微滴无保护剂玻璃化法冷冻附睾微量精子的研究

Microdrop-vitrification for epididymal spermatozoa without cryoprotectants

目的:探讨微滴无保护剂的玻璃化法冷冻经皮附睾穿刺抽吸术(PESA)所获微量精子的可行性及安全性。方法:通过比较微滴法冷冻(微滴组)和常规慢速冷冻(冷冻管组)这两种冷冻方法的复苏率、回收率及精子核DNA断裂发生率,评价两种冷冻方法的冷冻效果。结果:22例患者附睾精子标本经两种方法冷冻后,仅冷冻管组中有1例患者附睾精子标本中未见活动精子,余均有活动精子,冷冻管组与微滴组的复苏率均低,分别为(18.16±9.38)%和(21.99±10.95)%,两者差异无显著性(P>0.05)。冷冻管组与微滴组的回收率分别为(58.39±12.67)%和(70.82±14.94)%,两者差异有显著性(P<0.01)。22例患者附睾精子冷冻前都存在一定比例核DNA断裂,经单细胞凝胶电泳后出现典型的彗星精子,精子核DNA断裂发生率为(26.68±9.45)%。复苏后冷冻管组和微滴组精子核DNA断裂发生率分别为(28.68±12.54)%和(27.64±10.70)%,与冷冻前比较均未增加,差异均无显著性意义(P>0.05)。结论:微滴可作为微量精子冷冻的合适载体,并且微滴法无保护剂的玻璃化冷冻附睾微量精子可能是一种简单、安全而行之有效的冷冻方法。

Objective：To explore the feasibility and safety of microdrop-vitrification for epididymal spermatozoa obtained by percutaneous epididymal sperm aspiration（PESA） without cryoprotectants.Methods：We treated the epididymal sperm samples from 22 patients by conventional freezing（Group 1） and microdrop-vitrification without cryoprotectants（Group 2）,and evaluated the effectiveness of the two methods by comparing their revival rate,retrieval rate and incidence of sperm nuclear DNA fractures.Results：Motile sperm were found in all but 1 case in Group 1.The revival rates of the frozen sperm were low in both Groups 1 and 2（[18.16 ± 9.38]% vs [21.99 ± 10.95]%,P〈0.05）,but statistically significant differences were shown between the two groups in the retrieval rate（[58.39 ± 12.67]% vs [70.82 ± 14.94]%,P〈0.01）.Before freezing,nuclear DNA fractures existed in the epididymal sperm samples of all the 22 patients,comet sperm were seen after unicellular gel electrophoresis,and the incidence of sperm nuclear DNA fracture was（26.68 ± 9.45）%.After freezing,no increase was observed in the incidence of sperm nuclear DNA fracture in either Group 1 or 2（[28.68±12.54]% vs [27.64±10.70]%,P〈0.05）.Conclusion：Microdrop can be used as a suitable freezing carrier for a low number of sperm,and cryoprotectant-free vitrification with microdrop may be a simple,safe and effective method for the cryopreservation of a low number of epididymal sperm.