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.

The DNA integrity of cryopreserved spermatozoa separated for use in assisted reproductive technology is unaffected by the type of cryoprotectant used but is related to the DNA integrity of the fresh separated preparation.

OBJECTIVE: To investigate and compare seven different commercially available cryoprotectant media in terms of the DNA integrity of spermatozoa recovered after cryopreservation and separation using density gradient centrifugation (DGC). DESIGN: A prospective clinical study. SETTING: Tertiary care fertility clinic. PATIENT(S): Three hundred twenty men presenting for infertility investigations. INTERVENTION(S): Each sample was randomly assigned to one of seven commercially available cryoprotectants or to no cryoprotectant. MAIN OUTCOME MEASURE(S): Percentage sperm DNA fragmentation after cryopreservation and preparation using DGC. RESULT(S): The mean percentage fragmentation was significantly higher post-thaw and post-DGC; however, some patients (26.3%) demonstrated a lower percentage fragmentation post-thaw. No single cryoprotectant was identified as the best at preserving DNA integrity. The difference in fragmentation after thawing and DGC was found to be highly dependent on the prefreeze fragmentation. Motility was also significantly correlated with the difference in fragmentation post-thaw (r = -0.161). CONCLUSION(S): Neither the presence nor type of cryoprotectant affects the DNA integrity of spermatozoa after cryopreservation and DGC. Individuals with lower prefreeze fragmentation in DGC-prepared spermatozoa have larger increases in fragmentation and are less likely to exhibit lower levels of fragmentation post-thaw. The reverse effect observed in those with higher prefreeze fragmentation gives rise to a possible novel method of reducing fragmentation in sperm used for assisted reproductive technology treatment cycles without the need for testicular sperm retrievals.