Global transcriptomic changes occur in uterine fluid-derived extracellular vesicles during the endometrial window for embryo implantation

STUDY QUESTION: Are uterine fluid-derived extracellular vesicles (UF-EVs) a ‘liquid biopsy’ reservoir of biomarkers for real-time monitoring of endometrial status? SUMMARY ANSWER: The transcriptomic cargo of UF-EVs reflects the RNA profile of the endometrial tissue as well as changes between the non-receptive and the receptive phase, possibly supporting its use for a novel endometrial receptivity test. WHAT IS KNOWN ALREADY: EVs have been previously isolated from uterine fluid, where they likely contribute to the embryo-endometrium crosstalk during implantation. Based on a meta-analysis of studies on endometrial tissue implantation-associated genes and the human exosomes database, 28 of the 57 transcripts considered as receptivity markers refer to proteins present in human exosomes. However, the specific transcriptomic content of receptive phase UF-EVs has yet to be defined. STUDY DESIGN, SIZE, DURATION: Two experimental series were set up. First, we simultaneously sequenced RNA species derived from paired UF-EVs and endometrial tissue samples collected from physiologically cycling women. Second, we analyzed RNA species of UF-EVs collected during the non-receptive (LH þ 2) and receptive (LH þ 7) phase of proven fertile women and from the receptive (LH þ 7) phase of a population of women undergoing ART and transfer of euploid blastocysts. PARTICIPANTS/MATERIALS, SETTING, METHODS: For paired UF—endometrial tissue sampling, endometrial tissue biopsies were obtained with the use of a Pipelle immediately after UF collection performed by lavage of the endometrial cavity. Overall, n ¼ 87 UF samples were collected and fresh-processed for EV isolation and total RNA extraction, while western blotting was used to confirm the expression of EV protein markers of the isolated vesicles. Physical characterization of UF-EVs was performed by Nanoparticle Tracking Analysis. To define the transcriptomic cargo of UF-EV samples, RNA-seq libraries were successfully prepared from n ¼ 83 UF-EVs samples and analyzed by RNA-seq analysis. Differential gene expression (DGE) analysis was used to compare RNA-seq results between different groups of samples. Functional enrichment analysis was performed by gene set enrichment analysis with g:Profiler. Pre-ranked gene set enrichment analysis (GSEA) with WebGestalt was used to compare RNA-seq results with the gene-set evaluated in a commercially available endometrial receptivity array. MAIN RESULTS AND THE ROLE OF CHANCE: A highly significant correlation was found between transcriptional profiles of endometrial biopsies and pairwise UF-EV samples (Pearson’s r ¼ 0.70 P < 0.0001; Spearman’s q ¼ 0.65 P < 0.0001). In UF-EVs from fertile controls, 942 gene transcripts were more abundant and 1305 transcripts less abundant in the LH þ 7 receptive versus the LH þ 2 nonreceptive phase. GSEA performed to evaluate concordance in transcriptional profile between the n ¼ 238 genes included in the commercially available endometrial receptivity array and the LH þ 7 versus LH þ 2 UF-EV comparison demonstrated an extremely significant and consistent enrichment, with a normalized enrichment score (NES)¼9.38 (P < 0.001) for transcripts up-regulated in LH þ 7 in the commercial array and enriched in LH þ 7 UF-EVs, and a NES ¼ −5.40 (P < 0.001) for transcripts down-regulated in LH þ 7 in the commercial array and depleted in LH þ 7 UF-EVs. When analyzing LH þ 7 UF-EVs of patients with successful versus failed implantation after transfer of one euploid blastocyst in the following cycle, we found 97 genes whose transcript levels were increased and 64 genes whose transcript levels were decreased in the group of women who achieved a pregnancy. GSEA performed to evaluate concordance in transcriptional