Human Implantation Process Research Articles

P-793 Human trophoblastic spheroid derived from human expanded potential stem cells (hEPSC) for the identification of adhesion molecules for embryo implantation.

Abstract Study question Are cell surface proteins on trophoblastic spheroid BAP-EB derived from hEPSC involved in the endometrial epithelial cell attachment? Summary answer E-cadherin (CDH1) and Neuropilin-1 (NRP1) are two potential embryonic surface molecules involved in the human implantation process. What is known already Study of the mechanisms of human implantation in vivo is challenging. The availability of high-quality human embryos for in vitro research is limited. We have recently derived morula-like hEPSC lines (hEPSC-em) from donated human embryos. Trophoblastic spheroids (em-BAP-EB) derived from hEPSC-em resemble human trophectoderm during the early implantation process. The em-BAP-EBs attach specifically onto receptive endometrial epithelial cells, suggesting its usefulness as human embryo surrogate for studying embryonic surface molecules involving in early implantation process. Study design, size, duration hEPSC-em was subjected to embryoid body formation and induced to differentiate into trophoblastic spheroids (em-BAP-EB) for 48h and 72h. Endometrial aspirates were collected from infertile women in their natural cycles 2 or 7 days after LH surge (LH + 2/+7). Endometrial epithelial cells (EEC) were isolated and cultured for the attachment assays. Biotin-labelled apical surface proteins on em-BAP-EB were identified by mass spectrometry. Antibody blocking assays were performed to confirm the biological functions. Participants/materials, setting, methods The attachment rates of em-BAP-EB onto EEC were compared between pre-receptive (LH + 2 day) and receptive (LH + 7 day) phases. The differentially expressed apical surface protein on em-BAP-EB-48h and -72h were subjected to plasma membrane proteins (PMP) identification and pathway analysis. The gene and protein expression patterns of the target proteins were analyzed by single cell RNA sequencing (scRNA-seq) and western blotting respectively. The functional roles of the target proteins were studied by antibody blocking during attachment Main results and the role of chance The attachment rates of em-BAP-EB-72h onto receptive stage (LH + 7, n = 17) EEC were significantly higher than those onto pre-receptive stage (LH + 2, n = 5) EEC. Gene Ontology analysis indicated that the PMP with induced expression levels on em-BAP-EB-72h when compared to -48h (72h>48h) were enriched for “protein binding” and “cadherin binding” in molecular functions, and “cell-cell adhesion” in biological processes. Among them, 28 PMP were trophectoderm-specific. CDH1 and NRP1 were selected for further analysis. Analysis based on a published dataset showed that significantly higher expressions of CDH1 and NRP1 were detected in polar trophectoderm as compared to mural trophectoderm of E7 human blastocyst. CDH1 and NRP1 were significantly up-regulated in trophectoderm-like em-BAP-EB by scRNA-seq analysis. Live-cell immunocytochemistry demonstrated more intense apical surface expression of CDH1 and NRP1 on em-BAP-EB-72h as compared to -48h. Antibody blocking experiments indicated that pre-treatment of the em-BAP-EB-72h with specific antibody individually against the ectodomains of CDH1 and NRP1 significantly reduced the attachment rates onto receptive endometrial epithelial cells. The data suggested that CDH1 and NRP1 are two potential apical surface molecules essential for blastocyst attachment. Limitations, reasons for caution The lack of embryonic compartment in the em-BAP-EB and the absence of other key players like decidualized stromal cells and immune cells in the coculture model might not fully represent the early attachment process. The limited sample size in this study may also limit the interpretation of the data. Wider implications of the findings Despite advancement in in vitro fertilization, the success rate remains unsatisfactory. It is not known if embryological factor(s) other than embryo aneuploidy are involved. The current data may suggest the potential roles of CDH1 and NRP1 as non-chromosomal embryological markers of its implantation potential. Trial registration number Not applicable

3D model of the maternal-fetal interface: challenges, recent advances and beyond.

Embryo implantation is a complex and highly coordinated process that involves an intricate network of factors establishing intimate contact at the maternal-fetal interface. Knowledge of the human implantation process is compromised by both ethical issues, which do not allow the study of this process in vivo, and by the accuracy and reproducibility of in vitro models of human endometrium. Effective and reliable embryo implantation models are, therefore, necessary to mimic the molecular event cascade that occurs in vivo. 3D models are considered a new step to foster precision medicine and an advanced tool for the study of endometrial biology, endometrium associated diseases and to understand the complex mechanisms surrounding endometrium-embryo crosstalk. In this review we explore the various methods by which 3D cultures of endometrium and trophoblast can be created, exploring targets and applications of these in vitro models.

Potential of Stem Cells in Overcoming Infertility Problems in Women

Stem cells are characterized as cells with undifferentiated kinds that have the ability to differentiate into a variety of various cell types in response to the environment in which they are growing and develop (niche). Stem cells are classified into three broad categories based on their origin: extra-embryonic stem cells, which are derived from the amniotic fluid, umbilical cord and placenta; adult stem cells, which are derived from adult tissues such as blood, fat, bone marrow and skin; and embryonic stem cells, which are derived from the blastocyst. Stem cells exposed to certain suitable conditions will differentiate into 3 germ layers and also primordial germ cells. This is the basis for the latest research to obtain mature haploid gametes capable of developing into normal embryos and fetuses. Somatic Nuclear Cell Transfer (SNCT) technique is used to produce mature gametes so that the resulting cells contain cell nuclei with new genetic material. Infertility is a common problem that occurs with a prevalence of 10-15% of couples of reproductive age. Causes of infertility in women include metabolic and hormonal disorders accompanied by interactions with environmental factors that reduce oocyte quality. This results in an increased rate of aneuploidy in the resultant oocytes and impairs the human implantation process. The main objective of this research is to enhance the understanding of stem cells in women's infertility. The method used in writing this review article is online literature studies obtained by accessing national and international scientific journals as well as scientific articles related to stem cells (Stem cells), infertility, women. From the results of this study, it can be seen the potential of stem cells (stem cells) in treating infertility in women. Further studies are needed, especially pre-clinical and clinical trials so that they can be widely applied.

APOE polymorphism status (E4) may help in predicting the risk of recurrent implantation failure.

To examine the association between apolipoprotein E (APOE) gene polymorphisms and incidence of recurrent implantation failure (RIF). In a case-control study, 100 women with RIF were compared with 100 women with at least one live child. DNA was extracted from the peripheral blood and APOE genotyping was performed through polymerase chain reaction, followed by restriction fragment length polymorphism. Statistical analysis was performed using Pearson's χ2 test. Our data revealed a significantly higher frequency for the E3/E4genotype and E4 allele in the RIF group compared with controls. Significant differences in frequencies of the E4 allele (odds ratio [OR] 2.176; 95% confidence interval [CI] 1.131-4.185; P=0.026) and E3/E4genotype (OR 2.203; 95% CI 1.092-4.443; P=0.038) were observed between the groups. The E4 polymorphism is correlated with RIF occurrence in women undergoing assisted reproductive treatment and potentially can be considered as a risk factor to the human implantation process.

The role of mediating factors involved in angiogenesis during implantation.

Angiogenesis is a critical component of normal implantation and placentation and underlines the importance of vascularization in early pregnancy. Differentiated expression of angiogenesis factors in different decision tissues during different stages of implantation, indicates their involvement in the regulation of vascular remodeling and angiogenesis. Disorders in vascular development may play a role in the pathogenesis of recurrent abortions. The success of implantation, placentation and subsequent pregnancy evolution requires coordination of vascular development and adaptations at both sides of the maternal–fetal interface. The human implantation process is a continuous process, which begins with the apposition and attachment of the blastocyst to the apical surface of the luminal endometrial epithelium and continues throughout the first trimester of pregnancy until the extravillous trophoblast invades and remodels maternal vascularization. Numerous regulatory molecules play functional roles in many processes, including preparation of the endometrial stroma (decidualization), epithelium for implantation, control of trophoblastic adhesion and invasion. These regulatory molecules include cytokines, chemokines, and proteases, many of which are expressed by different cell types, having slightly different functions as the implant progresses

TROPHOBLAST INVASION AND UTERIOIMPLANTATION GROWTH IN EARLY RAT GESTATION REQUISITE ADRENOMEDULLIN

Objective: Adrenomedullin (ADM) is a currently exposed hypotensive peptide that is articulated in a variety of cell and tissue types. The existence of ADM has been immunohistochemically demonstrated in pathologic uterioimplantation regions, but no systematic study of ADM expression in early pregnancy in uterioimplantation region has been reported.Methods: Rats on the gestational day 2 were implanted subcutaneously with osmotic (Alzet) minipumps delivering 125 and 250 μg rat/day/of AM22–52 and were killed on the gestational day 9. We have ascertained the hypothesis that ADM, a multiregulatory ubiquitous peptide hormone, works as a trophoblast pro-invasion factor.Results: We confirmed ADM significance in in vitro assay using ACH-3P cell line, which is first-trimester trophoblast cell line.Conclusion: Our data support the conception that ADM is involved in the human implantation process through regulating trophoblast proliferation and differentiation.

Role of selectins and their ligands in human implantation stage.

Selectins are a family of calcium-dependent, type I transmembrane, carbohydrate-binding glycoproteins. Selectins and their ligands are not only involved in physiological processes such as leukocyte homing and pathological processes such as cancer, but also play an essential role in the human implantation. L-selectin and its ligands participate in the adhesion of the blastocyst to the endometrium at the maternal-fetal interface. P-selectin and E-selectin are involved in immune recognition of maternal decidua to the embedded embryo as well as trophoblast migration within decidual spiral arterioles. Moreover, altered expression of selectins and their ligands are found to be associated with some abnormal pregnancies and infertilities. This review focuses on the current progress of research on the role of selectins and their ligands in the human implantation process.

Effets antiprolifératifs et proapoptotiques du bisphénol A dans les cellules trophoblastiques humaines JEG-3

Summary Different studies performed in rodents revealed that Bisphenol-A (BPA), an environmental compound, altered early embryonic development. However, little is known concerning the direct effects of BPA on human implantation process. Thus, we decided to study in vitro BPA's effects on proliferative capacities of the human trophoblastic cell line, JEG-3. For this purpose, we first have shown that JEG-3 cells express the specific BPA receptor, namely estrogen-related receptor γ1 (ERRγ1). Secondly, once the non-toxic effect of BPA on JEG-3 cell viability was verified, [3H]-thymidine incorporation experiments were performed and revealed that BPA significantly reduced cell proliferation. The results also showed that BPA induced JEG-3 apoptosis as reflected by DNA fragmentation experiments. In conclusion, we describe here the direct impact of BPA on trophoblastic cell number mediated through both antiproliferative and proapoptotic effects and could then participate in the impact of BPA on placental development.

Annexin A2 is critical for embryo adhesiveness to the human endometrium by RhoA activation through F‐actin regulation

Annexin A2 (ANXA2) is present in vivo in the mid- and late-secretory endometria and is mainly localized in the luminal epithelium. Our aim was to evaluate its function in regulating the human implantation process. With an in vitro adhesion model, constructed to evaluate how the mouse embryo and JEG-3 spheroids attach to human endometrial epithelial cells, we demonstrated that ANXA2 inhibition significantly diminishes embryo adhesiveness. ANXA2 is also implicated in endometrial epithelial cell migration and trophoblast outgrowth. ANXA2 was seen to be linked to the RhoA/ROCK pathway and to regulate cell adhesion. We noted that ANXA2 inhibition significantly reduces active RhoA, although RhoA inactivation does not alter the ANXA2 levels. RhoA inactivation and ROCK inhibition also moderate embryo adhesiveness to endometrial epithelial cells. We corroborated that the induction of constitutively active RhoA partially reverses the effects of ANXA2 inhibition on endometrial adhesiveness. These molecules colocalize on the plasma membrane of endometrial epithelial cells, and a large proportion of ANXA2 and RhoA are colocalized in the F-actin networks. The functional effects of ANXA2 inhibition and RhoA/ROCK inactivation are associated with significant alterations in F-actin organization and its depolymerization. ANXA2 may act upstream of the RhoA/ROCK pathway by regulating F-actin remodeling and is a key factor in human endometrial adhesiveness.

Antiproliferative and proapoptotic effects of bisphenol A on human trophoblastic JEG-3 cells

Different studies performed in rodents revealed that bisphenol-A (BPA), an environmental compound, altered early embryonic development. However, little is known concerning the direct effects of BPA on human implantation process. Thus, we decided to study in vitro BPA's effects on proliferative capacities of the human trophoblastic cell line, JEG-3. For this purpose, we first have shown that JEG-3 cells express the specific BPA receptor, namely estrogen-related receptor γ1 (ERRγ1). Secondly, we demonstrated that BPA did not exert any cytotoxic action in JEG-3 cells up to 10 −6 M. Moreover [ 3H]-thymidine incorporation experiments revealed that BPA significantly reduced cell proliferation. The results also showed that BPA induced JEG-3 apoptosis capacity as reflected by DNA fragmentation experiments. In conclusion, we describe here the direct impact of BPA on trophoblastic cell number mediated through both anti-proliferative and pro-apoptotic effects.

Cytokines and Growth Factors in Implantation

Implantation failure is the most important rate limiting factor in the success of assisted reproductive techniques like In vitro fertilization–embryo transfer (IVF-ET). Cytokines are multifunctional signaling molecules having an implicit role in the human implantation process. This review focuses on the redundant roles of cytokines during the various stages of implantation. It also indicates that levels of cytokines in biological fluids like serum and follicular fluid obtained during oocyte retrieval might act as determinants of implantation potential of the blastocyst. Thus a holistic, metabolomic approach of analyzing biological fluids may provide a simpler approach to study the hitherto enigmatic process rather than the proteomic and genomic approach.

Update on the role of leukemia inhibitory factor in assisted reproduction

To review the recent literature on the involvement and importance of leukemia inhibitory factor (LIF) in the human implantation process, and the attempts using LIF-based interventions to improve assisted reproductive technologies (ARTs) outcome in women with recurrent implantation failure. High LIF expression is an indicator of receptive endometrium in fertile women. However, in infertile individuals, the data on endometrial LIF expression and secretion are controversial. Even after ruling out other causes of infertility, such as tubal, endocrine, male factor, and endometriosis, LIF-only detection is not sufficient for assessment of implantation potential in women with unexplained infertility. This is obviously in contrast to evidence of the crucial role of LIF in mouse endometrial physiology. In a large multicenter study, recombinant human LIF failed to improve the outcome of IVF treatment in women with recurrent implantation failure. A better comprehension of the mechanisms underlying endometrial receptivity and implantation should guide clinicians through proper management and treatment of infertility and implantation failure, and may eventually enable widespread adherence to single embryo transfer practices.

HB-EGF but Not Amphiregulin or Their Receptors HER1 and HER4 Is Altered in Endometrium of Women With Unexplained Infertility

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and its receptors (HER1 and HER4) play a role in the human implantation process. Amphiregulin is a member of the EGF family but with unknown function in human fertility. It has been suggested that some women with unexplained infertility have defective endometrial development. The aim of this study is to determine the presence of amphiregulin and the receptors HER1 and HER4 in normal human endometrium throughout the menstrual cycle. In addition, the present study aims to compare endometrium from women with unexplained infertility with endometrium from women with male factor infertility and healthy fertile controls. Immunohistochemistry and real-time polymerase chain reaction were used to determine the expression of HB-EGF, HER1, HER4, and amphiregulin. The stromal staining of HER1 and the epithelial staining of HER4 were most intense in the mid- and late-secretory-phase endometrium. Amphiregulin did not vary during the menstrual cycle. In the mid-secretory phase, the protein expression of HB-EGF was lower in endometrium from women with unexplained infertility versus normal endometrium and endometrium from women with male factor infertility. HB-EGF and HER4 mRNA expression in mid-secretory endometrium of women with unexplained and male factor infertility were increased compared with normal controls. Impaired endometrial expression of certain members of the EGF family may contribute to infertility in some women with unexplained infertility.

Adrenomedullin Enhances Invasion by Trophoblast Cell Lines1

We have tested the hypothesis that adrenomedullin (ADM), a multifunctional peptide hormone, works as a trophoblast proinvasion factor. Our results showed that ADM receptor components-the mRNA and proteins of calcitonin receptor-like receptor (CALCRL) and receptor activity modifying proteins (RAMPs)-were expressed by human choriocarcinoma JAr cells and first-trimester cytotrophoblast HTR-8/SV neo cells. ADM stimulates both JAr and HTR-8/SV neo cell proliferation. The invasion capabilities of JAr cells and HTR-8/SV neo cells were also enhanced by ADM, and this was associated with increased gelatinolytic activity and reduced plasminogen activator inhibitor-1 mRNA expression (SERPINE1). Our data support the notion that ADM may be involved in the human implantation process via regulating trophoblast proliferation and differentiation.

Co-existence of heparin-binding epidermal growth factor-like growth factor and pinopodes in human endometrium at the time of implantation.

Pinopodes have been suggested to be markers of uterine receptivity. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is expressed in increasing amounts in the secretory phase endometrium and is considered to be important for the human implantation process. The aim of this study was to investigate a possible co-existence of pinopodes and HB-EGF in the normal human endometrium. Endometrial biopsies were obtained from women with normal menstrual cycles. The biopsies were examined by scanning electron microscopy for the detection of pinopodes, by immunohistochemistry for the expression of HB-EGF protein, and by confocal microscopy to determine if HB-EGF was present on the surface of the pinopodes. The expression of HB-EGF in luminal and glandular epithelium was highest when fully developed pinopodes were present. Using confocal microscopy it was shown that HB-EGF was present both inside the luminal epithelial cells and on the surface of pinopodes. These findings suggest that HB-EGF might play a role in both the attachment and penetration steps in the human implantation process. Furthermore, the immunohistochemical staining demonstrates that HB-EGF can be used as a marker for the implantation window.

Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst.

Embryonic implantation is a crucial event for the human reproductive function. Cytokines and paracrine molecules have been proposed as putative local regulators of this process. The leptin or the OB protein has been linked to the reproductive function and inflammatory response. In the present study, we describe for the first time the expression of leptin and leptin receptor (long form) in the secretory endometrium and that endometrial leptin secretion is regulated in vitro by the human blastocyst. Leptin and leptin receptor messenger RNA and protein were identified in secretory endometrium and in cultured endometrial epithelial cells (EECs) by RT-PCR, Western blot, and immunohistochemistry. The concentrations of immunoreactive leptin secreted by human embryos alone or cocultured with EECs were also assessed. We found that human blastocysts secrete significantly higher levels of leptin than arrested embryos. In contrast, leptin concentrations secreted by arrested embryos cocultured with EECs were significantly higher than blastocysts cocultured with EECs. These findings suggest that the human endometrium is a site for local production and a target tissue for circulating leptin. Expression of leptin and its functional receptor in the endometrium and regulation of endometrial leptin secretion by the human embryo suggests that the leptin system may be implicated in the human implantation process.

The Adhesion Molecule CEACAM1 (CD66a, C-CAM, BGP) Is Specifically Expressed by the Extravillous Intermediate Trophoblast

CEACAM1 (CD66a, C-CAM, BGP) is an adhesion molecule of the carcinoembryonic antigen family which has been shown to be normally expressed at the apical pole of epithelial cells, including the apical pole of endometrial surface and glandular epithelia. The purpose of the present study was to investigate its expression pattern at the maternal-fetal interface, and thus to determine whether CEACAM1 could be implicated in the human implantation process. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody (mAb) as well as flow cytometry and Western blot on isolated trophoblast populations. On the maternal side of the maternal-fetal interface, CEACAM1 was present in epithelial cells of pregnancy endometrium as well as in small endometrial vessels, whereas it was absent from decidual cells. On the fetal side, CEACAM1 was strongly expressed by the extravillous (intermediate) trophoblast at the implantation site, as well as by extravillous trophoblast cells with invasive phenotype in primary culture, as shown by flow cytometry and Western blot. Expression was also observed in placental villous core vessels but was absent from both villous cyto- and syncytiotrophoblasts throughout the pregnancy. We conclude that, given its specific expression pattern, CEACAM1 can be a useful marker for extravillous intermediate trophoblast and might be functionally implicated in mediating trophoblast/endometrial and/or trophoblast/endothelial interactions during the trophoblastic invasion of the endometrium.

Clinical approaches to increasing uterine receptivity during human implantation.

An improvement in clinical pregnancy rate follows a natural or induced period of amenorrhoea. The cause is unknown, but it could be related to the period of uterine 'rest' which may restore full function to the steroid-sensitive systems, such as the pinopodes, after a prolonged period of constant menstrual cycling. This may result in the restoration of uterine capacity for embryo implantation to levels typical of younger women. Some other avenues of research on the unusual aspects of human implantation are worth further study. An impaired uterine blood flow could lower the chances of implantation. Immunological factors may emerge as major elements in the human implantation process, because the neutralization of antiphospholipid antibodies may enhance the activity of early trophoblast.