Age-related changes in extracellular vesicles of seminal plasma isolated by asymmetric depth-filtration

Background . Biological fluids contain diverse extracellular vesicle (EV) populations differing in size, origin, composition, and function. This study pioneers asymmetric depth filtration for isolating seminal plasma (SP) EVs, bypassing ultracentrifugation (current gold standard). Objective . Validate the novel asymmetric depth filtration method for SP EV isolation as an alternative to ultracentrifugation. Design and methods. Twelve samples of seminal plasma were collected from young men and patients of advanced reproductive age with mild teratozoospermia. EVs were isolated from the SP using the proposed method. Western blot analysis for vesicle membrane markers CD9, CD63, and CD81 confirmed the presence of SP EVs in the studied samples. Transmission electron microscopy further validated the presence of SP EVs. Morphological characteristics of SP EVs from men of different age groups were described. Quantitative characteristics of the SP EV samples were assessed using Nanoparticle Tracking Analysis (NTA). Results . The obtained particle size and concentration distributions revealed age-related changes in SP EVs: in the advanced reproductive age group, a shift in the dis tribution peak toward larger particles was observed compared to the peak distribution in the young men group. Conclusion . The results provide a basis for further investigation of SP EVs and exploration of their potential clinical applications in assisted reproductive technologies, such as in vitro fertilization programs.