miR-519d-3p released by human blastocysts negatively regulates endometrial epithelial cell adhesion by targeting HIF1 alpha

Successful embryo implantation requires a competent embryo, a receptive endometrium and synchronized communication between them. The selection of embryos with the highest implantation potential remains a challenge in the field of assisted reproductive technology. Moreover, little is known about the precise molecular mechanisms underlying embryo-endometrium crosstalk. MicroRNAs (miRNAs/miRs) have been detected in the spent embryo culture medium (SCM); however, their functions at the preimplantation stage remain unclear. In the present study, human SCM samples were collected during in vitro fertilization/intracytoplasmic sperm injection-embryo transfer and divided into implanted and not-implanted groups according to the clinical pregnancy outcomes. Total RNA was extracted and six miRNAs (miR-372-3p, miR-373-3p, miR-516b-5p, miR-517a-3p, miR-519d-3p and miR-520a-3p) were selected for reverse transcription-quantitative PCR (RT-qPCR) analysis. The results revealed that miR-372-3p and miR-519d-3p were markedly increased in SCM from blastocysts that failed to implant compared with in blastocysts that implanted. The receiver operating characteristic curve analysis revealed that miR-519d-3p was superior to miR-372-3p in predicting pregnancy outcomes. In vitro miRNA uptake and cell adhesion assays were performed to determine whether miR-519d-3p could be taken up by endometrial epithelial cells and to examine the biological roles of miR-519d-3p after internalization. Potential targets of miR-519d-3p were verified using a dual-luciferase reporter system. The results demonstrated that miR-519d-3p was taken up by human endometrial epithelial cells and that it may inhibit embryo adhesion by targeting HIF1 alpha. Using RT-qPCR, western blot analysis and flow cytometry assay, HIF1 alpha was shown to inhibit the biosynthesis of fucosyltransferase 7 and sialyl-Lewis X (sLex), a cell-surface oligosaccharide that serves an important role in embryonic apposition and adhesion. In addition, a mouse model was established and the results suggested that miR-519d-3p overexpression hampered embryo implantation in vivo. Taken together, miRNAs in SCM may serve as novel biomarkers for embryo quality. Furthermore, miR-519d-3p was shown to mediate embryo-endometrium crosstalk and to negatively regulate embryo implantation by targeting HIF1 alpha/FUT7/sLe(x) pathway.