Functional and Structural Determinants of Human Sperm Cryoresistance: Insights from Motility, Mitochondrial and Chromatin Integrity Analyses

Sperm cryopreservation is essential for fertility preservation and assisted reproduction; however, post-thaw sperm quality varies markedly between individuals despite standardized protocols. This exploratory study investigated whether good and poor freezers differ in cellular, sub-cellular, and molecular features associated with cryoresistance. One ejaculate from each of 100 normozoospermic donors was assessed before cryopreservation and after thawing. Samples were classified after thawing as good freezers (GFs; n = 50) or poor freezers (PFs; n = 50) according to post-thaw motility performance, using total motility ≥ 42% and progressive motility ≥ 30% as thresholds. Sperm quality was evaluated using computer-assisted sperm analysis, membrane and acrosome integrity assays, mitochondrial membrane potential, capacitation-associated patterns, membrane lipid disorder and DNA/chromatin integrity tests. Post-thaw thermoresistance was assessed at 37 °C for 60 and 120 min. An exploratory Western blot panel was performed only on fresh pre-freeze samples from a selected subset of 4 GF and 4 PF samples. Cryopreservation reduced sperm quality in both groups; however, PF samples showed significantly greater declines in total and progressive motility, sperm viability, membrane and acrosome integrity, mitochondrial membrane potential and thermoresistance. Exploratory Western blot data showed GF-favoring patterns for proAKAP4, SPAG6, proACR and the proACR/ACR ratio, whereas PF samples showed relatively higher ACR abundance; these molecular findings require validation in larger cohorts. Our data indicate that human sperm cryoresistance is a coordinated multi-level phenomenon involving membrane, mitochondrial, acrosomal, chromatin and flagellar resilience. This study identifies candidate functional and molecular indicators associated with cryoresistance and supports future development of pre-freeze screening strategies rather than providing a validated predictive model or clinical cut-off values.