MicroRNAs in small extracellular vesicles indicate successful embryo implantation during early pregnancy

Abstract Background : Synchronous communication between the developing embryo and the receptive endometrium is required for successful embryo implantation. Assessing of uterine receptivity is important for overcoming the recurrent implantation failure (RIF). Although the potential roles of small extracellular vesicles (sEVs) miRNAs in pregnancy have repeatedly mentioned, the systematic study of sEVs derived from endometrium and its cargoes during the implantation have not yet been reported. Methods : In this study, sEVs were isolated from mouse uterine cavity on D2 (pre-receptive phase), D4 (receptive phase) and D5 (implantation) of pregnancy using ultracentrifugation and analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The miRNAs expression profiles of sEVs were identified by RT-qPCR. The datasets analysis of RIF were employed by an integrative bioinformatics analysis. The protein markers of sEVs were examined by western blotting. The effects of miRNAs in embryo implantation were determined by an agomir injection test. Results : Here we show that the number of multivesicular bodies (MVBs) in the endometrium is increased during the window of implantation (WOI). Meanwhile, the expression of CD63 is mainly located in the luminal and glandular epithelium. sEVs miRNAs profile reveal that miR-34c-5p, miR-210, miR-369-5p, miR-30b and miR-582-5p are enriched during WOI. By further integrating the database analysis results of RIF, we found that miR-34c-5p regulates GAS1 to involve normal process of embryo implantation. It is interesting that miR-34c-5p is down-regulated during implantation but enriched in sEVs. An implication of this is the possibility that sEVs miR-34c-5p could be used to evaluate uterine states. Conclusion : Our study identified that mouse endometrium release sEVs during the early stage of pregnancy, especially during WOI. We found that miR-34c-5p in sEVs affects embryo implantation by targeting to GAS1. Furthermore, the sEVs miRNAs suggest potential biomarkers for the choose of suitable period for embryo implantation. This study also has a number of important implications for future practice.