Isolation and profiling of extracellular vesicles in uterine fluid to determine novel markers of endometrial receptivity

Uterine fluid contains endometrium-derived extracellular vesicles (EVs), which are membrane-bound cell-cell mediators containing lipids, proteins and nucleic acids. MicroRNAs (miRNA), the small non-coding RNA molecules directing post-transcriptional gene silencing, are a prominent cargo of EVs. Analysis of the miRNAs in EVs may offer insights into the endometrial secretome and endometrial-embryonic cross-talk around the time of implantation. This study is designed to characterize differentially expressed miRNAs from endometrium-derived EVs during the receptive phase versus pre-receptive phase in both natural and stimulated in-vitro fertilization (IVF) cycles, with the goal to identify novel markers of endometrial receptivity. Healthy fertile women (Group 1, N=22) with regular menstrual cycles, and women under 40 years of age (Group 2, N=36) undergoing their first or second stimulated IVF cycles were recruited with informed consent. In Group 1, uterine fluid aspiration (UFA) sampling was performed on the day of LH+2 (pre-receptive) and LH+7 (receptive) in natural cycles. In Group 2, UFA sampling was performed on the day of hCG+2 (pre-receptive, day of egg retrieval) and hCG+7 (receptive, day of blastocyst transfer) in IVF cycles. Cellular pellet was removed from the aspirated uterine fluid by centrifugation. EVs in the supernatant were then isolated by two steps of ultracentrifugation at 100,000g for 70 minutes. Isolated EVs were characterized by Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and flow cytometry. RNAs were extracted from the isolated EVs and profiling of miRNAs was carried out by next-generation sequencing. Resulting sequencing counts were mapped to miRNAs and bioinformatic analysis was performed on all paired samples to determine differentially expressed miRNAs between conditions (adjusted p value < 0.05). We confirmed the presence of EVs in human uterine fluid, which were characterized as 30-200 nm vesicles with bilayer membranes by TEM. NTA confirmed the high yield of EVs isolated by ultracentrifugation and demonstrated that the majority of these EVs were between 100-200 nm in size. Flow cytometry validated the successful isolation of EVs by their biomarkers CD9 and CD63. Through profiling of the miRNAs in EVs, over 100 miRNAs were found to be differentially expressed (≥ 2-fold change) between receptive phase and pre-receptive in either natural cycles or IVF cycles. Cross-referencing the differentially expressed miRNAs in both cycles, we identified three overlapping miRNAs (hsa-mir-331, hsa-mir-382, hsa-mir-505), all of which were up-regulated during the receptive phase, as potentially important miRNAs related to the establishment of endometrial receptivity. This study validates that endometrium-derived EVs can be isolated and characterized from human uterine fluid by ultracentrifugation. It is the first study to comprehensively profile the miRNAs in these EVs and it has identified a small cohort of candidate miRNAs that may be the novel biomarkers of endometrial receptivity.