气相冷冻法和液相冷冻法对人类精子运动能力、形态学及超微结构的影响

目的研究液氮蒸汽熏蒸法(简称气相冷冻法)和直接投入液氮冷冻法(简称液相冷冻法)对人类精子冷冻复苏后运动能力、形态学及超微结构的影响。方法选择25例捐精者的精液标本,按照1∶1的比例添加改良甘油-卵黄-枸橼酸钠(GYC)作为冷冻保护剂。每份精液标本等分为2份,分别进行气相法冷冻和液相法冷冻。复苏后,利用IVOS精子分析仪测定精子总活动率、前向运动精子百分率、运动参数,巴氏染色观察精子形态并计算形态正常率,透射电子显微镜观察精子超微结构。结果气相法冷冻从28℃降温到-185℃用时468 s,再经过1 h后降温到-196℃;液相法冷冻从30℃降温到-196℃用时90 s。两种方法冷冻复苏后精子总活动率、前向运动精子百分率、运动参数、形态正常率与冷冻前比较均明显降低(P<0.05)。气相法冷冻复苏后精子的复苏率、总活动率、前向运动精子百分率均高于液相法,差异有统计学意义(P<0.05);两种冷冻方法复苏后精子形态正常率、运动参数比较,差异均无统计学意义(P>0.05)。通过透射电子显微镜观察,使用GYC作为冷冻保护剂,两种方法冷冻复苏后,精子头部质膜和顶体膜超微结构未出现明显的损伤。结论改良GYC保护剂对精子超微结构具有保护作用;气相冷冻法冷冻人类精子的复苏结果优于液相法。

Effects of vapor nitrogen freezing method and direct liquid nitrogen freezing method on motility,morphology and ultrastructure of human sperm

Objective To investigate the effects of vapor nitrogen freezing method and direct liquid nitrogen freezing method on the motility,morphology and ultrastructure of post-thaw human sperm.Methods Twenty-five semen samples from semen donors were selected,and glycerol-yolk-sodium citrate(GYC) was added as cryoprotectant with the proportion of 1∶ 1.Each semen sample was equally distributed to two cryovials,and were cryopreserved with vapor nitrogen freezing method and direct liquid nitrogen freezing method respectively.The total motility rate,progressive motility rate and motility parameters of post-thaw sperm were examined by IVOS sperm analyzer.The morphology of post-thaw sperm was observed with papanicolaou staining,and the rate of post-thaw sperm with normal morphology was calculated.Besides,the ultrastructure of post-thaw sperm was observed by transmission electron microscope.Results It cost 468 s to drop in temperature from 28 ℃ to-185 ℃ by vapor nitrogen freezing method,and an hour later the temperature went to-196 ℃.However,it only cost 90 s to drop in temperature from 30℃ to-196 ℃ by direct liquid nitrogen freezing method.The total motility rates,progressive motility rates,motility parameters and rates of post-thaw sperm with normal morphology in two cryopreservation methods were significantly lower than those before cryopreservation(P<0.05).The revival rate,total motility rate and progressive motility rate in vapor nitrogen freezing method were significantly higher than those in direct liquid nitrogen freezing method(P<0.05).There was no significant difference in motility parameters and rates of post-thaw sperm with normal morphology between two cryopreservation methods(P>0.05).Transmission electron microscopy indicated that no obvious damage was found on the ultrastructure of serosa and acrosome membrane at the head of post-thaw sperm in two different cryopreservation methods by using GYC as cryprotectant Conclusion GYC has a protective effect on the ultrastructure of sperm.Vapor nitrogen freezing method may yield better post-thaw results than direct liquid nitrogen freezing method in cryopreservation of human sperm.