue arrows illustrate the directions of folding of the cut uterine wall (E). Four isolated decidual capsules are shown in (F). The part of the decidual capsule attached to the mesometrium is slightly wider than the antimesometrial tip. A vertical cut (blue line in (G)) can be made slightly off-center of the decidual capsule to expose the embryo ((G) and to the left capsule half in (H)). Alternatively, a horizontal cut (blue line in (I)) can be made to isolate the decidua basalis from the placental primordium, embryo, and rest of the decidual capsule (J). The mesometrial end of the red streak that defines the embryonic crypt is the site for placement of the cut that isolates only the decidua basalis.

One difficulty often experienced during first attempts at these dissections arises from the use of too much medium (usually saline). While you do not want the tissue to dry out, it is impossible to dissect it if it is floating! We find that the use of three small Petri dishes works well. In the first, a large volume of medium is present, and the dish contains the uterine horns, usually divided at the cervix. The second dish is essentially dry and is used for transection of the uterine horn into individual implant sites if this approach is used or to open the uterine horn along the antimesometrial surface. Dampness of the tissue is maintained by carryover medium only. The third dish holds sufficient medium to float the dissected materials as they are prepared. We additionally find that for some dissections, the dry dish is a better platform if it is inverted and the tissue is dissected on the bottom of the dish. The reason for this is that there is no dish edge, and cuts with scalpels or razor blades are not impeded by the wall of the dish getting in the way of the dissector’s motions.

Whether a single implant site or the length of the uterine horn is being dissected, the initial cut is along the antimesometrial surface, as illustrated in Plates 3(A) and 3(C) (dashed lines). For studies that are the focus of this book, the tissues of interest are on the mesometrial side. The holding forceps are placed on the myometrium to avoid damage internally within the implantation site, and the cutting instrument is inserted into the small circular opening of the uterus, where the horn was removed from the cervix or the uterus was transected across the nondecidualized, interimplant site. This may sound easy but is likely the most important part of a successful dissection. This is because timid insertion leads to shedding the layers of the uterine wall because the antimesometrial cut is not deep enough, whereas excessive insertion will damage the decidual capsule. This dissection is done under the magnification of a dissection microscope, so be sure to grasp the full thickness of the antimesometrial muscle, which is clearly visible. We then release the left forceps, bring it beside the right one, and take a grasp of the full thickness of the uterine wall. By gently retracting both instruments in opposite directions, the uterine wall will tear open. The forceps are then advanced one at a time (the second one is used for holding and stabilization) forward along the cut, and when the forceps are again opposite each other in the new position, the next retraction in opposite directions is completed. This procedure is repeated until the entire length of the antimesometrial wall musculature has been opened.

Because the uterine musculature is contractile and now at a temperature lower than physiological, it contracts away from the antimesometrial incision line, exposing the decidual capsule around each conceptus. Using the left forceps again as a holding instrument, the right forceps are placed along the internal mesometrial surface of the uterus, and the decidual capsule is gently brushed away from its site of slight attachment to the uterus (see Plate 3(E) for illustration, and Plates 1(C) and 2(B) for the appearance expected through this stage of dissection). This attachment area, which is the junction between the decidua basalis and inner circular smooth muscle and the location of transit for the maternal blood supply to the implant site, is often the area of interest for the research question. Thus, care should be exercised not to damage tissue relationships in this region. Plates 2(C), 3(F), 4(B), and 4(C) show the resulting individual, intact decidual capsules with or without a small region of retained uterine wall. Retention of the tag of the uterine wall can be an important landmark in certain studies.

Plate 4 Appearance of dissected implantation sites at gestation day (gd) 8.5.

A series of photographs illustrate the appearance of implantation sites at gd 8.5. An isolated implantation site together with the uterine wall is shown in (A). Blood vessels of the uterine wall are prominent. After opening the uterine horn along its antimesometrial edge and retracting its wall mesometrially, an isolated implantation site is obtained, as illustrated in (B). A lighter area at the base of the developing placenta (∗) is the site of development for the mesometrial lymphoid aggregate of pregnancy (MLAp). Uterine wall tissue is attached mesometrially to the decidual capsule at the site of MLAp development in (B) and has been removed in (C). The red areas in (B) and (C) represent healthy development of decidual blood vessels. The embryo, seen before development of its cardiovascular system (blue arrow), is viewed through incision of the antimesometrial decidua. The embryo is located above the ectoplacental cone region of trophoblast that is exposed as the red area in (E), but is not dissected in (D) and remains surrounded by maternal decidua basalis. The maternal surface of the developing placenta is shown in (F), whereas the fetal surface is shown in (G). A vertical cut is made at the center of the developing placenta. The placenta is viewed at the cut surface with the maternal surface at the top and fetal surface at the bottom (H).

Next, the dissected decidual capsules (that should now be in a dish of clean medium) are opened. Again, using a dry inverted Petri dish and placing the newly dissected materials into dishes of fresh medium are recommended. When the gd 7.5 or gd 6.5 decidual capsule is examined, two features provide anatomic orientation. One is shape, and the other is color. The structure is pear shaped, with one end, the mesometrial end, slightly wider and with fuzzy edges. In contrast, the antimesometrial side is narrower and has a distinct smooth edge. The color gradient is similarly distributed, with the mesometrial end being relatively paler than the antimesometrial end. For the authors, who are right-handed, the decidual capsule is oriented transversely across the field of the dissection microscope with the mesometrial side to the left and the antimesometrial side to the right. Using well-sharpened #5 watchmaker’s forceps, the mesometrial end is stabilized by placing spread forceps tips on the lateral bottom edges of the mesometrial decidua. This somewhat flattens the decidual capsule and in healthy implant sites, brings into prominence and focus a vivid red line running from the antimesometrial to mesometrial regions in the center of the capsule. The red region is the embryonic implantation chamber, and it is completely surrounded by decidua. For many applications, such as a decidual genome expression study, or to recover conceptus components, the conceptus-derived material needs to be removed by further dissection.

To remove the conceptus, the right-hand forceps are again rotated 90° to the stabilizing left-hand forceps and used for cutting. A shortcut is made through the entire decidual thickness at the mesometrial pole. This gives access to the right forceps to now cut parasagittally to the red line but only on the upper layer of the decidual capsule (Plate 3(G)). The cut must extend through the mesometrial pole of the decidua. The right forceps is then rotated back to a