| I. Objective and purposeEmbryo implantation is a complex process during which the active embryo orients, adheres, invades and finally implants into the receptive uterine endometrium. The invasive ability of embryo and endometrial receptivity are two key factors of successful embryo implantation. With the development of assisted reproductive technology (ART), individualized controlled ovarian hyperstimulation and advanced fertilization and embryo culture technology has significantly improved the embryo quality, but the low implantation rate is always one of the unresolved problems in the field of Reproductive Medicine. The impaired receptivity of endometrium maybe the main factor leading to low implantation rate, while unable to diagnose and treat the impaired endometrial receptivity is a major obstacle to the success of ART. Therefore, assessment and improvement of endometrial receptivity is of extremely important clinical significance to improve the success rate of ART, exploring markers of endometrial receptivity and study the mechanism is the basis to improve endometrial receptivity and pregnancy rate. Integrin alphavbeta3(ITGAVB3) specifically expresses in the window of implantation (WOI), its abnormal expression leads to impaired endometrial receptivity and closely relates to the incidence of infertility. Integrin alphavbeta3is now recognized as one of the important biological markers of endometrial receptivity, but the specific mechanism in the process of embryo implantation remains unclear.In this study, the integrin alphav (ITGAV) overexpression vector and integrin beta3(ITGB3) overexpression vector were constructed, and the endometrial epithelial cells were transfected to observe the effects on the adhesion of embryonic spheroids to the integrin alphavbeta3overexpressed endometrial epithelial cell monolayer, investigate the roles of integrin alphavbeta3in embryo adhesion, and provide a research basis for further elucidation of its function and the molecular mechanism in embryo implantation and reproductive process. Ⅱ. Methods1. Constructed the pcDNA3.1-3FLAG-ITGAV、pcDNA3.1-3FLAG-ITGB3overexpression vectors, and transfected into the human endometrial epithelial cells(RL95-2cells).2. The mRNA expression of integrin alphav, integrin beta3and osteopontin (OPN) were detected by the methods of real time RT-PCR.3. The protein expression of integrin alphav and integrin beta3were detected by the methods of indirect immunofluorescence and flow cytometry assay.4. Established the Bewo cell spheroids and RL95-2cell monolayer co-culture system as the in vitro embryo implantation model, detected the effects of integrin alphavbeta3overexpressed RL95-2cellls on adhesion and extension of Bewo spheroids, observed the protein expression of integrin alphav, integrin beta3and OPN by indirect immunofluorescence.Ⅲ. Results1. Compared with RL95-2cells transfected with pcDNA3.1-3FLAG-ITGAV or pcDNA3.1-3FLAG-ITGB3individually, co-transfected with ITGAV and ITGB3overexpression vector significantly improved the expression of integrin alphavbeta3in RL95-2cells.2. Integrin alphavbeta3overexpression of RL95-2cells significantly improved the adhesion rate of Bewo spheroids to RL95-2cell monolayer (74.44±4.51%vs53.31±3.14%, P<0.01), promoted Bewo spheroids extension to RL95-2cell monolayer (118.26±6.00μm vs111.88±3.72μm, P<0.05).3. In Bewo spheroids and RL95-2cell monolayer co-culture system, integrin alphavbeta3overexpression in RL95-2cells promoted integrin alphav and OPN expression of Bewo cells.Ⅳ. Conclusions1. Successfully constructed the ITGAV and ITGB3overexpression vector.2. ITGAV and ITGB3co-transfection makes integrin alphavbeta3ovexpression in RL95-2cells. 3. Integrin alphavbeta3overexpression of endometrial cells promotes embryo adhesion and extension.4. Integrin alphavbeta3and OPN mediate embryo adhesion and invasion to endometrial cells and play an important role in embryo implantation. |
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