Long-term cryostorage does not impair human sperm motility: evidence from a three-decade cohort study

PURPOSE: To evaluate whether long-term cryostorage duration affects post-thaw sperm motility and progressive motile concentration (PMC) in samples cryopreserved for up to 30 years. METHODS: A retrospective cohort study of 629 donor sperm samples cryopreserved between 1990 and 2020 in a single university-affiliated andrology laboratory was conducted. Motility and PMC were assessed immediately after thawing (T0) and after extended storage (TES). Associations between storage duration and post-thaw changes were evaluated using correlation analysis, multivariable linear regression, and multilevel mixed-effects models with donor-level random effects. K-means clustering was applied to identify sperm quality phenotypes. RESULTS: Total motility declined significantly following the freeze-thaw process (p < 0.001) but remained within clinically usable ranges after extended storage. Storage duration showed only a weak association with relative motility change (r = 0.169, p < 0.001), without a consistent duration-dependent trend in absolute post-thaw motility. PMC changes were modest and non-monotonic across storage groups. Multilevel models indicated that storage duration explained only a small proportion of variance in long-term outcomes. Cluster analysis identified distinct sperm quality phenotypes independent of storage duration. CONCLUSIONS: Long-term cryostorage of up to 30 years is not associated with substantial impairment of sperm motility or PMC. These findings support the stability of cryopreserved spermatozoa under prolonged storage conditions.