Potential Role of Menstrual Fluid‐Derived Small Extracellular Vesicle Proteins in Endometriosis Pathogenesis

Endometriosis, a chronic debilitating disease affects 1 in 7-10 girls and women, who have symptoms of severe chronic pain and subfertility and significantly impacts the overall quality of life. Currently, no effective early diagnostic methods are available for early stages of endometriosis. We used menstrual fluid-derived small extracellular vesicles (MF-sEVs) from women with self-reported endometriosis (laparoscopically diagnosed, n = 8) and self-reported without endometriosis and no painful periods (n = 9). MF-sEVs were separated using differential ultracentrifugation and characterised using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western Blot, flow cytometry, mass-proteomics analysis and functional assays. Spherical-shaped sEVs were identified with a median diameter of ∼120 nm, expressing sEV marker proteins. The MF-sEV proteins were classified as endometrial origin. Over 5000 proteins were identified, ∼77% of which were decreased whilst only 22 proteins (largely comprising immunoglobulins) were increased in endometriosis/MF-sEVs compared to control/MF-sEVs. Decreased proteins were involved in nitrogen compound metabolism, immune response, intracellular signal transduction, regulation of programmed cell death, maintenance of cell polarity and actin cytoskeleton organisation. Flow cytometry demonstrated a significant increase in CD86 expression (immune activation marker) in endometriosis/MF-sEVs. Mesothelial cells showed a significant decrease in cellular resistance and junctional protein expression. MF-sEVs are possible contributors to the pathogenesis of endometriosis and may have the potential for early detection of the disease.