Impact of Cooling Curve Type on Sperm DNA Integrity During Cryopreservation

To assess whether sperm DNA fragmentation is affected differently by cryopreservation techniques using different cooling curves before immersion in liquid nitrogen (LN₂).

A paired case-control study was conducted with 21 semen samples from men undergoing fertility evaluation. Each sample (Fresh Group) was initially analyzed for sperm concentration, vitality, motility, and DNA fragmentation. The remaining semen was divided into two groups and subjected to different cryopreservation protocols: 1) preliminary refrigeration for 20 min at a cooling rate of -0.9°C/min, by exposure to liquid nitrogen vapor (Refrigerated Group), 2) direct exposure to liquid nitrogen vapor at a cooling rate of -20.5°C/min (Vapor Group).

Both cooling protocols (Refrigerated and Vapor) caused a significant reduction compared with the Fresh Group in progressive motility (49.0% vs. 30.4% and 27.4%, p<0.001) and sperm vitality (85.1% vs. 64.9% and 64.1%, p<0.001). No significant differences were observed between the Refrigerated and Vapor Groups in terms of recovery of vitality and motility (p=0.361 and p=0.718, respectively). DNA fragmentation increased from 27.4% in the Fresh Group to 39.6% in the Refrigerated Group and to 36% in the vapor Group (p<0.001). However, the Vapor Group showed a lower mean DNA fragmentation rate than the Refrigerated Group (p=0.001).

Cryopreservation using direct exposure to liquid nitrogen vapor was associated with lower sperm DNA damage compared with the protocol involving pre-refrigeration, suggesting that faster cooling may better preserve DNA integrity during freezing.