Does the duration of cryopreservation impact human sperm dna integrity and apoptosis following thawing?

Abstract

OBJECTIVE: Sperm cryopreservation is widely used in ART procedures and male fertility preservation. Although cryopreserved sperm may remain frozen for long periods, it is not known whether the duration of cryopreservation affects sperm DNA fragmentation and/or apoptosis, which in turn could have an impact on ART success and the health of the offspring. The objective of this study was to identify if the duration of cryopreservation has an effect on the induction of DNA fragmentation and apoptosis in human sperm. DESIGN: Prospective study. MATERIALS AND METHODS: This study was conducted on cryopreserved semen samples (n=61) collected from 26 proven fertile donors. After thawing, sperm samples were assessed for % progressive motility. Flow cytometric assays were used to evaluate DNA fragmentation, apoptosis and necrosis in conjunction with acridine orange, annexin V and propidium iodide staining, respectively. Spearman's correlation test and linear models with generalized estimating equations were used for statistical analysis. RESULTS: The average duration of cryopreservation was 10.9 years (range: 1-22), while the average donor age was 32.1 (range: 20-41). DNA fragmentation was associated with donor age (r=0.26, p=0.03). Following adjustment for donor age as covariate, there was no correlation between duration of cryopreservation and DNA fragmentation (r=0.11, p=0.31) or percentage of necrotic sperm (r=-0.18, p=0.55). Mean DNA fragmentation within 1-5, 5-10, 11-15, and 16-22 year groups was 15.7 ± 8.6, 12.5 ± 7.8, 15.0 ± 6.2, and 17.9 ± 10.1. In contrast, there was a significant correlation between duration of cryopreservation and percentage of apoptotic sperm (r=0.48, p=0.002) as well as post-thaw motility (r-0.23, p=0.005). CONCLUSION: The duration of cryopreservation and long term freezing does not affect sperm DNA integrity or necrosis. Although there is a statistically significant negative association between the duration of cryopreservation and sperm apoptosis and motility; the association does not appear to be strong.