Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase

The effect of intraoviductal embryos on endometrial receptivity was studied by intraendometrial and intrauterine embryo transfer. Five-week-old female ICR mice were mated after superovulation; a vaginal plug confirmed day 1 of pregnancy. On day 4 (90 h after hCG injection), blastocysts were collected and transferred to pseudopregnant female mice and to recipient mice in which the uterotubal junction had been ligated bilaterally on day 1 of pregnancy. Three embryos per uterine horn, a total of six embryos per recipient mouse at days 1-6, were transferred to the endometrium or uterine cavity and implantation and pregnancy rates were calculated. The implantation rate for intraendometrial embryo transfer to recipients of days 3, 5 and 6 was significantly higher for uterotubal junction-ligated mice (72.2, 20.8 and 9.7%, respectively) than for pseudopregnant mice (55.0, 8.3 and 0.0%, respectively). The implantation rate for intrauterine embryo transfer to recipients at days 2, 5 and 6 was significantly higher for uterotubal junction-ligated mice (11.1, 25.0 and 8.3%, respectively) than for pseudopregnant mice (0.0, 3.3 and 0.0%, respectively). Uterotubal junction-ligated mice achieved implantation and bore neonates by intrauterine embryo transfer on days 2 and 6, whereas no implantation was achieved in pseudopregnant mice. The difference in implantation rate could not be explained by a difference in progesterone concentration between the groups. The distribution of proliferating cells in the endometrium was also studied immunohistochemically by use of anti-proliferating cell nuclear antigen (PCNA) antibody in the recipient mice. PCNA-positive cells were more abundant in uterotubal junction-ligated mice and demonstrated a marked extension from the epithelium to the stroma over time, in contrast to those in pseudopregnant mice. These findings indicate that an intraoviductal embryo exerts a biological effect by sending a signal to the endometrial epithelium and stroma, thus facilitating endometrial receptivity to the embryo and improving the rate of implantation.     

Effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation

Mouse embryo implantation depends on the complex interaction between the embryo trophoblast cells and the uterine environment, which deposits an extracellular matrix with abundant amounts of laminin. Intrauterine injection and blastocyst or ectoplacental cone culture models were used to study the effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation in vivo and in vitro. Intrauterine injection of 32/67 kDa laminin-binding protein antibody (0.4 mg in 1 ml Ham's F-10 medium, 5 microl per mouse) into the left uterine horns of mice (n = 22) on day 3 of pregnancy inhibited embryo implantation significantly (P < 0.001) compared with the contralateral horns that had been injected with normal rabbit IgG. A continuous section study on day 5 after injection showed that the embryos in the control uteri implanted normally and developed healthily, but there were no embryos or the remaining embryos had disintegrated in the uteri injected with 32/67 kDa laminin-binding protein antibody. Blastocysts or ectoplacental cones were cultured in media containing 32/67 kDa laminin-binding protein antibody (0.2 mg ml(-1)) on laminin-coated dishes with normal rabbit IgG at the same concentration as in the controls. The 32/67 kDa laminin-binding protein had no effect on blastocyst or ectoplacental cone attachment, but prohibited the blastocyst or ectoplacental cone outgrowth and primary or secondary trophoblast giant cell migration. These results indicate that 32/67 kDa laminin-binding protein antibody blocked mouse embryo implantation by preventing embryo trophoblast cell invasion and migration through the uterine decidual basement membrane-like extracellular matrix which has a high laminin content.     

Decidualization and implantation: embryo-uterine bioinformatics at work

The implantation of the blastocyst into a nurturing endometrium involves two overlapping steps: 1. The blastocyst-endometrial luminal epithelial attachment. 2. The decidualization of the endometrial stroma. An intriguing question is how does the blastocyst identify the uterine implantation site. Current research is focused on hypothetical soluble signaling molecules released by the blastocyst for conditioning a discrete uterine luminal epithelial domain for implantation. A still unresolved issue is the functional significance of receptor autophosphorylation following binding of uterine epithelial cell-derived heparin-binding epidermal growth factor-like growth factor to the epidermal growth factor receptor on trophoectodermic cell surfaces. With recent results hinting at the role of signaling proteins associated with the bone morphogenetic protein, fibroblast growth factor, WNT and hedgehog families to enable embryo implantation, the dynamics of uterine-embryo interaction becomes linked to fundamental cellular pathways of growth, differentiation and apoptosis.     

Paracrine effects of bFGF and KGF on the process of mouse blastocyst implantation

Implantation is a complex process that requires the interaction of the blastocyst, and subsequently, that of the developing embryos with the endometrium. Several growth factors and cytokines are involved in implantation, but the details of their actions as related to the regulation of blastocyst implantation remain unclear. In the present study, the RT-PCR method was used to determine the gene expression of basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1 (FGFR1), FGF receptor 2 (FGFR2), and KGF receptor (KGFR) in mouse embryos and in the stromal and epithelial cells of the uterine endometrium. Basic FGF and KGF mRNA were expressed in the endometrial cells, but were not expressed in the embryos. The mRNAs of receptors for bFGF and KGF were expressed in the blastocysts and in the in vitro implanting embryos, suggesting that bFGF and KGF may exert paracrine effects on blastocyst implantation. In this mouse model of blastocyst implantation, it was found that transforming growth factor α (TGF-α) at the concentrations of 1 ng/ml and 10 ng/ml significantly enhanced the blastocyst attachment and trophoblast spreading and increased trophoblast surface area. Relatively high concentrations of bFGF (100–500 ng/ml) significantly enhanced the rates of blastocyst attachment and of trophoblast spreading and promoted the expansion of the surface area of the implanting embryos. Unlike the rates of blastocyst attachment and trophoblast spreading, the surface area of the spreading embryos was significantly increased by addition of KGF (1–100 ng/ml). These results suggest that the bFGF and KGF derived from the endometrial cells exert paracrine effects on the process of implantation by stimulating trophoblast outgrowth through their cognate receptors. Mol. Reprod. Dev. 50:54–62, 1998. © 1998 Wiley-Liss, Inc.     

Development of a high-throughput assay for human proprotein convertase 5/6 for detecting uterine receptivity

Embryo implantation requires a healthy embryo and a receptive uterus. In women, the inner lining of the uterus, the endometrium, remains in a hostile state and becomes receptive for embryo implantation for only a short period during each menstrual cycle. Determining endometrial receptivity is vital in in vitro fertilization (IVF) treatment because the timing of embryo transfer needs to be synchronized with endometrial receptivity. We have previously demonstrated that proprotein convertase 5/6A (PC6) is highly expressed in the receptive endometrium and that PC6 is critical for receptivity establishment in women. Furthermore, endometrial PC6 is secreted into the uterine fluid, and levels correlate with receptivity status. Detection of PC6 in uterine fluids, therefore, would provide a nonsurgical assessment of endometrial receptivity. However, to date no assays are available for human PC6. In this study, we produced three PC6 monoclonal antibodies (mAbs) and developed a sandwich enzyme-linked immunosorbent assay (ELISA) for PC6 detection in human uterine fluids. The PC6 mAbs were confirmed to be highly specific to PC6, and the ELISA detected PC6 in human uterine fluids with a significantly higher level during the receptive phase. This newly established PC6 ELISA provides an important tool in the development of noninvasive strategies to detect endometrial receptivity in women.     

Bmp2 is critical for the murine uterine decidual response

The process of implantation, necessary for all viviparous birth, consists of tightly regulated events, including apposition of the blastocyst, attachment to the uterine lumen, and differentiation of the uterine stroma. In rodents and primates the uterine stroma undergoes a process called decidualization. Decidualization, the process by which the uterine endometrial stroma proliferates and differentiates into large epithelioid decidual cells, is critical to the establishment of fetal-maternal communication and the progression of implantation. The role of bone morphogenetic protein 2 (Bmp2) in regulating the transformation of the uterine stroma during embryo implantation in the mouse was investigated by the conditional ablation of Bmp2 in the uterus using the (PR-cre) mouse. Bmp2 gene ablation was confirmed by real-time PCR analysis in the PR-cre; Bmp2fl/fl (termed Bmp2d/d) uterus. While littermate controls average 0.9 litter of 6.2+/-0.7 pups per month, Bmp2d/d females are completely infertile. Analysis of the infertility indicates that whereas embryo attachment is normal in the Bmp2d/d as in control mice, the uterine stroma is incapable of undergoing the decidual reaction to support further embryonic development. Recombinant human BMP2 can partially rescue the decidual response, suggesting that the observed phenotypes are not due to a developmental consequence of Bmp2 ablation. Microarray analysis demonstrates that ablation of Bmp2 leads to specific gene changes, including disruption of the Wnt signaling pathway, Progesterone receptor (PR) signaling, and the induction of prostaglandin synthase 2 (Ptgs2). Taken together, these data demonstrate that Bmp2 is a critical regulator of gene expression and function in the murine uterus.     

Effects of leukaemia inhibitory factor on embryo implantation in the mouse

Leukaemia inhibitory factor (LIF) is a pleiotrophic cytokine. Recent reports indicate that LIF is relevant to murine embryo implantation. In this work, results of indirect immunofluorescence under a confocal microscope illustrated that LIF was mainly located in the uterine lumen and uterine epithelial cells in pregnant mice on day 4. The number of embryos implanted in pregnant mice on day 8 decreased significantly after injection of 3 microg LIF antibodies into a uterine horn (P<0.001), which demonstrated again that LIF is a critical factor for embryo implantation. In a co-culture system, LIF (0.1 ng/ml, 1 ng/ml, 10 ng/ml and 100 ng/ml) significantly enhanced the blastocyst outgrowth after 24, 48 or 72 h of co-culture, and outgrowth areas after 72 h of co-culture. Conversely, 5 microg/ml and 10 microg/ml, but not 1 microg/ml, LIF antibodies decreased the percentage of blastocysts with outgrowth; only 10 microg/ml LIF antibody inhibited blastocyst outgrowth area significantly (P<0.001). However, neither LIF nor its antibodies changed embryo attachment. Analysis of correlation showed that the effects of LIF or its antibodies on the blastocyst outgrowth were dose-dependent. In summary, different pathways may exist to regulate the blastocyst attachment and outgrowth on a monolayer of uterine epithelial cells. LIF protein from the maternal uterus exerts an essential role in embryo implantation in the mouse, which is mediated by stimulating trophoblast outgrowth, but not by promoting the attachment.     

Rabbit endometrium in organ culture: Morphological evidence for progestational differentiation in vitro

Summary This communication describes conditions for long-term organotypic culture of rabbit endometrium allowing progesterone-induced transformation, as typcial for early pregnancy, to continue in vitro. This system appears to compare favorably with in vitro models so far proposed for the study of hormonal control of uterine function or for the investigation of cell-biological aspects of embryo implantation. The specific aim in the presented system is to provide approximate normal epithelium-stroma interrelationships. Fragments of endometrium consisting of epithelium and stroma were obtained during early pseudopregnancy and cultured on a gyratory shaker. Morphology was investigated by light microscopy, transmission and scanning electron microscopy. During the first two days the epithelium grows over the exposed stroma regenerating a complete epithelial lining. No central necrosis is found in the stroma for up to 6 days, and the tissue keeps its organotypic architecture although certain morphological differences can be observed between regenerated versus original epithelium. In the regenerating portion a stage-specific cell differentiation and the reformation of a basal lamina are missing. Progesterone substitution preserves cell morphology and allows to maintain, in vitro, the stage-specific pattern of cell organelles. Most characteristic is the induction of extensive fusion of epithelial cells. These large symplasms are comparable in size and structure to those formed in pregnancy in the implantation chamber in vivo. Only the superficial parts of the original (not the regenerated) epithelium are capable of progesterone-induced large-scale fusion. This organotypical culture system appears to be of potential value for in vitro studies on hormone action and on endometrial receptivity for embryo implantation.Abbreviations d.p.c. dies post coitum - d p. hCG days after injection of hCG (dies post injectionem hCG) - FBS fetal bovine serum - hCG human chorionic gonadotropin Preliminary results were presented by Denker et al. (1984) and by Hohn et al. (1984)     

Annexin A2 Acts as an Adhesion Molecule on the Endometrial Epithelium during Implantation in Mice

To determine the function of Annexin A2 (Axna2) in mouse embryo implantation in vivo, experimental manipulation of Axna2 activities was performed in mouse endometrial tissue in vivo and in vitro. Histological examination of endometrial tissues was performed throughout the reproduction cycle and after steroid treatment. Embryo implantation was determined after blockage of the Axna2 activities by siRNA or anti-Axna2 antibody. The expression of Axna2 immunoreactivies in the endometrial luminal epithelium changed cyclically in the estrus cycle and was upregulated by estrogen. After nidatory estrogen surge, there was a concentration of Axna2 immunoreactivities at the interface between the implanting embryo and the luminal epithelium. The phenomenon was likely to be induced by the implanting embryos as no such concentration of signal was observed in the inter-implantation sites and in pseudopregnancy. Knockdown of Axna2 by siRNA reduced attachment of mouse blastocysts onto endometrial tissues in vitro. Consistently, the number of implantation sites was significantly reduced after infusion of anti-Axna2 antibody into the uterine cavity. Steroids and embryos modulate the expression of Axna2 in the endometrial epithelium. Axna2 may function as an adhesion molecule during embryo implantation in mice.     

A proteomic atlas of ligand–receptor interactions at the ovine maternal–fetal interface reveals the role of histone lactylation in uterine remodeling

Well-orchestrated maternal–fetal cross talk occurs via secreted ligands, interacting receptors, and coupled intracellular pathways between the conceptus and endometrium and is essential for successful embryo implantation. However, previous studies mostly focus on either the conceptus or the endometrium in isolation. The lack of integrated analysis impedes our understanding of early maternal–fetal cross talk. Herein, focusing on ligand–receptor complexes and coupled pathways at the maternal–fetal interface in sheep, we provide the first comprehensive proteomic map of ligand–receptor pathway cascades essential for embryo implantation. We demonstrate that these cascades are associated with cell adhesion and invasion, redox homeostasis, and the immune response. Candidate interactions and their physiological roles were further validated by functional experiments. We reveal the physical interaction of albumin and claudin 4 and their roles in facilitating embryo attachment to endometrium. We also demonstrate a novel function of enhanced conceptus glycolysis in remodeling uterine receptivity by inducing endometrial histone lactylation, a newly identified histone modification. Results from in vitro and in vivo models supported the essential role of lactate in inducing endometrial H3K18 lactylation and in regulating redox homeostasis and apoptotic balance to ensure successful implantation. By reconstructing a map of potential ligand–receptor pathway cascades at the maternal–fetal interface, our study presents new concepts for understanding molecular and cellular mechanisms that fine-tune conceptus–endometrium cross talk during implantation. This provides more direct and accurate insights for developing potential clinical intervention strategies to improve pregnancy outcomes following both natural and assisted conception.     

Ubiquitin-specific protease 7 (USP7) is essential for endometrial stromal cell decidualization in mice

Ubiquitin-specific protease 7 (USP7), a member of the deubiquitinating (DUB) enzyme family, regulates protein stability and has a well-characterized function in tumorigenesis. Given its critical role in growth and development, it was speculated to be involved in modulating processes in the female reproductive system but its exact role has not been elucidated. Decidualization is one of the key processes in pregnancy and aberrant decidualization is a cause of pregnancy failure. The uterine endometrium layer undergoes significant structural and functional changes during decidualization in preparation for and after embryo implantation. Here, we hypothesized that USP7 could be involved in mediating endometrial stromal cell (ESC) decidualization and set out to determine its function with a primary stromal cell culture. Using in situ hybridization and immunohistochemical techniques, we observed increased USP7 expression during uterine decidualization and found that it was predominantly localized to the decidual zone in the post-implantation uterus. Since the ovarian hormones, progesterone (P4) and estrogen (E2), function in promoting stroma decidualization, we investigated their relationship with USP7 expression and found that they exert minimal influence. Moreover, increased USP7 expression observed during deciduoma development was found to be independent of blastocyst attachment. Using a specific USP7 inhibitor, HBX19818, we demonstrated an additional novel role for USP7 in endometrial stroma decidualization in mice during early pregnancy. Our findings could potentially be applied towards future research and development in female infertility.     

Aberrant expression of retinol-binding protein, osteopontin and fibroblast growth factor 7 in the porcine uterine endometrium of pregnant recipients carrying embryos produced by somatic cell nuclear transfer

The technique of somatic cell nuclear transfer (NT) is a useful tool to produce cloned animals for various purposes, but the efficiency to generate cloned animals using this technique is still very low. To improve the low efficiency in production of cloned pigs it is critical to understand the reprogramming process during development of cloned embryos, but it is also essential to understand the uterine function interacting with the transferred cloned embryos during implantation and placentation. Thus, to understand the uterine responsiveness to NT cloned embryos during pregnancy, we investigated expression of retinol-binding protein (RBP), osteopontin (OPN) and fibroblast growth factor 7 (FGF7), which play important roles in implantation and/or maintenance of pregnancy as a transport protein, an extracellular matrix protein and a growth factor, respectively, in the uterine endometrium in pigs. The uterine tissue samples were obtained by C-section from pigs with NT cloned normal (NT-normal) embryos and NT cloned abnormal (NT-abnormal) embryos and pigs with non-NT (Non-NT) embryos at term. Immunoblot analysis showed that expression of RBP and FGF7 decreased in the uterine endometrium of recipient gilts carrying NT embryos than in the endometrium of gilts carrying Non-NT embryos. Levels of OPN protein of 70 and 45kDa were not different in between the uterine endometrium of gilts carrying Non-NT and NT-normal embryos, but in the uterine endometrium of gilts carrying NT-abnormal embryos 70 and 45kDa OPN proteins increased compared to those in the endometrium of gilts carrying Non-NT embryos. Immunohistochemistry results showed that RBP expression was lower in the endometrial glandular epithelial cells, while OPN expression was higher in the endometrial luminal epithelial cells of the uterus of gilts carrying NT embryos than in the uterus of gilts carrying Non-NT embryos. Results of this study showed that maternal uterine genes were aberrantly expressed in the uterine endometrium of gilts carrying NT cloned embryos in varying degrees depending on the normality of the developing embryos. These results indicate that abnormal maternal-fetal interactions of the uterus carrying the developing NT cloned embryos may cause problems in development of cloned embryos.     

Early implantation stages in the marmoset monkey (Callithrix jacchus)

The morphology of the initial stages of implantation in the marmoset monkey (Callithrix jacchus) was studied by obtaining embryos and associated endometrium at timed intervals after ovulation. Estrus cycles were detected by measuring daily levels of plasma progesterone. Following a short follicular phase, circulating levels of progesterone above 20 ng/ml were taken as representing day 1 after ovulation. On this basis, single, twin, and triplet embryos were recovered from six perfused-fixed females on days 13, 16, 19, 23, and 29 after ovulation and prepared in resin for light microscopy. Early implantation stages, 13 and 16 days after ovulation, were characterized by the intrusion of syncytial trophoblast between epithelial cells of the endometrium with minimal cellular damage. Some hyperplasia of epithelium at the margin of the implantation site was evident. The consolidation of the initial attachment was achieved by an increase in syncytial trophoblast underlying the inner cell mass of the embryo which rapidly surrounded and breached maternal capillaries. Although initially separate, the chorions of twin or triplet embryos started to fuse by day 19 after ovulation. This process was complete by day 29 such that embryos shared a common uterine exocoelom surrounded by continuous trophoblast. It was concluded that implantation in the marmoset monkey commenced on days 11-12.5 after ovulation and involved an intrusive mechanism. Although trophoblast penetration of endometrium was superficial, maternal capillaries were tapped at an early stage of implantation. The fusion of chorions of twins and triplets first occurred around day 19 after ovulation.     

Omics insights into extracellular vesicles in embryo implantation and their therapeutic utility

Implantation success relies on intricate interplay between the developing embryo and the maternal endometrium. Extracellular vesicles (EVs) represent an important player of this intercellular signalling through delivery of functional cargo (proteins and RNAs) that reprogram the target cells protein and RNA landscape. Functionally, the signalling reciprocity of endometrial and embryo EVs regulates the site of implantation, preimplantation embryo development and hatching, antioxidative activity, embryo attachment, trophoblast invasion, arterial remodelling, and immune tolerance. Omics technologies including mass spectrometry have been instrumental in dissecting EV cargo that regulate these processes as well as molecular changes in embryo and endometrium to facilitate implantation. This has also led to discovery of potential cargo in EVs in human uterine fluid (UF) and embryo spent media (ESM) of diagnostic and therapeutic value in implantation success, fertility, and pregnancy outcome. This review discusses the contribution of EVs in functional hallmarks of embryo implantation, and how the integration of various omics technologies is enabling design of EV-based diagnostic and therapeutic platforms in reproductive medicine.     

Differential effects of p-nitrophenyl-D-xylosides on mouse blastocysts and uterine epithelial cells

A number of studies have implicated glycoconjugates in cell recognition events associated with implantation of mammalian blastocysts into the uterus. We have found that p-nitrophenyl-D-xylosides inhibit mouse embryo attachment and outgrowth on monolayers of uterine epithelial cells when cocultured in vitro. Inhibition of attachment and trophoblast formation by alpha- and beta-xylosides was observed in embryos cultured on tissue culture plastic in serum containing medium or on monolayers of epithelial cells. The biochemical basis for this inhibition has been investigated. Consistent with their accepted mode of action, beta- but not alpha-D-xylosides greatly stimulated glycosaminoglycan chain production by uterine epithelial cells and likewise reduced proteoglycan assembly. In contrast, both alpha- and beta-anomers selectively inhibited embryo attachment and outgrowth without stimulating glycosaminoglycan chain production by embryos. The inhibitory effect of the xylosides on embryos was reversible and did not require concentrations that reduced the rate of protein synthesis. Both alpha- and beta-D-xylosides inhibited the synthesis of proteoglycans including heparan sulfate as well as certain other glycoconjugates by embryos. Collectively, these data indicate that proper assembly of glycoconjugates, including proteoglycans, is required for implantation-related processes, although the inhibition of embryo outgrowth by xylosides may be by an as yet uncharacterized mechanism.     

Zonula occludens-1 and E-cadherin are coordinately expressed in the mouse uterus with the initiation of implantation and decidualization

Two-way interactions between the blastocyst trophectoderm and the uterine luminal epithelium are essential for implantation. The key events of this process are cell-cell contact of trophectoderm cells with uterine luminal epithelial cells, controlled invasion of trophoblast cells through the luminal epithelium and the basement membrane, transformation of uterine stromal cells surrounding the blastocyst into decidual cells, and protection of the "semiallogenic" embryo from the mother's immunological responses. Because cell-cell contact between the trophectoderm epithelium and the luminal epithelium is essential for implantation, we investigated the expression of zonula occludens-1 (ZO-1) and E-cadherin, two molecules associated with epithelial cell junctions, in the mouse uterus during the periimplantation period. Preimplantation uterine epithelial cells express both ZO-1 and E-cadherin. With the initiation and progression of implantation, ZO-1 and E-cadherin are expressed in stromal cells of the primary decidual zone (PDZ). As trophoblast invasion progresses, these two molecules are expressed in stroma in advance of the invading trophoblast cells. These results suggest that expression of these adherence and tight junctions molecules in the PDZ serves to function as a permeability barrier to regulate access of immunologically competent maternal cells and/or molecules to the embryo and provide homotypic guidance of trophoblast cells in the endometrium.