Menstrual blood serum extracellular vesicles reveal novel molecular biomarkers and potential endotypes of unexplained infertility

Several biomolecules have been previously associated with unexplained infertility (uIF) in blood and uterine samples, immune cells and their secreted factors, endometrial tissue, menstrual blood, serum, and stromal cells, however they do not comprehensively represent different uIF endotypes and their isolation/detection involves invasive diagnostic methods and lacks precision. This ex-vivo study was performed on extracellular vesicles (EVs) from menstrual blood collected on cycle day 2 from 9 fertile volunteers and 8 women with uIF. Menstrual blood serum (MBS) EVs were isolated from fertile and uIF MBS using Iodixanol Density Gradient Centrifugation and quantified by flow cytometry. EVs were characterized according to MISEV2023 guidelines. Comprehensive proteomic analysis of MBS EVs and EV-depleted MBS showed significant changes in uIF proteome, mostly affecting cell adhesion, immune response, apoptosis, response to oxidative stress and lipid metabolism. These processes were previously linked to pathologies of the female reproductive system but never investigated in uIF and were used to stratify patients into distinct molecular endotypes. Area under the curve (AUC) analysis was used to determine the optimum set of biomarkers for each of the uIF molecular endotypes. These findings provide new insights into uIF that could facilitate personalised treatment approaches.