Protocol for isolation and characterization of small extracellular vesicles from human menstrual fluid using size-exclusion chromatography

Human menstrual fluid (MF) is rich in extracellular vesicles originating from peripheral blood components and local endometrial and immune cells. Here, we present a protocol to obtain MF-derived small extracellular vesicles (MF-sEVs) using dilution, low-speed centrifugation, filtration, and size-exclusion chromatography. We describe steps for characterizing pooled fractions by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. This workflow yields high-quality MF-sEVs suitable for reproducible omics and functional assays, enabling robust biomarker discovery and therapeutic exploration in uterine disorders.