Menstrual fluid-derived small extracellular vesicles: a novel reservoir with distinct molecular signatures and implications for endometriosis etiopathology

Abstract STUDY QUESTION Can the transcriptomic profile of small extracellular vesicles (sEVs) derived from menstrual fluid (MF) provide preliminary insights into their potential roles as biomarkers and mediators in endometriosis (EM) progression? SUMMARY ANSWER MF-derived sEVs from EM patients display altered molecular signatures and enhanced proangiogenic activity in vitro, suggesting their potential involvement in EM pathophysiology. WHAT IS KNOWN ALREADY EM is a chronic gynecological disorder affecting approximately 10% of reproductive-age women, associated with pain, infertility and delayed diagnosis. Small extracellular vesicles carry cargo that resembles their cellular origin, making them promising biomarkers for various diseases. While sEVs have previously been isolated from MF and their protein content has been analyzed, their transcriptomic content and biological function remain unexplored. STUDY DESIGN, SIZE, DURATION Two cross-sectional studies were conducted. The first compared MF-sEVs between nulliparous (n = 10) and multiparous (n = 12) women. The second compared MF-sEVs from endometriosis patients (n = 10) and Controls (n = 11). Participants were recruited between 2021 and 2023 from a tertiary care center in Santiago, Chile. PARTICIPANTS/MATERIALS, SETTING, METHODS MF from healthy (nulliparous and multiparous) and endometriosis patients was self-collected using a menstrual cup on day 2 of menstruation, and peripheral blood of a subset of healthy women was obtained by venipuncture. sEVs were isolated from the plasma fraction of MF and from peripheral blood plasma. Nanoparticle tracking analysis, transmission electron microscop, and flow cytometry were used to assess sEVs concentration, size, morphology and tetraspanin expression. Next-generation sequencing was performed to analyze MF-derived sEVs transcriptomic profiles, and endothelial tubulogenesis assays using human umbilical vein endothelial cells (HUVECs) assessed angiogenic function in vitro. MAIN RESULTS AND THE ROLE OF CHANCE MF-derived sEVs of healthy women were significantly more abundant than those isolated from their peripheral blood (p = 0.008), and they exhibited typical morphology and tetraspanin expression. sEVs. Neither parity nor disease affected MF-sEVs concentration or size; however, CD63 expression was significantly reduced in sEVs from nulliparous women and EM patients (p = 0.02). Transcriptomic analysis revealed unique mRNAs and long noncoding RNAs (lncRNAs) signatures that reflect their potential contribution to disease etiopathology. Functionally, MF-derived sEVs from endometriosis patients (MF-EM-derived sEVs), significantly enhanced tubule formation in vitro (p 0.05), indicating proangiogenic potential in EM. LARGE SCALE DATA The raw sequencing data have been deposited in the NCBI GEO database under the accession number GSE310627. LIMITATIONS, REASONS FOR CAUTION The sample size limits the generalizability of findings. The RNA-seq component should be interpreted as a discovery-phase analysis; while it reveals potential molecular differences in MF-derived sEVs from women with EM, these results are preliminary and require validation in larger, independent cohorts. Functional validation was restricted to in vitro assays; in vivo studies are needed to confirm biological effects and mechanisms underlying the MF-EM-derived sEVs proangiogenic effects. WIDER IMPLICATIONS OF THE FINDINGS The non-invasive isolation and disease-associated transcriptomic signatures of MF-sEVs-enriched isolates highlight their potential as biomarkers for EM although validation is required in an independent and larger cohort to assess their robustness and clinical utility. Currently, EM diagnosis relies on invasive laparoscopic surgery, often resulting in diagnostic delays. The proangiogenic effect of MF-sEVs observed in vitro suggests that their cargo may reflect disease progression. These findings are in line with a recent report that described the proteome of MF-sEVs from EM patients, demonstrating their involvement in mesothelial barrier disruption, which reinforces the role of MF-sEVs in EM etiopathology. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Agency for Investigation and Development-ANID, FONDECYT Regular #1230932, #1230875, #1241103, FONDECYT POSTDOCTORADO #3230201, FAIN-UANDES #202201, and ANID-Basal funding for Scientific and Technological Center of Excellence, IMPACT #FB210024. The authors declare no conflicts of interest.