The effect of hypoxia in development

There is increasing evidence that the oxygen supply to the human embryo in the first trimester is tightly controlled, suggesting that too much oxygen may interfere with development. The use of hypoxia probes in mammalian embryos during the organogenic period indicates that the embryo is normally in a state of partial hypoxia, and this may be essential to control cardiovascular development, perhaps under the control of hypoxia-inducible factor (HIF). A consequence of this state of partial hypoxia is that disturbances in the oxygen supply can more easily lead to a damaging degree of hypoxia. Experimental mammalian embryos show a surprising degree of resilience to hypoxia, with many organogenic stage embryos able to survive 30-60 min of anoxia. However, in some embryos this degree of hypoxia causes abnormal development, particularly transverse limb reduction defects. These abnormalities are preceded by hemorrhage/edema and tissue necrosis. Other parts of the embryo are also susceptible to this hypoxia-induced damage and include the genital tubercle, the developing nose, the tail, and the central nervous system. Other frequently observed defects in animal models of prenatal hypoxia include cleft lip, maxillary hypoplasia, and heart defects. Animal studies indicate that hypoxic episodes in the first trimester of human pregnancy could occur by temporary constriction of the uterine arteries. This could be a consequence of exposure to cocaine, misoprostol, or severe shock, and there is evidence that these exposures have resulted in hypoxia-related malformations in the human. Exposure to drugs that block the potassium current (IKr) can cause severe slowing and arrhythmia of the mammalian embryonic heart and consequently hypoxia in the embryo. These drugs are highly teratogenic in experimental animals. There is evidence that drugs with IKr blockade as a side effect, for example phenytoin, may cause birth defects in the human by causing periods of embryonic hypoxia. The strongest evidence of hypoxia causing birth defects in the human comes from studies of fetuses lacking hemoglobin (Hb) F. These fetuses are thought to be hypoxic from about the middle of the first trimester and show a range of birth defects, particularly transverse limb reduction defects.     

Important aspects of placental-specific gene transfer

The placenta is a unique and highly complex organ that develops only during pregnancy and is essential for growth and survival of the developing fetus. The placenta provides the vital exchange of gases and wastes, the necessary nutrients for fetal development, acts as immune barrier that protects against maternal rejection, and produces numerous hormones and growth factors that promote fetal maturity to regulate pregnancy until parturition. Abnormal placental development is a major underlying cause of pregnancy-associated disorders that often result in preterm birth. Defects in placental stem cell propagation, growth, and differentiation are the major factors that affect embryonic and fetal well-being and dramatically increase the risk of pregnancy complications. Understanding the processes that regulate placentation is important in determining the underlying factors behind abnormal placental development. The ability to manipulate genes in a placenta-specific manner provides a unique tool to analyze development and eliminates potentially confounding results that can occur with traditional gene knockouts. Trophoblast stem cells and mouse embryos are not overly amenable to traditional gene transfer techniques. Most viral vectors, however, have a low infection rate and often lead to mosaic transgenesis. Although the traditional method of embryo transfer is intrauterine surgical implantation, the methodology reported here, combining lentiviral blastocyst infection and nonsurgical embryo transfer, leads to highly efficient and placental-specific gene transfer. Numerous advantages of our optimized procedures include increased investigator safety, a reduction in animal stress, rapid and noninvasive embryo transfer, and higher a rate of pregnancy and live birth.     

High Fat Diet Induced Developmental Defects in the Mouse: Oocyte Meiotic Aneuploidy and Fetal Growth Retardation/Brain Defects

Background

Maternal obesity is associated with poor outcomes across the reproductive spectrum including infertility, increased time to pregnancy, early pregnancy loss, fetal loss, congenital abnormalities and neonatal conditions. Furthermore, the proportion of reproductive-aged woman that are obese in the population is increasing sharply. From current studies it is not clear if the origin of the reproductive complications is attributable to problems that arise in the oocyte or the uterine environment.

Methodology/Principal Findings

We examined the developmental basis of the reproductive phenotypes in obese animals by employing a high fat diet mouse model of obesity. We analyzed very early embryonic and fetal phenotypes, which can be parsed into three abnormal developmental processes that occur in obese mothers. The first is oocyte meiotic aneuploidy that then leads to early embryonic loss. The second is an abnormal process distinct from meiotic aneuploidy that also leads to early embryonic loss. The third is fetal growth retardation and brain developmental abnormalities, which based on embryo transfer experiments are not due to the obese uterine environment but instead must be from a defect that arises prior to the blastocyst stage.

Conclusions/Significance

Our results suggest that reproductive complications in obese females are, at least in part, from oocyte maternal effects. This conclusion is consistent with IVF studies where the increased pregnancy failure rate in obese women returns to the normal rate if donor oocytes are used instead of autologous oocytes. We postulate that preconceptional weight gain adversely affects pregnancy outcomes and fetal development. In light of our findings, preconceptional counseling may be indicated as the preferable, earlier target for intervention in obese women desiring pregnancy and healthy outcomes.     

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.     

Immunohistochemical localization of epidermal growth factor, transforming growth factor-alpha and growth factor-beta s in the caprine peri-implantation period

Control over the action of steroid hormones in the uterus and conceptus during the initial period of gestation appears to be regulated locally by growth factors. This study involved immunohistochemical detection of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and transforming growth factor-beta s (TGF-beta s), to determine their role in the caprine peri-implantation period. Epidermal growth factor was expressed in the luminal and glandular endometrial epithelium of goats on all days studied (Days 22 to 30 post coitum), but it was not detected in trophoblastic cells or in other embryonic structures. Between Days 22 and 30 post coitum, TGF-alpha was detected in the epithelial cells and superficial stroma of the uterus and in the trophoendodermic cells of the embryo. Transforming growth factor-beta s expression, observed in the endometrium, embryo and extraembryonic membranes on Day 22 post coitum, decreased by Day 24 post coitum and disappeared in the embryo by Day 30 post coitum, while remaining in the other structures. The presence of these growth factors during the peri-implantation period in the goat suggests their participation in proliferation and differentiation phenomena which occur during implantation and embryonic development.     

Uterine asynchrony: a cause of embryonic loss

During early gestation, hormonal events associated with corpora lutea formation and embryonic synthesis of proteins, prostaglandins, and steroids result in synthesis and release of endometrial secretory products into the uterine lumen. The embryo, inherently and in response to secretory products of the uterus, develops and grows. However, considerable embryonic mortality occurs when uterine secretions become altered in such a manner that they are asynchronous to the developing embryo. Factors that advance or retard development of the uterus and embryo have been utilized to document utero-embryonic asynchrony, and it has been observed that the uterus will not "wait" for embryos to become synchronous. However, the reverse is possible: embryonic development can be accelerated or decelerated. Furthermore within the uterus, localized areas might also exist that favor development of some embryos at the expense of others. This review will consider causes of utero-embryonic asynchrony and offer models of embryonic loss associated with an asynchronous environment in cattle, sheep, and swine.     

Aging and reproductive potential in women.

Reproductive potential in women declines with age. Age-related changes in the ovary account for most of this loss of reproductive function. Oocytes, all of which are present at birth, decline in number and quality with age. The endocrine function of the ovary also declines with age, and the ovary becomes unable to sustain its normal function in the neuroendocrine axis. The neuroendocrine axis may be further affected by primary changes occurring in the hypothalamus and pituitary during aging, although this has not been established in humans. Aging also affects the function of the uterus as the endometrium loses its ability to support implantation and growth of an embryo. Diminished uterine function during aging may be due to changes in the uterine vasculature or to changes in the hormone-dependent development of the endometrium. Finally, aging increases a woman's risk of developing medical, gynecologic or obstetric conditions that may impair her fertility. Knowledge of these affects of aging on a woman's reproductive function is essential to advise and treat the growing number of women seeking pregnancy at advanced reproductive age.     

Prostaglandin F and E levels in the conceptus, uterus and plasma during early pregnancy in the ewe

Concentrations of prostaglandins E and F (PGE and PGF) were measured in the embryo or fetus, extra embryonic or fetal membranes (membranes), intercaruncular and caruncular endometrium and plasma collected from uterine and ovarian arterial and venous vessels from separate groups of ewes laparotomized at 5 day intervals from day 10 to day 55 of pregnancy. Our purpose was to investigate the role of prostaglandins E and F in the maternal recognition of pregnancy, implantation and early placental function. Our data suggest that the initial maintenance of the corpus luteum in the pregnant ewe does not involve a reduction in PGF production, compared to pregnant ewes; but a change in the pattern of PGF secretion. This is accompanied by an elevation in PGE production of similar magnitude to that observed in non pregnant ewes. The extra embryonic/fetal membranes appear to be the major source of elevated PGF levels in the maternal circulation prior to day 30 of pregnancy. Between days 35 and 55 of gestation the rising PGF levels in maternal serum probably come from the fetus. Over the same period PGE levels rise in the fetus and intercaruncular endometrium, but PGE secretion into the maternal circulation is not enhanced. A role for PGF and PGE in fetal, placental and uterine growth is suggested; placental and uterine endocrine function may also be targets.     

Prostaglandin F and E levels in the conceptus, uterus and plasma during early pregnancy in the ewe

Concentrations of prostaglandins E and F (PGE and PGF) were measured in the embryo or fetus, extra embryonic or fetal membranes (membranes), intercaruncular and caruncular endometrium and plasma collected from uterine and ovarian arterial and venous vessels from separate groups of ewes laparotomized at 5 day intervals from day 10 to day 55 of pregnancy. Our purpose was to investigate the role of prostaglandins E and F in the maternal recognition of pregnancy, implantation and early placental function. Our data suggest that the initial maintenance of the corpus luteum in the pregnant ewe does not involve a reduction in PGF production, compared to pregnant ewes; but a change in the pattern of PGF secretion. This is accompanied by an elevation in PGE production of similar magnitude to that observed in non pregnant ewes. The extra embryonic/fetal membranes appear to be the major source of elevated PGF levels in the maternal circulation prior to day 30 of pregnancy. Between days 35 and 55 of gestation the rising PGF levels in maternal serum probably come from the fetus. Over the same period PGE levels rise in the fetus and intercaruncular endometrium, but PGE secretion into the maternal circulation is not enhanced. A role for PGF and PGE in fetal, placental and uterine growth is suggested; placental and uterine endocrine function may also be targets.     

An early endometrial vascular indicator of completed orientation of the embryo and the role of dorsal endometrial encroachment in mares

The spherical equine embryonic vesicle is mobile throughout the uterine lumen for several days before becoming fixed in the caudal segment of a uterine horn on Day 16 (ovulation = Day 0). Orientation refers to the position of the embryo proper at the periphery of the vesicle relative to the position of the mesometrial attachment. In mares, the embryonic pole of the vesicle is antimesometrial after completion of orientation. Day of vesicle fixation, differential thickening of the endometrium near the mesometrial attachment, and orientation of the embryonic vesicle were studied in 30 ponies, using B-mode and color-Doppler transrectal ultrasonography. The thickness of the endometrium at the mesometrial aspect of the vesicle divided by the thickness at the antimesometrial aspect was termed the encroachment ratio. At the future site of fixation, the first increase (P < 0.05) in the encroachment ratio occurred between 4 and 1 days before fixation. An early vascular indicator of the future position of the embryo proper was discovered by color-Doppler imaging and consisted of a colored spot in the image of the endometrium close to the wall of the embryonic pole. The early indicator was detected in each mare 0.5 +/- 0.1 days after fixation and 2.5 +/- 0.2 days before first detection of the embryo proper. The position of the early indicator when first detected at the periphery of the embryonic vesicle was not significantly different from the position of the embryo proper when first detected. Results supported the hypothesis that differential thickening of the endometrium precedes orientation and indicated that orientation occurs immediately after fixation.     

Gamete origin in relation to early embryo development

Fertilization in vivo requires a complex series of selection events to occur in order to guarantee that only the fittest gametes take part in the fusion process and give rise to a viable embryo. Conventional practice in bovine in vitro fertilization however is to select oocytes and sperm by quite crude procedures. It is therefore not inconceivable that essentially unfit gametes may drive aberrant embryo development in vitro. Abnormal embryonic cells are being removed by apoptosis, which is a physiological process in embryos. Only an excess or a lack of apoptosis can lead to embryonic death or abnormal development. Suboptimal culture conditions undoubtedly contribute to undue embryonic apoptosis, but the intrinsic quality of the oocyte may also be a causative factor. It is generally accepted that the oocyte is in control of early embryogenesis, but is it also in control of future embryonic suicide? Is a compromised follicular environment predestining the oocyte to a dire fate? What is the contribution of the cumulus cells to oocyte quality, and can they rescue it from early demise? And what can be said about the origin of the spermatozoa? Research in human in vitro fertilization has definitely shown that factors such as paternal age, smoking and other sperm stressors can contribute to abnormal embryo development and even diseased offspring. This review will address the questions raised above, and will describe what is known about the cellular and molecular biology that may account for abnormal bovine embryo development caused by gamete origin.     

Molecular mechanisms of membrane interaction at implantation

Successful pregnancy is dependent upon the implantation of a competent embryo into a receptive endometrium. Despite major advancement in our understanding of reproductive medicine over the last few decades, implantation failure still occurs in both normal pregnancies and those created artificially by assisted reproductive technology (ART). Consequently, there is significant interest in elucidating the etiology of implantation failure. The complex multistep process of implantation begins when the developing embryo first makes contact with the plasma membrane of epithelial cells within the uterine environment. However, although this biological interaction marks the beginning of a fundamental developmental process, our knowledge of the intricate physiological and molecular processes involved remains sparse. In this synopsis, we aim to provide an overview of our current understanding of the morphological changes which occur to the plasma membrane of the uterine endothelium, and the molecular mechanisms that control communication between the early embryo and the endometrium during implantation. A multitude of molecular factors have been implicated in this complex process, including endometrial integrins, extracellular matrix molecules, adhesion molecules, growth factors, and ion channels. We also explore the development of in vitro models for embryo implantation to help researchers investigate mechanisms which may underlie implantation failure. Understanding the precise molecular pathways associated with implantation failure could help us to generate new prognostic/diagnostic biomarkers, and may identify novel therapeutic targets. Birth Defects Research (Part C) 108:19–32, 2016. © 2016 Wiley Periodicals, Inc.     

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.     

Developmental biology of uterine glands.

All mammalian uteri contain endometrial glands that synthesize or transport and secrete substances essential for survival and development of the conceptus (embryo/fetus and associated extraembryonic membranes). In rodents, uterine secretory products of the endometrial glands are unequivocally required for establishment of uterine receptivity and conceptus implantation. Analyses of the ovine uterine gland knockout model support a primary role for endometrial glands and, by default, their secretions in peri-implantation conceptus survival and development. Uterine adenogenesis is the process whereby endometrial glands develop. In humans, this process begins in the fetus, continues postnatally, and is completed during puberty. In contrast, endometrial adenogenesis is primarily a postnatal event in sheep, pigs, and rodents. Typically, endometrial adenogenesis involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. This process requires site-specific alterations in cell proliferation and extracellular matrix (ECM) remodeling as well as paracrine cell-cell and cell-ECM interactions that support the actions of specific hormones and growth factors. Studies of uterine development in neonatal ungulates implicate prolactin, estradiol-17 beta, and their receptors in mechanisms regulating endometrial adenogenesis. These same hormones appear to regulate endometrial gland morphogenesis in menstruating primates and humans during reconstruction of the functionalis from the basalis endometrium after menses. In sheep and pigs, extensive endometrial gland hyperplasia and hypertrophy occur during gestation, presumably to provide increasing histotrophic support for conceptus growth and development. In the rabbit, sheep, and pig, a servomechanism is proposed to regulate endometrial gland development and differentiated function during pregnancy that involves sequential actions of ovarian steroid hormones, pregnancy recognition signals, and lactogenic hormones from the pituitary or placenta. That disruption of uterine development during critical organizational periods can alter the functional capacity and embryotrophic potential of the adult uterus reinforces the importance of understanding the developmental biology of uterine glands. Unexplained high rates of peri-implantation embryonic loss in humans and livestock may reflect defects in endometrial gland morphogenesis due to genetic errors, epigenetic influences of endocrine disruptors, and pathological lesions.     

Mammalian prenatal development: the influence of maternally derived molecules

Normal fetal development is dependent upon an intricate exchange between mother and embryo. Several maternal and embryonic elements can influence this intimate interaction, including genetic, environmental or epigenetic factors, and have a significant impact on embryo development. The interaction of the genetic program of both mother and embryo, within the uterine environment, can shape the development of an individual. Accumulating data from animal models indicate that prenatal events may well initiate long‐term changes in the expression of the embryo genetic program, which persist, or may only become apparent, much later in the individual's life. Also, environmental conditions during prenatal development may prompt the adoption of different developmental pathways, leading to alternative life histories. This review focuses on environmental factors, specifically maternally derived molecules, to illustrate how they can influence in utero embryonic development and, by extension, adult life.     

Late embryo-defective mutants of Arabidopsis

The embryo-defective (emb) mutants of Arabidopsis constitute a large and diverse group of mutants disrupted in a broad range of embryonic processes, including morphogonesis, cell differentiation, and maturation programs. This report describes a subset of these mutants, the late embryo defectives, which develop beyond the globular stage of embryogenesis but fail to complete normal morphogenesis. A representative sample of 12 late mutants was chosen for this study, patterns of morphogenesis were characterized, the germination potential of mutant seeds was investigated, and additional mutant alleles within the collection were identified. Morphological defects in mutant embryos became apparent during the heart stage of development, when embryos normally begin the rapid cell division and expansion required for the completion of morphogenesis. Despite their morphological abnormalities, mutant embryos often germinated from dry seed, demonstrating that genetic programs required for the establishment of desiccation tolerance remained intact. Mutant seedlings displayed a wide range of developmental abnormalities, including altered morphology, lack of pigmentation, dwarfism, and disorganized vegetative growth. One late mutant was found to be allelic to an early embryo defective that arrests at the globular stage. These results suggest that a number of late EMB genes encode basic cellular and metabolic functions needed for cell division, enlargement, and embryonic growth. The rapid growth and metabolic changes that occur at the heart stage may present a barrier to normal development in the late mutants, resulting in altered embryo morphology and other developmental defects. It is proposed that many Arabidopsis mutants with abnormal embryo and seedling morphology are not defective in the regulation of pattern formation or morphogenesis, but rather in fundamental physiological and cellular processes required for the completion of normal growth and development. © 1995 Wiley-Liss, Inc.     

Computational hemodynamic optimization predicts dominant aortic arch selection is driven by embryonic outflow tract orientation in the chick embryo

In the early embryo, a series of symmetric, paired vessels, the aortic arches, surround the foregut and distribute cardiac output to the growing embryo and fetus. During embryonic development, the arch vessels undergo large-scale asymmetric morphogenesis to form species-specific adult great vessel patterns. These transformations occur within a dynamic biomechanical environment, which can play an important role in the development of normal arch configurations or the aberrant arch morphologies associated with congenital cardiac defects. Arrested migration and rotation of the embryonic outflow tract during late stages of cardiac looping has been shown to produce both outflow tract and several arch abnormalities. Here, we investigate how changes in flow distribution due to a perturbation in the angular orientation of the embryonic outflow tract impact the morphogenesis and growth of the aortic arches. Using a combination of in vivo arch morphometry with fluorescent dye injection and hemodynamics-driven bioengineering optimization-based vascular growth modeling, we demonstrate that outflow tract orientation significantly changes during development and that the associated changes in hemodynamic load can dramatically influence downstream aortic arch patterning. Optimization reveals that balancing energy expenditure with diffusive capacity leads to multiple arch vessel patterns as seen in the embryo, while minimizing energy alone led to the single arch configuration seen in the mature arch of aorta. Our model further shows the critical importance of the orientation of the outflow tract in dictating morphogenesis to the adult single arch and accurately predicts arch IV as the dominant mature arch of aorta. These results support the hypothesis that abnormal positioning of the outflow tract during early cardiac morphogenesis may lead to congenital defects of the great vessels due to altered hemodynamic loading.     

Hormonal asynchrony and embryonic development

Luteinizing hormone (LH), progesterone and estradiol profiles in peripheral blood serum were compared among parous and nonparous females with normal, abnormal or no embryonic development. Hormonal profiles between parous and nonparous females of the same embryonic status did not differ and the data were combined. Estrous cycle length was longer (P<.05) in parous (22.3±.4 days) than nonparous females (21.0±.4 days). Females with normal developing embryos had a higher serum progesterone concentration at Days 3 and 6 and a lower ratio of estradiol to progesterone than did females with abnormal embryonic development. Females with a normal embryo had higher (P<.05) preovulatory LH peaks than females with abnormal development or no recovery of an oocyte or embryo (34.3±4.7, 11.8±6.8 and 13.3±2.5 ng/ml, respectively). The interval from onset of estrus to LH peak was 8.9±2.1, 13.7±3.7 and 13.5±6.2 hr for females with normal, abnormal or no recovery of an embryo. The lower LH peak, the longer interval from onset of estrus to LH peak, and lower progesterone concentration in peripheral blood serum in females with abnormal embryos or no recovery indicated that these females had a hormonal asynchrony. The hormonal asynchrony may produce an undesirable uterine environment for male and female gametes or embryos which resulted in fertilization failure or embryonic death. In the second experiment, more transferable embryos were obtained when superovulated females received prostaglandin F_2α (PGF_2α) intravenously rather than intramuscularly. Administering PGF₂α intravenously rather than intramuscularly may have caused the demise of the corpus luteum sooner and thereby produced a more normal uterine environment which allowed more embryos to develop normally.     

A model for implantation: coculture of blastocysts and uterine endometrium in mice

One of the limitations in embryo implantation research is the lack of an available in vitro model that faithfully replicates embryo-uterine interactions. In previous studies, embryos were cultured on a monolayer of either uterine epithelial cells or extracellular matrix substratum on which embryos could adhere and outgrow. However, these models failed to display embryonic invasion, primarily because of the shortage of critical structural and molecular supports that are available in vivo. In the present study, we used intact mouse uterine endometrium collected on Day 4 of pregnancy and placed in contact with blastocysts to initiate coculture experiments in a defined medium at the air-liquid interface. The culture medium was composed of Ham F-12/Dulbecco modified Eagle medium (1:1), 30% fetal calf serum, 63.5 nmol/L of progesterone, 7.14 nmol/L of estradiol-17beta, 100 mug/ml of insulin, and 20 ng/ml of epidermal growth factor, whereas the incubation condition was mixed air of 50% oxygen, 5% carbon dioxide, and 45% nitrogen with a humidity of greater than 90% at 37 degrees C. Our observations from 24 h of culture clearly demonstrated that embryos were capable of attachment to the uterine endometrium and displayed partial invasion into the endometrial stroma. Interestingly, no outgrowth of trophoblasts on the surface of uterine endometrium was seen, but embryos exhibited a pole-specific attachment. Overall, this model is capable of demonstrating a true invasion of embryo within the endometrial stroma and may be suitable in studies related to early embryo implantation.