Free apical surface hetero-cellular CDH1 homophilic binding is a major mediator of blastocyst-endometrium interaction in humans.

study questionIs E-cadherin (CDH1) expressed on the free apical surface of endometrial epithelial cells (EECs) involved in embryo attachment? SUMMARY ANSWER: Embryonic signals induced apical expression of endometrial CDH1, which facilitated attachment via hetero-cellular CDH1 homophilic binding. WHAT IS KNOWN ALREADY: Understanding human blastocyst-endometrium interaction helps fertility treatment by facilitating the evaluation of endometrial receptivity and blastocyst quality. CDH1 is an adhesion molecule commonly known for the maintenance of epithelial integrity through adherens junction formation on the lateral membranes of adjacent epithelial cells. The apical region of some epithelia also expressed CDH1 of unknown function. STUDY DESIGN, SIZE, DURATION: Laboratory experimental study. PARTICIPANTS/MATERIALS, SETTING,

methodsThe potential apical cell plasma membrane proteins participating in adhesion between blastocysts and EEC were identified by comparing the surface proteomes of polar trophectoderm-like trophoblastic spheroids (BAP-EB) and receptive EEC. The apical surface expression of CDH1 in human blastocysts and primary EEC was confirmed by live-cell immunofluorescent staining. Antibody blocking and gene knockdown approaches were used to study the functional roles of hetero-cellular homophilic binding of CDH1. The adhesiveness of EEC and polar trophectoderm-like cells was confirmed by atomic force microscopy upon gene knockdown. The embryonic signals induced endometrial apical surface relocations of CDH1 and its stabilizer delta-1 catenin (CTNND1) were studied by treatments with conditioned media of BAP-EB and human blastocysts, as well as with HCG. The correlations of endometrial CDH1 and CTNND1 with pregnancy outcomes were confirmed by western blotting analysis of their protein expressions in EEC and immunofluorescent staining of endometrium collected from women who gave live birth, those who failed to become pregnant after IVF, and those with repeated implantation failure. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 27 pairs of potential interactive cell plasma membrane transmembrane proteins between BAP-EB and EEC, and CDH1 homophilic binding was one of them. CDH1 was localized to the apical surface of receptive EEC and the polar trophectoderm of human blastocysts. The CDH1 expression in EEC was higher during the window of implantation than those at the pre-receptive phase. With the use of atomic force microscopy, we revealed that the adhesiveness of the apical surface of polar trophectoderm-like cells and EEC decreased when CDH1 was silenced. The attachment rates of BAP-EB onto EEC were decreased when the surface CDH1 of BAP-EB and/or the EEC was blocked by an anti-CDH1 antibody, indicating that homophilic binding of trophectodermal CDH1 with endometrial CDH1 mediated the attachment of BAP-EB onto EEC. Besides cell surface adhesiveness, knockdown of CDH1 or CTNND1 in EEC also reduced expression of adhesion-related molecules and BAP-EB attachment. The conditioned media of BAP-EB and human blastocysts and HCG enhanced apical expression of CDH1 in EEC, suggesting embryonic signals, in particular, modulated the expression of CDH1 and stimulated the adhesion of EEC. The protein expression of CDH1 was significantly higher in EEC isolated from patients who gave live birth as compared to those with repeated implantation failure. Importantly, the expression of CDH1 in the apical region of the luminal epithelium of the endometrium from fertile women was also significantly higher than that from women with repeated implantation failure. LARGE SCALE DATA: none. LIMITATIONS, REASONS FOR CAUTION: This study only includes in vitro experiments. Due to the limited availability of human blastocysts and difficulty in long-term expandable culture of primary EEC, embryo surrogates BAP-EB and endometrial adenocarcinoma cell lines were used for most of the experimental work. The sample size of the primary EEC for protein analysis in this study was small, and the 2D culture of primary EEC in vitro might have impaired its native epithelial polarity. WIDER IMPLICATIONS OF THE

findingsEndometrial factors can lead to infertility, but the exact molecules involved in endometrial receptivity remain unclear. Human endometrial receptivity is best assessed by determining whether a human blastocyst can attach and implant onto the endometrium of interest. With the use of a human embryo surrogate named BAP-EB, we demonstrated the important roles of CDH1 and CTNND1 in human embryo attachment. In particular, we showed that homophilic binding between trophectodermal CDH1 and epithelial CDH1 was one of the major mediators of the first contact in embryo-maternal interaction. Embryo-derived factors induced apical redistribution of CTNND1 and CDH1 in the EEC. Most importantly, the expression of CDH1 on the apical region of EEC represented endometrial receptivity. The results strengthen our understanding of embryo-maternal interaction in humans, and CDH1 could potentially be used for predicting IVF outcomes, which helps clinicians to better counsel the couples who fail with ART. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grant for Fertility Innovation 2016 from Merck; Health and Medical Research Fund (grant numbers: HMRF 04151546; 10212996; 11222296) from the Food and Health Bureau, Government of the Hong Kong Special Administrative Region; InnoHK initiative of the Innovation and Technology Commission of the Hong Kong Special Administrative Region Government (Health@InnoHK); Shenzhen Science and Technology Program (Grant No. KQTD20190929172749226); General Research Fund (grant number: 17212922); Collaborative Research Fund C7100-22GF from the Research Grants Council of Hong Kong; and Shenzhen Sanming Project of Medicine (SZSM202211014). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.