Human Endometriotic Lesion‐Derived Small Extracellular Vesicles Impair Macrophage Function in the Peritoneal Microenvironment

Endometriosis (EM) is a chronic inflammatory disease that affects ∼10% of women during reproductive age. It is characterised by ectopic (ECT) growth of endometrial-like tissue mainly in the pelvic cavity. Small extracellular vesicles (sEVs) mediate cellular interactions, but their function remains poorly understood in the pathogenesis of EM. 3D endometrial epithelial organoids (EEOs) from ECT lesions and eutopic (EUT) endometrium from EM patients and controls were established to investigate sEVs. Multiplex bead-based flow cytometry revealed CD133/1 and EpCAM as dominant markers on EEO-sEVs, with ECT EEO-sEVs showing upregulation of CD44, CD29 and downregulation of EpCAM compared to EUT EEO-sEVs. Peritoneal fluid (PF)-sEVs displayed high and correlated CD133/1 and EpCAM expression, indicating a major contribution from endometrial epithelial (EE) cells, alongside sEVs of lymphocyte and endothelial origin. Functionally, both ECT EEO-sEVs and PF-sEVs from EM patients significantly suppressed macrophage phagocytosis, as assessed by pH-sensitive fluorescent bioparticles. The effect was reversed by CD47 blockade. The coexpression of CD47 with CD133/1 and EpCAM on PF-sEVs indicates the involvement of EE cell-derived sEVs in CD47/SIRP-α mediated suppression. This study provides the first thorough characterisation of EE-derived sEVs utilising EEO models in EM and demonstrates their potential immunomodulatory role in the peritoneal microenvironment via CD47/SIRP-α signalling.