Changes in Cryotolerance of Spermatozoa in Men with Teratozoospermia Under the Influence of Extracellular Vesicles from Donor Seminal Plasma Isolated by Depth Filtration

Currently, there is a need to improve the cryopreservation process for male gametes, especially for patients with low cryotolerance during sperm cryopreservation. Methods: The content and size of donor extracellular vesicles (EVs) in seminal plasma (SP) were assessed using nanoparticle tracking analysis (NTA), CD marker analysis, and transmission electron microscopy (TEM). Patient ejaculates were exposed to cryopreservation with or without prior co-culture with SP EVs and were not exposed to cryopreservation. The interaction of SP EVs with spermatozoa was assessed by TEM. Apoptotic, necrotic and late apoptotic cells, and mitochondrial functional activity were detected by flow cytometry. Results: NTA showed the highest concentration of SP EVs with a size of 80 nm, corresponding to small EVs. The binding of SP EVs to spermatozoa occurred along the entire plasma membrane, with an increased concentration of SP EVs at the neck and upper third of the sperm head. A significant increase in sperm motility was observed in the EVs co-culture group after cryopreservation/thawing. Flow cytometry showed a significant difference in the JC-1 Red/JC-1 Green ratio, indicating a higher mitochondrial membrane potential in the EVs exposure group. Conclusions: SP EVs have a protective function during human sperm cryopreservation. © 2025 by the authors.