Regulation Of Embryo Implantation By The Exosomal MiRNAs Derived From Endometrium In The Early Stage Of Pregnancy

Embryo implantation,a complex and vital step in pregnancy establishment of mammals,occurs in a limited period.Successful embryo implantation requires synchronous communication between the maternal uterus and blastocyst during the window of implantation(WOI).Most of the pregnancy failures occur in the period of implantation during early pregnancy.The errors of embryo implantation can cause many adverse consequences,such as spontaneous abortion and other pregnancy diseases.Therefore,understanding the mechanism of maternal-fetal communication during embryo implantation is critical for successful pregnancy.Exosomes,a subtype of extracellular vesicles,are coated with a lipid bilayer of 40-150 nm diameter.Exosomes are released by almost all types of cells.Accumulating evidence suggests that the generation and release of exosomes is a crucial strategy in cellular communication.Meanwhile,exosomal contents also could be biomarkers that reflect the physiological and pathological state of the body.Although many studies have suggested that exosomes play crucial roles during early pregnancy or other biological processes,their roles in regulating embryo implantation during early pregnancy remain unclear.In this study,we isolated receptive(Ishikawa)and non-receptive(HEC-1-A)endometrial cells-derived exosomes by ultracentrifugation and investigated the effects of exosomes on the potency(migration,invasion,and proliferation)of HTR8/SVneo trophoblast cells,as well as exosomal effect on embryo implantation in vivo.Results showed that receptive endometrial cells significantly secreted more exosomes than non-receptive cells.In addition,the endometrial cells-derived exosomes can be delivered to trophoblast cells.The experiment of uterine horn injection in mice showed that HEC-1-A-derived exosomes significantly inhibited embryo implantation.Notably,receptive endometrial cell-derived exosomes activate the phosphorylation level of FAK and JNK in trophoblast cells,which significantly promotes the migration,invasion,and proliferation of trophoblast cells.Using in vitro angiogenesis experiments,we found that receptive endometrial cell-derived exosomes markedly promote angiogenesis.Next,we screened differentially expressed miRNAs in exosomes derived from receptive and non-receptive endometrial cells through high-throughput sequencing of small RNAs.We found that miR-100-5p was highly enriched in the exosomes derived from receptive endometrial cells when compared to non-receptive cells-derived exosomes.Meanwhile,the expression of miR-100-5p was significantly up-regulated in the endometrium during the period of WOI.Then,HTR8/SVneo trophoblast cells HUVEC cells were transfected with miR-100-5p.The results demonstrate that miR-100-5p promotes the migration,proliferation,and invasion of trophoblast cells,and miR-100-5p also promotes the migration of HUVEC cells and angiogenesis in vitro.Altogether,these results indicate that exosomal miR-100-5p plays an important role in the regulation of embryo implantation.Additionally,we isolated the exosomes derived from mouse uterus during the period of pre-implantation(D2),WOI(D4),and post-implantation(D5)in the early stage of pregnancy.Consistent with the results in cell lines,we observed that the secretion of exosomes significantly increased during the period of implantation.Meanwhile,we analyzed the miRNA expression profile of mouse uterine-derived exosomes using qRT-PCR.We found that the expression of miR-34c-5p,miR-210,miR-369-5p,miR-30b,and miR-582-5p in exosomes were highly enriched during WOI.Then,R basic was used to perform the analysis of human recurrent implantation failure(RIF)data sets.We found a key gene GAS1,which significantly associated with successful implantation.Further study indicated that miR-34c-5p targets GAS1 to regulate its expression and has important effects on embryo implantation.MiR-34c-5p is almost not expressed in the endometrium during implantation,but it is significantly upregulated in uterine-derived exosomes during this period,suggesting that endometrial-derived exosomes miR-34c-5p can be the biomarker for indicating the receptive uterus.In summary,our results showed that receptive endometrial cell-derived exosomal miR-100-5p provides a new insight for understanding the regulatory mechanism of embryo implantation.Meanwhile,we identified the possibility of uterus-derived exosomal miRNA as a biomarker for successful implantation.These findings shed new light on understanding the maternal-embryo communication and regulatory mechanisms during embryo implantation.This study also has several important implications for future practice,including therapy of infertility.