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

study questionCan 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 WHAT IS KNOWN ALREADY: EM is a chronic gynecological disorder affecting ∼10% of reproductive-age women, associated with pain, infertility, and delayed diagnosis. sEVs 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 EM 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

methodsMF from healthy (nulliparous and multiparous) and EM 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 microscopy, 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 MAIN RESULTS AND THE ROLE OF CHANCE: MF-derived sEVs of healthy women were significantly more abundant than those isolated from their peripheral blood ( 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 WIDER IMPLICATIONS OF THE

findingsThe 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 STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Agency for Investigation and Development-ANID, FONDECYT Regular No. 1230932, No. 1230875, No. 1241103, FONDECYT POSTDOCTORADO No. 3230201, FAIN-UANDES No. 202201, and ANID-Basal funding for Scientific and Technological Center of Excellence, IMPACT No. FB210024. The authors declare no conflicts of interest.