The potential roles of endometrial cell-derived extracellular vesicles on embryo implantation and dormancy

Abstract

Extracellular vesicles (EVs) are membrane bound vesicles secreted by many cell types. The contents of EVs vary among cell types and in different physiological conditions. EVs fuse with and modulate biological activities of their target cells. EVs have been identified in uterine luminal fluid (ULF) of mice and humans. This project hypothesized that the endometrial cell derived ULF-EVs contribute to embryo implantation and dormancy, two opposite phenomena crucial to reproduction. Specifically, ULF-EVs mediated transfer of microRNAs from endometrium to embryos where the transferred microRNAs regulated expression of target genes important for implantation/dormancy. The hypothesis was tested with 3 objectives. Objective 1 compared four EVs isolation methods, namely ultracentrifugation, ultra-filtration, polymer-based precipitation (PBP), and PBP with size-based purification using EVs from human endometrial Ishikawa cells as model. The EVs isolated by these methods had similar size and expressed EVs-enriched proteins CD63, TSG101 and HSP70. Among the four methods, PBP could best preserve the biological activities of EVs and was able to isolate EVs from a small volume of sample. Thus the method was used in subsequent experiments. Objective 2 studied the EVs as a means of endometrium-embryo communication. Mouse ULF-EVs were successfully isolated by PBP. Electron microscopy showed that they had a diameter of 30-300 nm, expressed TSG101, CD63 and HSP70, and were localized to uterine lumen and surface of blastocysts in vivo. Fluorescence-labeled human endometrial cells-derived EVs were internalized into trophoblast cells and embryos. The results showed that the ULF-EVs were derived from the endometrium and target on the blastocysts. Objective 3 determined the small RNA content of ULF-EVs by RNA-sequencing. The specific microRNA profiles of ULF-EVs during implantation, dormancy and estradiol-induced reactivation of the delayed implantation model were consistent with functional roles of microRNAs in implantation/dormancy. Bioinformatics analyses showed up-regulation of 204 microRNAs in the dormant ULF-EVs that were associated with biological process related to implantation/dormancy. MicroRNA Let-7a was up-regulated in the dormant ULF-EVs and was studied further because of its known actions on implantation. The high expression of let-7a in the dormant endometrial epithelial cells, their ULF-EVs and dormant embryos confirmed the transfer of let-7a from the epithelial cells to the embryos via ULF-EVs. Coincubation of let-7a-enriched endometrial cells-derived EVs suppressed the expression of let-7a target gene c-Myc in the mouse blastocysts. Moreover, inhibition of production of EVs suppressed attachment of trophoblast spheroids onto endometrial cells in vitro and number of implantation sites in vivo. Fifteen microRNAs were commonly upregulated in both dormant ULF-EVs and dormant blastocysts relative to their activated counterparts. These microRNAs were likely to be involved in maintenance of embryo dormancy. Consistently, bioinformatics analyses showed that these microRNAs could potentially target on a significant portion of molecules in the mTOR signaling, Myc signaling and polyamines biosynthesis pathways. Inhibition of these pathways was known to induce embryo dormancy. In conclusion, PBP could isolate mouse ULF-EVs. Endometrial microRNAs were transferred to the embryos via endometrial cell derived ULF-EVs and affected embryo implantation/dormancy. The study demonstrates that the ULF-EV could mediate embryo-maternal communication during implantation/dormancy.