P-313 Unlocking fertility secrets: endometrial extracellular vesicles in endometriosis and their impact on embryo implantation and development

Abstract Study question Is the cargo of extracellular vesicles (EVs) secreted by the eutopic endometrium of women with endometriosis associated with their fertility problems? Summary answer EVs secreted by the eutopic endometrium of endometriosis women contain miRNAs involved in endometrial receptivity, embryo implantation and embryo development, potentially contributing to fertility problems. What is known already Our group previously demonstrated that endometrial EVs carry miRNAs associated with endometrial receptivity, embryo implantation process and embryo development, serving as key mediators in the embryo-endometrial communication. In pathological conditions, we previously reported that the eutopic endometrium from women with adenomyosis releases EVs containing miRNAs involved in disease progression, impaired embryo development, and pregnancy complications. Given that endometriosis has also been linked to impaired embryo implantation and adverse pregnancy outcomes, we are now investigating whether EVs secreted by the endometrium of women with endometriosis contain miRNAs implicated in endometriosis-related infertility. Study design, size, duration Conditioned culture from endometrial secretory organoids of women with endometriosis (n = 15) was collected and pooled (4 biopsies/pool). EVs were isolated by ultracentrifugation and characterized by transmission electron microscopy (TEM), Exoview, and Nanoparticle Tracking Analysis (NTA). miRNAs were extracted, and miRNA-seq was performed. Target genes of the most expressed miRNAs were obtained from mirDIP database and functional enrichment analysis was performed with G Profiler. Participants/materials, setting, methods Endometrial biopsies were obtained by hysteroscopy from endometriosis women (stages III and IV) at IVI Valencia Clinics and Hospital La Fe. Patients were between 18≤40 years old and BMI≤30 kg/m2, had at least 6 month of infertility and were free of other uterine pathologies. Endometrial organoids were derived from epithelial endometrial cells of biopsies of eutopic endometrium from women with endometriosis (n = 15) and differentiated into the secretory phase by supplementation with estrogens, progesterone and cAMP. Main results and the role of chance EVs exhibited a size range of 100-350 nm and expressed the EV protein markers CD63, CD9, and CD81. TEM corroborated EV cup-shaped morphology. miRNA-seq identified 262 miRNAs specifically expressed in endometriosis secretory EVs. The top 20 most abundantly expressed miRNAs had all previously been linked to endometrial receptivity or early embryo development and included, among others, miR-3201-3p, miR-10a-5p, the let 7 and miR- 30 family. This top 20 most expressed miRNAs targeted 3860 genes. Regarding some of them with a high integrated score, PAPPA levels is reduced in maternal serum of women with recurrent pregnancy loss, ZEB1 modulates endometrial receptivity, FGFR1 regulates trophectoderm development and facilitates blastocyst implantation and USP25 whose suppression inhibits trophoblast migration and invasion. GO enrichment analysis of miRNAs target genes identified 886 dysregulated biological processes (FDR < 0.05) Majority of them were involved in embryonic development and mesenchymal-epithelial and epithelial-mesenchymal transitions and cell to cell communication, processes that are crucial for embryo apposition, invasion and implantation. KEGG pathway analysis revealed 67 dysregulated pathways (FDR < 0.05) including TGF-beta and PI3K-Akt signaling pathways that are implicated in the development of preimplantation embryos, the process of decidualization and the pathogenesis of preeclampsia. Limitations, reasons for caution This is a descriptive analysis so future functional experiments are necessary. Although organoid models recapitulate the physiological and morphological characteristics of native tissue, they remain an in vitro model. Wider implications of the findings Endometrial EVs from endometriosis contain miRNAs involved in endometrial receptivity, embryo implantation and development, potentially linking them to fertility issues. This study opens further research insight to define potential targets to solve endometriosis-related infertility problems by ameliorate epithelial endometrium receptivity and embryo development. Trial registration number No