Cargo small non-coding RNAs of extracellular vesicles isolated from uterine fluid associate with endometrial receptivity and implantation success.

OBJECTIVE:To optimize a method of isolating extracellular vesicles (EVs) from uterine fluid and to characterize small non-coding RNAs (sncRNAs) from the EVs, with the goal of identifying novel receptivity-associated biomarkers. DESIGN:Longitudinal study comparing sncRNA expression profiles from endometrial EVs. SETTING:University-affiliated, hospital-based fertility clinic. PATIENT(S):Healthy volunteers with no history of infertility (Group A) and women receiving controlled ovarian stimulation (COS)-in vitro fertilization treatment (Group B). INTERVENTIONS(S):In Group A, EVs were isolated from uterine fluid obtained on luteinizing hormone (LH)+2 and LH+7 in one natural menstrual cycle. In Group B, EVs were isolated from uterine fluid obtained on human chorionic gonadotropin (hCG)+2 and hCG+7 in one COS cycle. RNAs extracted from EVs were profiled using next-generation sequencing. MAIN OUTCOME MEASURE(S):Differential EV-sncRNAs between LH+2 and LH+7 (Group A), between hCG+2 and hCG+7 (Group B), and between pregnant and nonpregnant in vitro fertilization cycles (Group B). RESULT(S):Ultracentrifugation was validated as the most efficient method to isolate EVs from uterine fluid. We identified 12 endometrial EV-sncRNAs (11 microRNAs and 1 piwi-interacting RNA) as receptivity-associated transcripts conserved in both natural and COS cycles. These sncRNAs were associated strongly with biological functions related to immune response, extracellular matrix, and cell junction. Within COS cycles, we also identified a group of EV-sncRNAs that exhibited differential expression in patients who conceived versus those who did not, with hsa-miR-362-3p most robustly overexpressed in the nonpregnant patients. CONCLUSION(S):This study is the first to profile comprehensively sncRNAs in endometrial EVs from uterine fluid and identify sncRNA biomarkers of endometrial receptivity and implantation success.