Cryoprotectant-free ultra-rapid freezing of human spermatozoa in cryogenic vials

摘要

The objective of this study was to investigate the effect of ultra-rapid freezing (direct immersion in liquid nitrogen) on human spermatozoa in cryogenic vials (>= 0.5 ml) at different concentrations of sucrose. After swim-up, the sperm suspensions (N = 58) were diluted with sperm preparation medium and divided into six aliquots: swim-up (fresh), conventional freezing group (slow freezing) and four ultra-rapid freezing groups containing sucrose at different concentrations (0.15 M, 0.20 M, 0.25 M and 0.30 M). Sperm motility, progressive motility, plasma membrane integrity, DNA stability and acrosome integrity of fresh and cooled-warmed spermatozoa were analysed. The progressive motility, plasma membrane and acrosome integrity of spermatozoa in the 0.20 M sucrose group were significantly higher than those of the slow freezing group (47.5 +/- 6.8% versus 36.4 +/- 8.7%, 73.2 +/- 6.9% versus 63.9 +/- 6.3%, 53.7 +/- 10.0% versus 35.9 +/- 9.7% respectively, P < 0.05). However, no differences were found in sperm motility or DNA stability (58.5 +/- 6.3% versus 54.2 +/- 5.3%, 90.1 +/- 2.8% versus 87.2 +/- 4.7%, P > 0.05 respectively) between the 0.20 M sucrose and the slow freezing group. No differences were found between the ultra-rapid and slow freezing group at the other concentrations of sucrose. Our findings suggest that the method of ultra-rapid freezing of human spermatozoa in cryogenic vials with a solution containing 0.20 M sucrose results in recovery of spermatozoon of superior qualities. In contrast to slow freezing, the ultra-rapid freezing technique of human spermatozoa seems to reduce cryoinjuries and maintain important physiological characteristics of the spermatozoa after warming.