Endothelial cell hyperproliferation and stratification in uteroplacental blood vessels of the black mastiff bat, Molossus rufus.

Placentation was studied histologically and immunocytochemically in black mastiff bats obtained at frequent intervals throughout pregnancy. These were bred in a captive colony or collected from a reproductively-synchronized wild population. During late pregnancy, the single fetus was largely sustained by a discoidal, hemochorial placenta located at the cranial end of the right uterine horn. This invariant positioning was determined by a vascular tuft that developed there both during early pregnancy and non-pregnant cycles. This provided a scaffold for early placental morphogenesis. As development proceeded, small arterioles and venules serving the tuft were converted to large uteroplacental vessels. Within the base of the placenta, these became lined by an unusual vascular epithelium composed of intermingled patches of multilayered endothelial cells and cytotrophoblast. Initially, the endothelium became multilayered by hypertrophy, proliferation, and infolding of its basal lamina. These created endothelial bilayers usually insinuated between basal laminae. The development of temporary gaps in the laminae then permitted further enlargement of the vessels and proliferation of the endothelial cells as monolayer sheets or chains. The latter were interconnected, forming a complex, stratified, cellular network associated with a prominent meshwork of basal laminae. Throughout much of pregnancy, these endothelial cells were cuboidal to columnar and possessed an abundance of basal glycoprotein granules presumably containing basal lamina precursors. The cells also expressed vimentin and frequently von Willebrand factor, but not cytokeratins or desmin. Pronounced thickening of the endothelia and amplification of their basal laminae likely evolved to greatly strengthen the walls of the uteroplacental vessels.

Genetic regulation of mammalian diversity.

Mammals have evolved a variety of morphological adaptations that have allowed them to compete in their natural environments. The developmental genetic basis of this morphological diversity remains largely unknown. Bats are mammals that have the unique ability of powered flight. We have examined the molecular embryology of bats and investigated the developmental genetic basis for their highly derived limbs used for flight. Initially, we developed an embryo staging system for a model chiropteran, Carollia perspicillata, the short-tailed fruit bat that has subsequently been used for staging other bat species. Expression studies focusing on genes that regulate limb development indicate that there are similarities and differences between bats and mice. To determine the consequences of these expression differences, we have conducted an enhancer switch assay by gene targeting in mouse embryonic stem cells to create mice whose genes are regulated by bat sequences. Our studies indicate that cis-regulatory elements contribute to the morphological differences that have evolved among mammalian species.

Altered trophoblastic differentiation and increased trophoblastic invasiveness during delayed development in the short-tailed fruit bat, Carollia perspicillata.

During pregnancy in the short-tailed fruit bat, lengthy post-implantational delays in conceptus development can occur in response to stress in captivity and seasonally in the wild. When comparisons were made between uteri carrying embryos in delay at the primitive streak stage and those growing more rapidly, many differences were noted. During delay the developing chorioallantoic placenta was generally smaller, contained a higher ratio of cytotrophoblast to syncytiotrophoblast, and had been invaded only to a limited extent on its embryonic side by mesoderm. Furthermore, much of the cytotrophoblast appeared relatively undifferentiated, randomly-oriented, linked primarily by primitive junctions, and lacked a basal lamina. In contrast, in placentae serving somite and limb-bud stage embryos, sizeable areas were noted that consisted only of more highly differentiated syncytiotrophoblast perforated by maternal vascular spaces (trophospongium). The first contact of the allantois with the developing placenta was also noted at the somite stage, and this initiated widespread invasion of the placenta by mesenchyme and allantoic blood vessels. Wherever this invasion had occurred, the cytotrophoblast between the mesenchyme and syncytiotrophoblast of the interhaemal barrier consisted of a single, polarized layer of more differentiated cells with an associated basal lamina. Eventually, all of the trophospongium was invaded by cytotrophoblast and vascularized fetal mesenchyme. These observations suggest that in addition to its germinal function, cytotrophoblast in this bat may play a major role in controlling mesenchymal invasion and angiogenesis on the embryonic side of the placenta. During the period of delay, highly invasive trophoblast is also released by the placenta. This invades the myometrium and sometimes extrauterine tissues via interstitial migration along maternal capillaries and veins.

Comparative studies on limb morphogenesis in mice and bats: a functional genetic approach towards a molecular understanding of diversity in organ formation.

The basis of species-specific morphogenesis has been a topic of fascination and speculation for centuries. In 1828, Karl Ernst von Baer noted that at the pharyngula stage of development all vertebrate embryos are morphologically very similar. Most subsequent hypotheses have proposed that the vertebrate body plan develops by a conserved mechanism, and that divergent forms develop by differential elaboration on this basic plan. Gene cloning and expression studies have largely confirmed that the genetic pathways of embryonic patterning are highly conserved. The finding that the proteins encoded by paralogous and orthologous genes within and between species can functionally replace each another is no longer novel; in most cases this is the expected result. How, then, does divergent morphology arise between species? One hypothesis that fits well with comparative data is that divergent morphogenesis arises from genetic differences in the timing, level and pattern of orthologous gene expression during development. This idea is being tested using a functional genetic approach comparing limb morphogenesis between the mouse and bat.

Unusual patterns of intermediate filament protein expression by the trophoblast and decidual cells of the short-tailed fruit bat, Carollia perspicillata.

In the short-tailed fruit bat (Carollia perspicillata) pregnancy can be prolonged by the occurrence of lengthy delays after implantation. This is associated with the development of highly invasive trophoblast that can penetrate the myometrium, mesenteries of the reproductive tract and the oviducts via perivascular (interstitial) routes. In order to confirm the identity and distribution of this trophoblast, intermediate filament protein immunocytochemistry was utilized. In some respects the expression of these proteins differed from what has been reported for more commonly-studied species. Cytotrophoblast in the placenta, its cytotrophoblastic shell and the highly invasive trophoblast strongly expressed cytokeratins. As pregnancy progressed, however, cytokeratin expression by syncytiotrophoblast lining much of the placental labyrinth became very weak. The cytotrophoblastic shell and highly invasive trophoblast also expressed vimentin. The highly invasive trophoblast was unusual in that it developed dendritic processes that sometimes extended out into adjacent tissues in great profusion. Decidual cells generally expressed desmin and vimentin; however, some also coexpressed cytokeratins. These observations indicate that some of the trophoblast in Carollia undergoes a significant epithelial-mesenchymal transformation. They also suggest that caution should be exercised in relying upon intermediate filament proteins as markers for cell identification purposes in exotic species, or when the patterns of protein expression by fetal and maternal cells might be altered in pathological or experimental situations.

Advanced oviductal development, transport to the preferred implantation site, and attachment of the blastocyst in captive-bred, short-tailed fruit bats, Carollia perspicillata.

The final stages of embryonic development in the oviduct, transport of the embryo to the uterus, and the initial stages of implantation have been examined in captive-bred Carollia perspicillata at the light and electron microscopic levels. Development progressed to the expanded, zona pellucida-free, blastocyst stage in the oviduct. The abundance of microvilli on the exterior of the trophoblast varied with the degree of blastocyst expansion and cell shape, and may function in part as a membrane reservoir. Cells of the blastocyst also typically contained many lipid droplets and prominent areas of cytoplasm occupied by finely granular material (probably glycogen) instead of organelles. In most females, closure of the uterine lumen occurred prior to, or around the time of, transport of the blastocyst to the usual implantation site and appeared to play a role in preventing transport of the blastocyst too far distally in the uterus. This was associated with increased endometrial edema, particularly in the fundic region of the simplex uterus, and the extravasation of many erythrocytes into the endometrial stroma. Both of these changes began while the blastocyst was still being held in the oviduct and became pronounced during implantation. Engulfment of these erythrocytes by processes of the endometrial stromal cells and their phagocytosis by macrophages was also observed. Implantation was usually initiated within narrow tubular segments, lined by endometrium, that were located between the end of each oviduct and the main cavity of the uterus, or from immediately adjacent areas of the main cavity. During the early stages of implantation, the blastocyst was clasped by the endometrium at the implantation site, and this was associated with extensive interdigitation of the microvilli of the trophoblast and adjacent uterine epithelial cells. Initial adhesion of the trophoblast, which was still cellular rather than synctial, occurred over the apical intercellular junctions of the uterine epithelial cells.

Relationships between orientation of the blastocyst during implantation, position of the chorioallantoic placenta, and vascularization of the uterus in the noctilionoid bats Carollia perspicillata and Noctilio sp.

In most eutherian mammals, the inner cell mass (ICM) of the blastocyst assumes an almost constantly specific orientation to the uterus at the time of implantation, and this is usually correlated with subsequent positioning of the fetal membranes and chorioallantoic placenta. Although these relationships tend to be conserved between closely related species, this is not the case in the noctilionoid bats. In Carollia perspicillata, which has a simplex uterus, the ICM of the single blastocyst becomes oriented towards the uterotubal junction on the side of ovulation, and the discoidal placenta develops in a fundic position. In Noctilio sp., which have partially bicornuate uteri, the ICM becomes oriented instead towards an endometrial ridge that runs along the antimesometrial to lateral side of the gravid horn. As development proceeds, however, the blastocyst rotates almost 180 degrees, and the discoidal placenta eventually assumes a mesometrial to lateral position. In these species, implantation and subsequent development of the discoidal placenta clearly seem to be targeting major maternal vessels supplying the uterus, rather than exhibiting a consistent pattern of orientation relative to its mesenteric attachments. This permits their chorioallantoic placentae to develop a dual maternal blood supply that may be essential for the development of relatively large, precocious infants.

The interhaemal barrier in the chorioallantoic placenta of the greater mustache bat, Pteronotus parnellii, with observations on amplification of its intrasyncytial lamina.

Chorioallantoic placentae were obtained from a reproductively synchronized wild population of greater mustache bats for ultrastructural and immunocytochemical examination. The single discoidal placenta was always located in a lateral to mesometrial position on the right side of the uterus, which in the non-pregnant state is partially bicornuate. The placenta was labyrinthine and haemodichorial in advanced pregnancy. The interhaemal barrier included syncytiotrophoblast that lined the maternal vascular spaces and an underlying, continuous layer of cytotrophoblast. The barrier also contained a discontinuous extracellular layer, the intrasyncytial lamina, that was usually completely surrounded by syncytiotrophoblast. Two lines of evidence suggest that the intrasyncytial lamina may serve, in part, to strengthen the interhaemal barrier: (1) the lamina became significantly thicker as the maternal vascular channels became larger; and (2) the syncytiotrophoblast in the walls of the smallest vascular tubules expressed only very limited amounts of cytokeratins, normally a major component of the cytoskeleton of cells of epithelial origin, while the cytokeratin-rich cytotrophoblast was often highly attenuated. It was not uncommon to see gaps between the ectoplasmic processes of syncytiotrophoblast which exposed portions of the intrasyncytial lamina to the maternal vascular space. As platelet adhesion was never observed in such areas, the intrasyncytial lamina may be augmented in part by material that is non-thrombogenic.

Co-expression of cytokeratins and vimentin by highly invasive trophoblast in the white-winged vampire bat, Diaemus youngi, and the black mastiff bat, Molossus ater, with observations on intermediate filament proteins in the decidua and intraplacental trophoblast.

Histological and immunocytochemical studies of gravid reproductive tracts obtained from the white-winged vampire bat (Diaemus youngi) and the black mastiff bat (Molossus ater) have established that both species develop unusually invasive trophoblast. This is released by the developing discoidal haemochorial placenta, expresses both cytokeratins and vimentin, and invades the myometrium and adjacent tissues (including the ovaries) via interstitial migration within the walls of maternal blood vessels. Hence, this trophoblast is noteworthy for the extent to which it undergoes an epithelial-mesenchymal transformation. In Molossus, it originates from the cytotrophoblastic shell running along the base of the placenta, is mononuclear, and preferentially invades maternal arterial vessels serving the discoidal placenta. This trophoblast may have a role in dilatation of these vessels when the discoidal placenta becomes functional. In Diaemus, the highly invasive trophoblast appears to originate instead from a layer of syncytiotrophoblast on the periphery of the placenta is multinucleated, and vigorously invades both arterial and venous vessels. During late pregnancy, it becomes extensively branched and sends attenuated processes around many of the myometrial smooth muscle fibres. In view of its distribution, this trophoblast could have important influences upon myometrial contractility and the function of blood vessels serving the gravid tract. Other aspects of intermediate filament expression in the uteri and placentae of these bats are also noteworthy. Many of the decidual giant cells in Molossus co-express cytokeratins and vimentin, while the syncytiotrophoblast lining the placental labyrinth in Diaemus late in pregnancy expresses little cytokeratin.

Giant, accordioned sperm acrosomes of the greater bulldog bat, Noctilio leporinus.

Sperm of the greater bulldog bat Noctilio leporinus display an architecture that is totally unique among mammalian spermatozoa. The sperm head of Noctilio is extraordinarily large and flat and lies eccentrically with respect to the sperm tail. The major portion of the atypically large acrosome lies anterior to the nucleus and is shaped into a dozen accordionlike folds that run parallel to the long axis of the sperm. The ridge of each fold is shaped into approximately 60 minute, evenly spaced rises that extend along the entire length of the fold. We speculate that acrosome ridges may serve to strengthen the sperm head during transport.

Formation of reticulated endoderm, Reichert's membrane, and amniogenesis in blastocysts of captive-bred, short-tailed fruit bats, Carollia perspicillata.

BACKGROUND AND METHODS: As part of an effort to develop the short-tailed fruit bat (Carollia perspicillata) as a new animal model for the study of interstitial implantation and trophoblast-uterine interactions, early embryogenesis was examined histologically and ultrastructurally in captive-bred females at different intervals after the first appearance of spermatozoa in daily vaginal smears (day 1 postcoitum [p.c.]). RESULTS: In most of the early uterine embryos examined on days 16-18 p.c., much of the endoderm appeared as a reticulated meshwork; however, a unilocular yolk sac was formed prior to the development of any mesoderm. Early blastocysts of Carollia were also unusual in that endoderm surrounded much of the inner cell mass (ICM), Reichert's membrane continued over the dorsal side of the ICM, and basal laminalike material was observed around many of the endoderm and epiblast cells. A primordial amniotic cavity was formed between days 19 and 26 p.c. by cavitation. The first mesoderm appeared between days 23 and 26 p.c., concommitant with the development of an embryonic shield. CONCLUSION: The unusual reticulated appearance of early endoderm in Carollia, which is reminiscent of that seen in early human blastocysts, may be attributable to constraints imposed on growth of the blastocyst by the site and mode of implantation, temporary retardation of trophoblastic invasion by the basal laminae of endometrial epithelial elements, and endodermal proliferation in anticipation of rapid yolk sac expansion. Reichert's membrane appears to play an important role in this species in tethering the ICM and embryonic shield to the developing placenta prior to the formation of significant amounts of mesoderm.

Delayed development in the short-tailed fruit bat, Carollia perspicillata.

Pregnancy was studied in short-tailed fruit bats, Carollia perspicillata, both maintained in a captive breeding colony and collected from a reproductively synchronized wild population on the island of Trinidad. Gestation periods for captive females that successfully reared their young varied as follows: mated at a regular oestrus during their first year in captivity (105-178 days) (mean +/- SD: 145 +/- 19 days); mated at a postpartum oestrus during their first year in captivity (110-158 days) (133 +/- 16 days); mated during their second year in captivity (113-169 days) (127 +/- 12 days); females born and mated in captivity (113-159 days) (119 +/- 9 days). Most females in the last group had gestation periods of 113-119 days; this may represent the normal (nondelayed) gestation period for the species. Histological studies established that most of the observed variation in duration of gestation was due to delays occurring after the completion of implantation. It seems likely that stress, rather than age, was responsible for the prolongation of pregnancy in some animals, because this occurred less frequently in both younger and older females. There may be stressful situations in the wild (for example, lack of sufficient food or roosting sites) in which the ability to delay pregnancies would be of considerable adaptive value. Evidence was obtained that under some circumstances Carollia can extend gestation even further. Many wildcaught females successfully gave birth at 160-229 days after being isolated from breeding males in captivity. These had been captured at the time of year when, based upon subsequent histological studies of field collected specimens, most adult females should have been in early pregnancy. The field studies have also provided evidence that females in the wild population exhibit a seasonal prolongation of pregnancy.

Unusual aspects of inner cell mass formation, endoderm differentiation, Reichert's membrane development, and amniogenesis in the lesser bulldog bat, Noctilio albiventris.

BACKGROUND AND METHODS: The early embryogenesis of the lesser bulldog bat, Noctilio albiventris (family Noctilionidae), was examined histologically in 59 pregnant females collected from a reproductively synchronized population in Colombia. RESULTS: Early blastocysts of Noctilio are unusual in lacking a typical inner cell mass. Instead, cells inside of the trophoblast are dispersed for a period as a monolayer. A typical inner cell mass (ICM) only forms and becomes properly oriented after the initiation of implantation. Several features of Reichert's membrane in this species are also noteworthy: it develops between the ICM and trophoblast and between the parietal endoderm and trophoblast; it becomes linked to a meshwork of basal laminalike material that extends into the ICM; and it appears to be continuous, or fused, with prominent basal laminae that develop within the cytotrophoblastic villi that radiate throughout the preplacenta. Amniogenesis occurs by cavitation and converts the ICM into a hollow epiblastic vesicle. Gastrulation commences before this vesicle exhibits obvious differentiation into an embryonic shield and amniotic ectoderm. CONCLUSIONS: Because development and proper orientation of a typical ICM in Noctilio occur after the initiation of implantation, these may involve the migration of cells on the interior of the blastocyst and/or an unusual method of early endoderm differentiation. The possibility exists that epiblast, endoderm, and cytotrophoblast may all contribute to the secretion of Reichert's membrane in this bat. Although the early embryogenesis of Noctilio exhibits many similarities to that in phyllostomid bats, substantial differences also exist between these closely related species.

Improved procedures for maintaining and breeding the short-tailed fruit bat (Carollia perspicillata) in a laboratory setting.

Improved methods have been developed for maintaining and breeding the neotropical short-tailed fruit bat, Carollia perspicillata, in an easily-reproduced, laboratory setting. Bipartite, all-metal cages have been designed which permit efficient, non-injurious handling of the animals. Also, a fruit-based liquid diet, which is simple and inexpensive to prepare, has been formulated from readily-available canned and powdered components. When efforts were initiated to breed wild-caught animals at 3-6 months after capture, this progressed slowly in most (but not all) cages. Breeding took place more rapidly when the bats had been in captivity for 11-24 months. Most females (122/144, 84.7%) then bred within 30 days of the introduction of a stud male, and the overall conception rate was 94.3% in those females which bred. This pattern of breeding activity, evidence that this bat is a spontaneous ovulator, and observed intervals between successive breeding periods (apparently representing much or all of non-pregnant cycles) in some individuals suggest that the cycle length in many of these bats probably falls between 20 and 30 days. Analysis of the breeding data also showed that certain patterns of breeding activity were frequently associated with a failure of females to establish ongoing pregnancies. Most laboratory-bred females (69/81) that were permitted to carry their pregnancies to term did so and successfully reared their young. The laboratory-reared young have generally exhibited excellent body condition, and many have proved to be fertile. The bats were found to exhibit a post-partum oestrus, which in nearly all cases (35/36) was fertile. The interval between the discovery of a new baby and the detection of a sperm-positive vaginal aspirate varied between 3-10 days, but most frequently was 3-6 days. Females which aborted non-term fetuses also had a post-partum oestrus with similar timing. These findings indicate that the short-tailed fruit bat, which is widely abundant in the lowland tropics of the New World, can be successfully maintained under controlled conditions, in cages of modest dimensions, for research purposes.

Pregnancy in chiroptera.

As with other aspects of their biology, bats exhibit considerable diversity in their reproductive characteristics. While early embryonic development in many species generally adheres to the pattern shown by more commonly studied eutherian mammals, others demonstrate advanced development in the oviduct (to the zona-free blastocyst stage) and a prolonged tubal journey of the embryo in a temporal sense. In some bats, the process for transporting ova through the oviducts can distinguish between living embryos and the remnants of dead ova. Implantation in bats is unusually diverse with respect to localization of the nidation sites (which in most species seem to be predetermined), orientation of the inner cell mass during implantation, depth of implantation, and trophoblastic invasiveness. Some species which can be bred in captivity would appear to be promising experimental models for studies of implantation because both this process and their endometrial biology in general exhibit many similarities to those of humans. Depending upon the species, bats develop either endotheliochorial or hemochorial placentas. Studies on black mastiff bats (Molossus ater) indicate that maternal endometrial endothelial cells probably play a very important morphogenetic role during placental development in this species. Similarities in placental structure suggest that this may also be the case in a number of other eutherian mammals.

Menstruation in short-tailed fruit bats (Carollia spp.).

Uterine function was assessed histologically in Carollia spp. removed from a laboratory breeding colony. The uteri of periovulatory bats examined on days 1-3 post coitum varied considerably depending upon whether regressing corpora lutea were present. In females lacking such corpora lutea, the endometrium was shallow and proliferative. In most of the bats having regressing corpora lutea, the endometrium was thicker and necrotic portions of the superficial lamina functionalis were being sloughed off with associated bleeding (i.e. in the form of a true menstrual process). The presence of regressing corpora lutea and well-developed endometria in these animals indicated that short-tailed fruit bats are spontaneous ovulators with a functional luteal phase to their cycles. Menstruation was also observed in some females that were not periovulatory, or that lacked regressing corpora lutea. The endometrium was not vascularized by spiral arterioles, but it did contain distinctive granulocytes similar to those observed in catarrhine primates and other menstruating bats. Most endometrial growth occurred after ovulation and, in pregnant animals, was associated with retention of the embryos in the oviducts for approximately 13-16 days. Carollia perspicillata normally produces one young at a time, after a gestation period of about 4 months, and is a seasonal breeder in the wild. Menstruation probably provides this bat with an efficient mechanism for eliminating a well-developed endometrium from its simplex uterus in the event of fertilization failure or early embryonic loss. This may give the female another chance to establish a pregnancy and produce an infant at a reasonably opportune time during the same breeding season.

Development of the discoidal hemochorial placenta in the black mastiff bat, Molossus ater: evidence for a role of maternal endothelial cells in the control of trophoblastic growth.

In an effort to define further the factors that can influence trophoblastic growth, development of the discoidal chorioallantoic placenta was examined histologically in laboratory-bred black mastiff bats, Molossus ater. This placenta normally forms only at the cranial end of the right uterine horn. Its positioning was found to be related to the development of an unusual vascular tuft in the endometrium at this site following ovulation. When a decidual reaction occurred, the endothelial cells of the tuft vessels hypertrophied and their basal laminae became more prominent in sections stained for glycoproteins. Cytotrophoblast then proliferated preferentially around the tuft in the right horn and its vessels became surrounded by trophoblastic cuffs. A functional placenta was formed when trophoblastic tubules grew out from these cuffs, became interconnected, developed lumina, and began to carry maternal blood. Maternal endothelial cells generally persisted only in portions of the larger vascular tubules. These observations suggest that the endothelial cells of the tuft vessels may be secreting factors that influence early trophoblastic growth and are at least partially incorporated into the basal laminae of the endothelial cells. Evidence is reviewed which raises the possibility that endometrial endothelial cells might have a similar morphogenetic role in many other mammals with invasive trophoblast. Mitotic activity in the trophoblastic tubules of M. ater ceased when the tubules became patent and began to carry blood. Further growth was then accomplished by hypertrophy of the existing cytotrophoblast cells. Electron microscopic examination of near-term placentas confirmed that the interhemal barrier was hemomonochorial and lacked a continuous layer of syncytiotrophoblast.

Spontaneous decidual reactions and menstruation in the black mastiff bat, Molossus ater.

Uterine function was assessed histologically in nonpregnant Molossus ater removed from a laboratory breeding colony. During the luteal phase of the cycle, bilateral decidual reactions were found to develop spontaneously in the absence of either embryos or experimental manipulation of the uterus. These included the formation of early decidual giant cells, closure of the uterine lumina, and morphological changes in the endometrial blood vessels. Some endothelial cell hypertrophy was noted in much of the decidua, but this was most pronounced in vessels associated with an unusual vascular tuft that formed in the endometrium surrounding the cranial end of the uterine lumen. These latter vessels also developed very prominent basal laminae. In pregnant bats, this tuft played a central role in the morphogenesis of the definitive discoidal chorioallantoic placenta. At the end of nonpregnant cycles, the decidua became necrotic and was sloughed off with associated bleeding. As in menstruating catarrhine primates, the endometrium of M. ater is vascularized by spiral arterioles and populated by distinctive granulocytes containing large, acidophilic granules. Increased coiling of these arterioles did not appear to be an essential element in the mechanism of mensturation in this species. M. ater is a monotocous, seasonal breeder, with a relatively long gestation period. Although it has a bicornuate uterus, ovulation and implantation appear to occur only on the right side of the tract. The ability to menstruate probably affords this bat an efficient mechanism for eliminating a highly differentiated endometrium from the usual implantation site in the event of a reproductive failure. In the wild, this may provide M. ater with another chance to establish a pregnancy at a still opportune time during the same breeding season.

Evaluation of leuprolide acetate and gonadotropins versus clomiphene citrate and gonadotropins for in vitro fertilization or gamete intrafallopian transfer.

A prospective randomized trial was conducted to compare the efficiency of two ovarian stimulation protocols for in vitro fertilization-embryo transfer or gamete intrafallopian transfer. Protocol 1 consisted of clomiphene citrate and human menopausal gonadotropin (hMG) with 55 cycles of 42 patients being evaluated. Protocol 2 had 38 cycles of 34 patients receiving a gonadotropin-releasing hormone agonist (GnRH-a) and hMG. The incidence of a spontaneous luteinizing hormone surge was 38.2% in protocol 1 and 0% in protocol 2. Both protocols had a similar cancellation rate. The total clinical pregnancy rates per oocyte retrieval for patients receiving protocol 1 and protocol 2 were 19.5% and 10.3%, respectively. The difference was not statistically significant. Therefore, as first-line ovulation induction agents, it cannot be concluded that either protocol demonstrates a clear superiority over the other and further trials of the GnRH-a/hMG combination are indicated.