The effect of repeated freezing and thawing on human sperm DNA fragmentation

Objective: To investigate the effects of repeated freezing and thawing on human sperm motility, vitality, and DNA integrity. Design: A prospective clinical study. Setting: Tertiary care fertility clinic. Patient(s): Twenty men presenting for infertility investigations. Intervention(s): Each sample was subjected to three cycles of freezing and thawing both with and without washing steps and the addition of fresh cryoprotectant between each cycle. Main Outcome Measure(s): Percentage sperm DNA fragmentation, motility, and vitality before and following repeated freezing and thawing. Result(s): The percentage sperm DNA fragmentation rose significantly following each freeze-thaw cycle; however, samples that were not washed and to which fresh cryoprotectant was not added after each cycle fared significantly better than their washed counterparts. Both motility and vitality decreased steadily following each cycle but cell survival was significantly greater in the unwashed samples. Conclusion(s): In terms of the level of sperm DNA fragmentation, up to three cycles of freezing and thawing can be performed with a level of risk comparable to that following a single cycle of freezing and thawing. This is provided that samples are refrozen in their original cryoprotectant and not washed or altered in any way in between, and provided that they are separated by density gradient centrifugation or swim-up before use in assisted reproduction technology.