Window Of Receptivity Research Articles

Temporal window of receptivity and intraspecific variability in the responsiveness to metamorphosis-stimulating cues in the megalopa of a semi-terrestrial crab, Sesarma curacaoense

Summary The larvae of numerous sessile and several motile marine invertebrate species respond to metamorphosis-stimulating cues, usually chemical signals from their benthic habitat. In three series of experiments (I-III), the megalopa larvae of a semi-terrestrial mangrove crab, Sesarma curacaoense de Man, were exposed for differential periods to adult-conditioned water (ACW), which had previously been found an effective cue. In the first experimental series (Exp. I), ACW was added on successively later days of the moulting cycle, while Exp. II comprised treatments in which the cues were provided from the first day (postmoult) and removed on sucessively later days. In Exp. III, the megalopae were exposed to ACW for 3 days at the beginning, in the middle, or near the end of the moulting cycle (days 1–3, 4–6, and 7–9, respectively). All experimental series also comprised a permanently exposed control group (C+) and another one kept in complete absence of the cue (C-), and all experiments were repeated with larvae originating from three different females (F1-F3 in Exps. I and II; F4-F6 in Exp. III). Both the timing and duration of exposure to ACW had a significant influence on the duration of development to metamorphosis, indicating an interaction between the extrinsic signal and the intrinsic (hormonal) control of the moulting cycle. While continuously exposed control megalopae (C+) required on average 7.6 ± 0.5 to 7.7 ± 0.5 d, those in group C- delayed their development by about 3–4 d (or on average by 48%), 7–9 d (119%), and 5–6 d (61%), respectively (F1-F3), indicating a great deal of intraspecific variability among hatches. In Exp. I, the cue had to have been added within about the first 6–7 d to become effective (no significant difference in development time between treatments and control C+), while a later addition of ACW had no more stimulating effect (no difference to C-). In Exp. II, metamorphosis was delayed in all treatments when the cue was removed after an initial exposure of less than 4–5 d, while later removal did not significantly reduce the stimulating effect of ACW. Together, the results from these two experimental series suggest that an exposure approximately during days 4–6 of the moulting cycle may be crucial as a temporal window of receptivity for the cue; this period coincides with the transition between intermoult and premoult (stages C-D0 of Drach's classification system). Its presumable importance as a critical point was confirmed in Exp. III where development time was consistently shortest in megalopae exposed only during days 4–6 of the moulting cycle (no significant difference to control C+), while an earlier or later exposure prolonged development to metamorphosis. Intraspecific variability in the responsiveness to ACW was tested by comparing development time in control groups (C+, C-) among megalopae from 14 different hatches (including F1-F6). These data showed a significant variability both in the delay due to complete absence of the cue and in the time of development in C+ groups. Twoway ANOVA indicated significant effects of exposure to ACW and of the female, as well as an interaction between these two factors, not only in development time, but also in the extent of individual variability among equally treated sibling larvae (measured as coefficient of variation). Analysis of molt-frequency distributions showed that the cue had a focussing (variabilityreducing) in addition to the accelerating effect. In conclusion, this study showed that the stage of the moulting cycle as well as intraspecific variability within and among hatches must be considered as potentially confounding factors interacting with effects of metamorphosisstimulating cues. The potential evolutionary significance of intraspecific variability in the extent of phenotypic plasticity is discussed in relation to different ecological scenarios.

Endometrial expression of calbindin (CaBP)-d28k but not CaBP-d9k in primates implies evolutionary changes and functional redundancy of calbindins at implantation.

The endometrium is hostile to embryo implantation except during the 'window of receptivity'. A change in endometrial gene expression is required for the development of receptivity. Calbindin-d9k (CaBP-d9k) and calbindin-d28k (CaBP-d28k) are proteins possessing EF-hand motifs which have high affinity for Ca2+ ions. Previously, it has been demonstrated that, in mouse endometrium, the expression of both calbindins is highly regulated during implantation and that both proteins play critical but functionally redundant roles at implantation. This study was the first to determine the expression of these two calbindins in the human and rhesus monkey endometrium. Initial RT-PCR analysis demonstrated that CaBP-d28k but not CaBP-d9k mRNA expression is detectable in the endometrium of both species. Western blot analysis confirmed the presence of immuno-reactive CaBP-d28k protein in the primate endometrium. Furthermore, the endometrial expression pattern of CaBP-d28k mRNA and protein was examined by Northern blot analysis and immunohistochemistry respectively in both species across the menstrual cycle and during early pregnancy. Semi-quantitative statistical analysis of the immunohistochemistry results revealed that, in the human, CaBP-d28k protein expression was maximal in luminal and glandular epithelium during the mid-secretory phase, coinciding with the time when the endometrium is receptive to embryo implantation. Expression in rhesus monkey showed a similar trend. These results suggest that, in the primate endometrium, only CaBP-d28k is expressed and that the specific regulation of this calbindin is potentially important for the establishment of uterine receptivity.

Stimulation of metamorphosis in an estuarine crab, Chasmagnathus granulata (Dana, 1851): temporal window of cue receptivity

The larvae of most benthic marine invertebrate species must develop for a minimum of time in the plankton before they become competent for settlement and metamorphosis in response to stimulating external cues. In an experimental laboratory study, we identified the temporal window of cue receptivity within the moulting cycle of the megalopa stage of an estuarine crab, Chasmagnathus granulata Dana. This species shows an export strategy including an early larval transport to coastal marine waters where zoeal development takes place, followed by the return of the megalopa stage to brackish habitats where the adults live. In two series of experiments (A, B), megalopae were exposed for differential periods to a combination of metamorphosis-stimulating cues which had previously been found effective (seawater conditioned with adult odor and presence of mud). In experimental series A, these cues were added on successively later days of the moulting cycle, while series B comprised treatments in which the cues were provided from the first day (postmoult) and removed on successively later days of the moulting cycle. Each series of experiments was repeated with larvae originating from two different females (F1, F2). The average development time of megalopae kept continuously in the presence of cues (control experiments, C1) ranged in the two hatches from 9.3 to 9.6 days. In the inverse controls where no cue was added at any time (C2), megalopal development to metamorphosis took on average 11.2–12.0 days. In series A, development duration in treatments with exposure to the cues commencing within 3–4 days after moulting was not significantly different from that in the permanently exposed C1 controls. Later beginning of the exposure, by contrast, had no stimulating effect (significant delay compared to C1, no significant difference from unexposed control, C2). In series B, no significant differences in development time were observed between the C1 controls and treatments with an initial exposure for a minimum of 4 or 6 days of the moulting cycle (F1, F2, respectively). Shorter initial periods of exposure had no metamorphosis-stimulating effects (no significant difference from C2). In conclusion, our results from both experiments suggest that the megalopa stage of C. granulata is most receptive of stimulating cues during a period lasting from ca. one third to one half of the moulting cycle, which coincides with the transition between stages C (intermoult) and D 0 (early premoult) of Drach's classification system. This suggests an interaction of extrinsic stimulating cues with intrinsic (hormonal) factors involved in the control of the moulting cycle.

17β-Estradiol induces nuclear translocation of CrkL at the window of embryo implantation

Crk family adaptors are widely expressed and mediate the timely formation of signal transduction protein complexes upon a variety of extracellular stimuli, including various growth and differentiation factors. The window of implantation is the favorable time period when the uterus develops a receptive approach to the invading embryo. Various signaling cascades are likely to become active at the window of implantation both in the uterus and the embryo. This helps create maternal embryo dialogue leading to successful embryo implantation. In this study we report for the first time the presence and nuclear translocation of the adaptor molecule CrkL both in the uterine and embryonic partners at the window of implantation. We also report that estrogen, which initiates and guides crucial changes in the uterus and the embryo at the window of receptivity, causes a massive surge in the expression and subsequent nuclear translocation of CrkL. We have also identified the existence of one LXXLL motif in the CrkL amino acid sequence and a single LXD is sufficient for activation by the estrogen receptor. This is suggestive that CrkL can bind to estrogen receptors and act as a coactivator.

Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle.

Relaxin (RLX) is a pregnancy-associated polypeptide hormone. In non-pregnant women, the peak of circulating relaxin coincides with the window of endometrial receptivity and both in vivo and in vitro experiments showed that it plays a role in the decidualization process. Recently, two receptors, LGR7 and LGR8, have been identified as high affinity receptors for relaxin. Here we describe LGR7 mRNA and protein expression in human endometrium using semi-quantitative and quantitative fluorescent PCR (Q-PCR) and immunohistochemical analyses. Three different experimental designs were used. First, endometrial biopsies from five different phases of the menstrual cycle were analysed. Secondly, we assessed the early luteal phase in more detail. Finally we analysed the expression at LH+2 (2 days after the natural LH surge, pre-receptive endometrium) versus LH+7 (receptive endometrium) within the same menstrual cycle from the same patient to avoid inter-cycle or inter-person variations in gene expression. Our results indicate that there is no consistent regulation of LGR7 mRNA expression, neither during the menstrual cycle nor during the early-mid-luteal phase. In general, we observed a large degree of variation in LGR7 mRNA expression levels between patients. LGR7 immunoreactive protein was identified in all stages of the menstrual cycle. LGR7 protein was localized in both the epithelial and the stromal compartments, except for the mid-luteal phase when the expression was restricted to the endometrial epithelium. We conclude that no consistent regulation of LGR7 mRNA expression can be detected in human endometrium during the menstrual cycle.

Uterine androgen receptors: roles in estrogen-mediated gene expressionand DNA synthesis.

The localization of androgen receptors (AR) and their ligands in the uterine microenvironment at early pregnancy suggest a role for AR in uterine physiology. We have evaluated AR expression in the pig uterine endometrium and examined whether AR ligands modulate peri-implantation uterine gene expression. Northern blot analysis demonstrated the approximately 10.5 kilobase AR transcript in endometrium. Endometrial levels of AR mRNA and protein were greater at early than at mid- or late pregnancy. Estrogen receptor-alpha mRNA levels showed similar maximal expression at early pregnancy. Immunocytochemical analysis of endometrium at early pregnancy localized AR to nuclei of glandular epithelial (GE) and stromal (ST) cells. To evaluate a role for AR in uterine gene regulation, the levels of mRNAs for insulin-like growth factor-I (IGF-I), proliferative cell nuclear antigen (PCNA), and AR itself were assessed in uterine endometrial explant cultures treated with estradiol-17beta (E), testosterone (T), and 19-nortestosterone (N). Induction by E of AR mRNA abundance occurred in endometrium from Day 10 but not from Day 12 pregnant animals and this was partially blocked by coaddition of N or T, although neither androgen alone had any effect. Abundance of IGF-I and PCNA mRNAs was increased by E and inhibited by coaddition of either T or N in Day 10 pregnant pig endometrium. In endometrium from Day 12 pregnant animals, addition of either N or T with E increased IGF-I mRNA levels over that of controls, although E alone was without effect. In contrast, PCNA mRNA abundance was suppressed by all steroid treatments in these explants. DNA synthesis in primary cultures of GE cells from endometrium at Days 10 and 12 of pregnancy was increased by E and was suppressed by T, the latter only at Day 12. E did not affect DNA synthesis in ST cells from endometrium at either pregnancy day, although T inhibited this process in an E-dependent manner in ST cells from pregnancy Day 12. Results identify AR in the pig endometrium during the window of maternal receptivity for implantation and demonstrate the functional, albeit complex, interactions of androgens and estrogens in the regulation of uterine endometrial gene expression and cell growth in vitro. Further elucidation of the role of androgens and their receptor in early pregnancy events may be relevant to an understanding of peri-implantation embryo loss.

Temporal regulation of gene expression during the expected window of receptivity in the rhesus monkey endometrium.

Progesterone has been shown to regulate a number of genes and gene networks in the primate endometrium. This action of progesterone is essential to provide an appropriate milieu for embryo-endometrial communication that can lead to implantation and the successful initiation of pregnancy. A temporal regulation of endometrial genes is most likely required to achieve an appropriate state of receptivity in the primate endometrium. Using simulated menstrual cycles in the rhesus monkey, endometrial tissue was harvested at days that encompass the expected window of receptivity (4-10 days after the estradiol surge) and subsequently converted to cycle day-specific cDNA populations. Using differential display reverse transcriptase-polymerase chain reaction, 12 cDNA fragments were isolated and sequenced whose mRNA levels were elevated during this time frame. The temporal expression patterns of these mRNAs were confirmed by semiquantitative polymerase chain reaction. Two of these fragments exhibited high homology to previously characterized human genes: 1) secretory leukocyte protease inhibitor, also known as antileukoprotease, an endometrial neutrophil elastase inhibitor with antibacterial and anti-inflammatory properties; and 2) syncytin, also known as endogenous retrovirus W envelope protein, a highly fusogenic membrane glycoprotein that induces formation of giant syncytia and is believed to be important in decidual and placental development. The temporal regulation of these genes by progesterone supports their likely role in the orchestration of molecular and cellular events that are required to achieve a state of receptivity in the primate endometrium.

The role of estrogen in uterine receptivity and blastocyst implantation

The endometrium is a specialized, hormonally regulated organ that is non adhesive for embryos throughout most of the reproductive cycle in mammals. Thus, the window of implantation is a self-limited period in which the endometrial epithelium (EE) acquires a functional and transient ovarian steroid-dependent status. The luminal EE initiates the adhesion of the developing blastocyst during this period, owing mainly to the presence of progesterone (P) after appropriate 17β-estradiol (E 2) priming in humans or because of the addition of E 2 after appropriate P priming in rodents. Wen-ge et al. have now demonstrated in mice that low levels of exogenous E 2 can maintain the window of receptivity for an extended period of time, whereas high doses of E 2 can rapidly initiate a refractory state. In summary, levels of E 2 within a very narrow range determine the duration of the window of implantation in uterine receptivity in mice. These outstanding results demonstrate the possibility of manipulating the receptivity window with the use of different doses of E 2.

The human placenta remodels the uterus by using a combination of molecules that govern vasculogenesis or leukocyte extravasation.

After fertilization, the next major hurdle for human reproduction is trophoblast differentiation, which is required for implantation, followed in lockstep by rapid assembly of these embryonic cells into a functional placenta. During this process, cytotrophoblast stem cells invade the uterus, anchoring the conceptus to the mother and establishing blood flow to the placenta. Cytotrophoblast invasion is actually a differentiation process that yields cells with many unusual attributes, that is, their tumor-like ability to invade the uterus and engraft maternal blood vessels. We discovered that once cytotrophoblasts commit to invasion they turn on expression of adhesion receptors characteristic of endothelium. They also begin to express and activate matrix metalloproteinase-9. Together, these phenotypic changes facilitate invasion while enabling cytotrophoblasts to present an endothelium-like surface to maternal blood. Currently, we are trying to understand the factors that play a role in cytotrophoblast differentiation/invasion. In keeping with the vascular characteristics that differentiated cytotrophoblasts assume, our recent data suggest that the key regulators include an unusual subset of vascular endothelial growth factor family members that play important roles in conventional vasculogenesis/angiogenesis. Surprisingly, we also discovered that these cells express functional l-selectin, which mediates neutrophil rolling and tethering, under shear stress, on inflamed endothelium. Trophoblast l-selectin likely interacts with carbohydrate selectin ligands that are upregulated on uterine glandular epithelium during the window of receptivity. Together, these data suggest that differentiating cytotrophoblasts have co-opted portions not only of vasculogenesis, but also of the process that facilitates leukocyte emigration from the blood into tissues, additional evidence of these cells' amazing plasticity.

Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation.

Many underlying causes of human infertility have been overcome by using in vitro fertilization (IVF) and embryo transfer (ET) techniques. Nevertheless, implantation rates in IVF programs remain low despite the transfer of apparently healthy embryos. This suggests that there are problems with the differentiation of the uterus to the receptive state in response to the ovarian hormones estrogen and progesterone. The molecular basis of this receptive state when the uterine environment is conducive to blastocyst acceptance and implantation remains poorly understood. Normally, the "window" of uterine receptivity lasts for a limited time. Using ETs and the progesterone-treated delayed-implantation model in mice, we demonstrate here that levels of estrogen within a very narrow range determine the duration of the window of uterine receptivity. Although estrogen at different physiological concentrations can initiate implantation, we find that the window of uterine receptivity remains open for an extended period at lower estrogen levels but rapidly closes at higher levels. The uterine refractoriness that follows the receptive state at high estrogen levels is accompanied by aberrant uterine expression of implantation-related genes. These results suggest that careful regulation of estrogen levels is one of the important factors for improvement of female fertility in IVFET programs.

Trophoblast L-selectin-mediated adhesion at the maternal-fetal interface.

Trophoblast adhesion to the uterine wall is the requisite first step of implantation and, subsequently, placentation. At the maternal-fetal interface, we investigated the expression of selectin adhesion systems that enable leukocyte capture from the bloodstream. On the maternal side, human uterine epithelial cells up-regulated selectin oligosaccharide-based ligands during the window of receptivity. On the fetal side, human trophoblasts expressed L-selectin. This ligand-receptor system was functional, because beads coated with the selectin ligand 6-sulfo sLe(x) bound to trophoblasts, and trophoblasts bound to ligand-expressing uterine luminal epithelium in tissue sections. These results suggest that trophoblast L-selectin mediates interactions with the uterus and that this adhesion mechanism may be critical to establishing human pregnancy.

31. Immunolocalisation of interleukin-11 and its receptor in endometrium of infertile women during the implantation window

The human endometrium is normally a hostile environment to embryo implantation except for a limited phase of the menstrual cycle known as the “window of receptivity”. Mice with a null mutation in the gene encoding for the interleukin (IL)-11 receptor alpha (Rα) are infertile due to a failure of embryo implantation. In the human endometrium IL-11 localises in a pattern suggesting a role in human fertility. The aim of this study was to examine the temporal and spatial location of IL-11 and IL-11Rα in endometrium from infertile women (infertile) and women with normal menstrual cycles and no known endometrial dysfunction (normal). Immunohistochemistry was performed on tissues collected between 5 and 10 days after ovulation. IL-11 and IL-11Rα immunoreactivity was absent in a subpopulation of tissues from infertile women. In the infertile tissues that exhibited staining, IL-11 stained minimally in glandular and luminal epithelial cells, stromal cells, and vascular smooth muscle and endothelial cells. Similarly, IL-11Rα immunoreactivity was minimal in all major cellular compartments, with the luminal epithelial cells and vascular smooth muscle cells showing the lowest staining. By contrast, IL-11 and IL-11Rα immunostaining was found in all normal tissues. Furthermore, staining for IL-11 and IL-11Rα was high in the glandular epithelial cells of the normal tissues. Moderate to low staining for IL-11 and IL-11Rα was seen in stromal and vascular endothelial cells, while low staining was apparent in the vascular smooth muscle and luminal epithelial cells. Staining for IL-11 and IL-11Rα was overall markedly higher in normal compared to infertile tissues. These data suggest a role for IL-11 in the preparation of a receptive endometrium, which is critical in the establishment of pregnancy. (1) Robb et al. (1998) Nat. Med. 3: 303–308. (2) Dimitriadis et al. (2000) Mol. Hum. Reprod. 6: 907–914.

Human endometrial receptivity: a genomic approach

The endometrium is a specialized tissue, hormonally-regulated, that is non-adhesive for embryos throughout most of the menstrual cycle in humans and other primates. Thus, endometrial receptivity is a self-limited period in which the endometrial epithelium (EE) acquires a functional and transient ovarian steroid-dependent status. The luminal EE acquires the ability to adhere (receptivity) the developing human blastocyst during this period due mainly to the presence of progesterone after appropriate 17β-oestradiol priming. This status is a key element for embryonic implantation and appears to be closely associated with morphological and biochemical changes of EE cells. This specific time window is thought to be open after 4–5 days and closes after 9–10 days of progesterone production or administration, creating a physiological window of receptivity limited to days 19–24 of the menstrual cycle in humans. The scientific knowledge of the endometrial receptivity process is fundamental for the understanding of the human reproduction, but, so far, none of the proposed biochemical markers for endometrial receptivity have been proved clinically useful. In this work new strategies are presented based on molecular biology technologies that aim to clarify the fragmented information in this field using differential display, quantitative PCR and cDNA microarray analysis of endometrial epithelial-derived cell lines and endometrial samples to investigate the hierarchy at the mRNA level of molecules implicated in the process of endometrial receptivity.

Progesterone induces calcitonin expression in the baboon endometrium within the window of uterine receptivity.

The mammalian uterus can accept a developing blastocyst for implantation only within a limited period of time, termed the receptive phase. Our previous studies showed that the expression of calcitonin, a peptide hormone that regulates calcium homeostasis, is induced by progesterone immediately preceding implantation, and is required for the generation of a receptive rat uterus. In this study, we investigated the expression and hormonal regulation of calcitonin in the baboon endometrium during the window of implantation. We monitored the spatio-temporal expression of calcitonin at various days of the menstrual cycle. Reverse transcriptase-polymerase chain reaction analysis of the baboon endometrium on Days 9 and 10 postovulation revealed stage-specific expression of calcitonin mRNA, which overlapped with the window of uterine receptivity. Immunocytochemical analysis of baboon endometrium sections localized calcitonin expression in the glandular epithelial and stromal cells. Treatment of animals with the antiprogestin ZK 137.316 dramatically reduced calcitonin expression, indicating that calcitonin expression in the baboon endometrium is under progesterone regulation. Collectively, these findings strongly suggest that the appearance of calcitonin in progesterone-dominated endometrium is conserved among species and may serve as a marker of uterine receptivity for embryo implantation.

Expression of uterine receptivity markers in natural cycles and in cycles with hormonal substitution: Preliminary results of 7 women with Tubal-Factor infertility

Objective: Uterine receptivity plays a major role in normal embryo implantation and successful pregnancy. Recent studies have suggested that implantation occurs in a narrow window of endometrial receptivity between 5 and 7 days after ovulation. However, the role of uterine receptivity in human infertility remains unclear. In this study, we report our preliminary results of pinopode expression and levels of estrogen receptors (ER) and progesterone receptors (PR) in natural cycles and in cycles after standardized hormonal substitution used for cryo-embryo transfer in a group of women with tubal-factor infertility.

Endometrial function: cell specific changes in the uterine environment

The uterus undergoes dynamic changes during the cycle and these events are largely driven by ovarian steroids. However, in the presence of an embryo, an additional series of changes that are not otherwise observed predominate. The ability of the embryo to modulate the uterine environment is restricted to a specific time of the cycle which is termed the ‘window of receptivity’. Changes that occur within this window of receptivity and immediately following implantation can be divided into three distinct phases. The first phase, regulated by estrogen and progesterone, is characterized primarily by changes in both the luminal and glandular epithelial cells in preparation for blastocyst apposition and attachment. If the action of progesterone is antagonized, these changes are inhibited and the uterus is maintained in a pre-receptive state. The second phase is in the further modulation of these steroids induced changes by embryonic signals. In the primate, infusion of chorionic gonadotropin in a manner that mimics blastocyst transit, results in the endoreplication and plaque formation in the luminal epithelium. The glandular epithelium responds by increasing transcriptional and post-translational modifications of secretory proteins and the stromal fibroblasts initiate their differentiation process into a decidual phenotype. The final phase is associated with trophoblast invasion and remodeling of the endometrial stromal compartment. The most dramatic effect is on the stromal fibroblasts, which in response to embryonic stimuli, differentiate into decidual cells, the major cell type of the gestational endometrium. Thus, during the window of receptivity, signals from the embryo can dramatically alter the morphological and functional characteristics of the uterine endometrium. We suggest that these changes are critical to ensure prolonged maintenance of endometrial function during gestation and facilitate trophoblast invasion.

Endometrial markers of uterine receptivity utilizing the donor oocyte model.

Ethical constraints limit the ability to study peri-implantation phase human endometrium. In this study, the donor oocyte model was used to study candidate endometrial markers of uterine receptivity. Archived, paraffin-embedded tissue obtained by endometrial biopsy during cycle days 21-23 of patients undergoing 'mock' hormonal treatment cycles were evaluated by standard histological criteria and immunohistochemical staining for alpha v beta 3 integrin and glycodelin. All of these patients (n = 101) had undergone a donor oocyte embryo transfer cycle utilizing the exact same hormonal protocol. Histological evaluation revealed 62 (61.3%) in-phase, 34 (33.7%) dyssynchronous, 2 (2.0%) immature and 3 (3.0%) advanced endometria. The clinical outcomes of patients with either in-phase or dyssynchronous endometria were similar. Very strong correlations were noted between endometrial glandular dating and either alpha v beta 3 integrin or glycodelin immunostaining intensity (P < 0.001 for both). Glycodelin and alpha v beta 3 integrin immunostaining intensities were also highly correlated with each other (P < 0.001). Throughout the time period corresponding to the putative window of maximal endometrial receptivity (cycle days 21-23) a dynamic process was observed in exogenous hormonal replacement cycles characterized by a rapid histological advancement of endometrial glandular elements as well as progressive alpha v beta 3 integrin and glycodelin expression.

Genes involved in implantation.

The endometrium is normally a hostile environment for an embryo, except for a short phase in each reproductive cycle known as the 'window of receptivity'. The precise molecular events involved in this transformation are not well understood. Application of state-of-the-art techniques of the 1990s has identified some of the genes involved, which are reviewed here. Mice with a null mutation in either the gene for leukemia inhibitory factor or the interleukin-11 receptor alpha chain are infertile, owing in both cases to a failure of embryo implantation. Both of these genes are expressed in the human endometrium with patterns suggesting a role in human fertility. The technique of RNA differential display has been applied to a comparison of the expression of genes at implantation sites v. inter-implantation sites in the mouse uterus on the first day of implantation, and has defined additional genes whose products may be important for this process. Among these are the calcium-binding protein D9K, the monoclonal non-specific suppressor factor beta, and the splicing factor SC35. The major challenge is to determine whether manipulation of such genes can increase or decrease endometrial receptivity in humans.

Pinopodes as markers of endometrial receptivity in clinical practice.

Clinical evidence indicates the existence in the human of a narrow window of uterine receptivity which opens during the mid-luteal phase. At the same time, formation of pinopodes on the apical membranes of the endometrial epithelial cells occurs. To develop a specific marker for receptivity in clinical practice, the kinetics of pinopode formation has been investigated through sequential endometrial biopsying in natural, hormone replacement and stimulated cycles. The results show that pinopodes last <48 h in all cycles, and the timing of their formation depends both on the hormonal treatment applied and on the patient's individual response. On average, pinopodes form earlier in stimulated cycles and later in hormone replacement cycles, compared with natural cycles. Pinopode expression is strongly correlated with implantation following embryo transfer and furthermore, detection of pinopodes in assessment cycles may be extremely useful for the assessment of receptivity on an individual basis to optimize embryo transfer, resulting in increased implantation rates. Finally, pinopodes seem to be correlated well with other cellular and molecular events occurring in the mid-luteal endometrium.

Implantation in the baboon: endometrial responses.

Blastocyst implantation in the baboon usually occurs between 8 and 10 days post ovulation. Changes that occur within this window of receptivity and immediately following implantation can be divided into three distinct phases. The first phase, regulated by estrogen and progesterone, is characterized primarily by changes in both the luminal and glandular epithelial cells in preparation for blastocyst apposition and attachment. The second phase is the further modulation of these steroid induced changes in both epithelial and stromal cells by embryonic signals. The final phase is associated with trophoblast invasion and the remodeling of the endometrial stromal compartment. During the initial phase, the actions of estrogen and progesterone are dependent on the presence of specific receptors. Estrogen up-regulates both its own receptor (ER) and the progesterone receptor (PR), while progesterone down-regulates this expression pattern. However, the pattern of progesterone-induced down-regulation of ER and PR is confined to the epithelial cells and demonstrates a gradient effect from the functionalis to the basalis. What is most intriguing is that the loss of epithelial PR is closely correlated with the establishment of uterine receptivity. Coincident with the changes in ER and PR expression, epithelial cells undergo alterations in their cytoskeletal architecture and secretory profile. These changes can be counteracted by PR antagonist treatment during the luteal phase. Although estrogen and progesterone play a critical role in establishing the initial phase of uterine receptivity, it is becoming increasingly evident that the embryo induces functional receptivity in ruminants and rodents. In our studies in the primate, we demonstrate that chorionic gonadotrophin when infused in a manner that mimics blastocyst transit, has physiological effects on the three major cell types in the uterine endometrium. The luminal epithelium undergoes endoreplication and distinct epithelial plaques are evident. The glandular epithelium responds by inducing transcriptional and post-translational modifications in the major secretory product, glycodelin. The stromal fibroblasts initiate their differentiation process into a decidual phenotype and are characterized by the expression of actin filaments. In phase three, blastocyst attachment to the surface epithelium and subsequent implantation is associated with local remodeling of the maternal stroma, smooth muscle, and endothelium of the blood vessels by the trophoblast. In addition, there is a gradual diminution of the epithelial plaques on the luminal surface although the glandular epithelium remains highly secretory. The most dramatic effect is on the stromal fibroblasts, which in response to embryonic stimuli, differentiate into decidual cells, the major cell type of the gestational endometrium. This differentiation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1) in these cells. The cytokine IL-1 beta is one possible embryonic signal. COX-2 is the rate-limiting enzyme for prostaglandin biosynthesis and transcription of this enzyme in response to the embryonic stimulus (IL-1 beta) results in an increase in prostaglandin biosynthesis in stromal fibroblasts at the site of implantation. Prostaglandins and PGE2 in particular, binds to its specific receptor (EP2 or EP4) and activates adenyl cyclase. The resulting increase in intracellular levels of cAMP can now activate IGFBP-1 gene transcription at the site of implantation. In summary, our studies have demonstrated that chorionic gonadotrophin, when infused into non-pregnant baboons during the window of uterine receptivity can induce epithelial responses that are similar to those observed in a fertile cycle. Stromal differentiation is initiated; however, decidualization requires a signal from the conceptus.