Rho-kinase inhibition reduces DNA fragmentation in human sperm cryopreservation

Human sperm cryopreservation is essential for assisted reproductive technology; however, the freeze-thaw process reduces motility and increases DNA fragmentation. Rho-associated coiled-coil kinase (ROCK) inhibitors improve post-thaw survival in several cell types, yet their application to human sperm remains underexplored. We evaluated whether ROCK inhibition reduces DNA fragmentation during cryopreservation by liquid nitrogen vapor slow freezing or vitrification. Each semen sample was divided into four aliquots: 2 were cryopreserved via liquid nitrogen vapor slow freezing with or without ROCK inhibitor and 2 underwent vitrification with or without ROCK inhibitor. After 1 week of cryostorage, samples were thawed at the room temperature, and DNA fragmentation was quantified using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. DNA fragmentation decreased with ROCK inhibitor versus control after liquid nitrogen vapor slow freezing (mean ± standard deviation [s.d.]: 10.47% ± 6.77% vs 13.54% ± 7.0%; P = 0.002) and after vitrification (mean ± s.d.: 12.51% ± 5.84% vs 16.5% ± 10.77%; P = 0.006). ROCK inhibition thus reduces post-thaw sperm DNA fragmentation with both cryopreservation methods, particularly with liquid nitrogen vapor slow freezing, supporting its potential to improve sperm storage for assisted reproductive technology.