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

doi:10.3969/j.issn.1674-8115.2012.08.003

›› 2012, Vol. 32 ›› Issue (8): 973-.doi: 10.3969/j.issn.1674-8115.2012.08.003

• 专题报道（不孕不育症及辅助生殖技术） • 上一篇下一篇

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

刘锋, 孟颖, 王峰, 刘勇, 王如瑶, 卢慧, 施文博, 李铮

上海交通大学医学院附属仁济医院泌尿外科 |上海市人类精子库 |上海市男科学研究所精子发育与遗传学实验室, 上海 200001

出版日期:2012-08-28 发布日期:2012-08-29
通讯作者: 李铮, 电子信箱: Lizhengboshi@163.com。
作者简介:刘锋（1956—）, 女, 副主任技师；电子信箱: lfyf2004@126.com；孟颖（1988—）, 女, 本科生；电子信箱: sjtu.mengying@gmail.com。
基金资助:
国家重点基础研究发展计划（“九七三”计划）资助项目（2011CB944504）和上海市科委基础研究重点项目（10JC1409900）

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

LIU Feng, MENG Ying, WANG Feng, LIU Yong, WANG Ru-yao, LU Hui, SHI Wen-bo, LI Zheng

Department of Urology, Shanghai Human Sperm Bank, Sperm Development and Genetics Laboratory, Shanghai Institute of Andrology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200001, China

Online:2012-08-28 Published:2012-08-29
Supported by:
National Basic Research Program of China, “973” program, 2011CB944504；Shanghai Science and Technology Committee Foundation, 10JC1409900

摘要/Abstract

摘要：

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。

关键词: 精子, 冷冻保存, 气相冷冻法, 液相冷冻法, 精子运动, 超微结构

Abstract:

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.

Key words: sperm, cryopreservation, vapor nitrogen freezing method, direct liquid nitrogen freezing method, sperm motility, ultrastructure

刘锋, 孟颖, 王峰, 等. 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[J]. , 2012, 32(8): 973-.

LIU Feng, MENG Ying, WANG Feng, et al. Effects of vapor nitrogen freezing method and direct liquid nitrogen freezing method on motility, morphology and ultrastructure of human sperm[J]. , 2012, 32(8): 973-.

[1]	黄晨辉，雷鸣. 端粒结合蛋白质影响哺乳动物精子发生过程的分子机制[J]. 上海交通大学学报（医学版）, 2018, 38(3): 241-.
[2]	卢克敏 1, 2，邹沙沙 2，陈向锋 2，刘强 1. 伐地那非预处理对精子冷冻效果的影响[J]. 上海交通大学学报（医学版）, 2018, 38(10): 1269-.
[3]	隋春华，陆颖理，郭郁郁. 胰高血糖素样肽1 类似物对2 型糖尿病大鼠肾脏功能及超微结构的影响[J]. 上海交通大学学报（医学版）, 2018, 38(1): 48-.
[4]	张时君，杜晶，吕仁华，田汝辉，李凤华. 非梗阻性无精子症患者睾丸局部生精功能的超声造影评估[J]. 上海交通大学学报（医学版）, 2017, 37(10): 1368-.
[5]	李朋，谭广兴*，黄煜华，陈慧兴，夏术阶，李铮 . 梗阻性无精子症显微外科重建策略分析[J]. 上海交通大学学报（医学版）, 2017, 37(03): 420-.
[6]	宋平平，邹沙沙,李铮，等. 精子鞭毛结构蛋白研究进展[J]. 上海交通大学学报（医学版）, 2015, 35(9): 1384-.
[7]	梅星星，李小勇，吴际. 一组miRNAs在睾丸发育中的表达及miR-125a对精原干细胞发育的调节作用[J]. 上海交通大学学报（医学版）, 2015, 35(5): 626-.
[8]	王鹰，沙天宇，周静哲，等. 青春前期十溴联苯醚暴露对雄性大鼠精子质量的影响[J]. 上海交通大学学报（医学版）, 2014, 34(7): 1063-.
[9]	唐龙，户宜，刘晓亮，等. 敌百虫亚急性染毒对雄性小鼠的生殖毒性作用[J]. 上海交通大学学报（医学版）, 2013, 33(12): 1587-.
[10]	高敏芝, 孙兆贵, 赵晓明, 等. 控制性超促排卵后不同卵巢反应对体外受精妇女妊娠结局的影响[J]. , 2013, 33(1): 39-.
[11]	卢慧, 施文博, 肖玉芳, 等. 温度和Hoechst 33258浓度对人活精子标记效果的影响[J]. , 2012, 32(8): 979-.
[12]	王鸿祥, 陈斌, 胡凯, 等. 梗阻性无精子症患者附睾精子DNA损伤与卵胞浆内单精子注射治疗结局的相关性研究[J]. , 2012, 32(8): 983-.
[13]	刘勇, 刘锋, 李铮, 等. 人类稀少精子细管法超快速冷冻的实验研究[J]. , 2012, 32(8): 988-.
[14]	夏兰, 赵晓明, 孙赟, 等. 卵胞浆内单精子注射失败后改行供精人工授精的时机和妊娠结局分析[J]. , 2012, 32(8): 1000-.
[15]	卢克敏, 卢慧, 李铮. PDE5抑制剂对精子活力及受精功能影响的研究进展[J]. , 2012, 32(8): 1010-.

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

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

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics