冷冻保存对人类精子线粒体DNA的影响  

Effect of sperm cryopreservation on mitochondrion DNA of human sperm

作　　者：傅龙龙 周芳[1] 安琪 张开舒 童越 许剑锋 王晓尉[1] 郭颖 李鸿 袁冬 卢文红 梁小薇 谷翊群 FU Long-long;ZHOU Fang;AN Qi;ZHANG Kai-shu;TONG Yue;XU Jian-feng;WANG Xiao-wei;GUO Ying;LI Hong;YUAN Dong;LU Weng-hong;LIANG Xiao-wei;GU Yin-gun(Human Sperm Bank/Department of Male Clinical Research,Key Laboratory of National Health ＆ Family Planning Commission for MaleReproductive Health,National Research Institute for Family Planning,Beijing 100081;Graduate School of Peking Union Medical College,Beijing 100730;Reproductive Department,the Afiliated Hospital of Qingdao University,Qingdao 266000)

机构地区：[1]国家卫生计生委科学技术研究所国家卫生计生委男性生殖健康重点实验室男性临床研究室/北京人类精子库,北京100081 [2]北京协和医学院研究生院,北京100730 [3]青岛大学附属医院生殖中心,青岛266000

出　　处：《生殖医学杂志》2018年第11期1126-1130,共5页Journal of Reproductive Medicine

基　　金：中华医学会临床医学科研专项资金-生殖医学青年医师研究与发展专项目(16020510667);国家卫生计生委科学技术研究所青年科技创新基金重点项目(2015GJZ08)

摘　　要：目的探讨常规精液冷冻技术对人类精子线粒体DNA的影响。方法收集符合精子库捐精条件的正式志愿者精液样品,将每份样品分为2份:1份进行精液冷冻复苏处理,为实验组;1份新鲜精液,为对照组。采用Markler计数板联合CASA法评估冷冻复苏前后精子活力;两组精液均采用实时荧光定量PCR和长链PCR技术,分别检测精子线粒体DNA的拷贝数和完整性。结果共收集22份精液标本,纳入志愿者年龄为(27.8±3.0)岁,禁欲天数(6.1±0.9)d;精液体积(5.0±1.5)ml,精子浓度(75.8±15.8)×106/ml,前向运动精子百分比为(68±6)%,总活动精子复苏率为(72±8)%。冷冻保存后,精子活力显著降低:前向运动精子百分比减少[(49.0±6.5)%vs.(68.0±6.1)%,P<0.05),平均路径速率(VAP)[(35.8±6.8)vs.(46.8±9.5),P<0.05]、直线速率(VSL)[(27.3±3.3)vs.(35.1±8.3),P<0.05]和曲线速率(VCL)[(57.6±6.9)vs.(91.8±10.2),P<0.05]较前下降。与新鲜精液相比,冷冻复苏后精子的线粒体DNA拷贝数显著增加[(10.12±8.41)vs.(5.66±5.53),P<0.05],完整性比较无统计学差异[(29.69±15.04)vs.(32.78±16.0),P=0.077]。结论在正式捐精志愿者人群内,常规精液冷冻技术降低精子活力,增加人类精子mtDNA的拷贝数,但未显著改变mtDNA的完整性。Objective： To detect the effect of routine semen cryopreservation on human sperm mitochondrial DNA. Methods： The semen samples ftom normal donors were collected in Beijing Human Sperm Bank. Each sample was divided into 2 parts：the cryopreservation experience group and the control group（fresh semen without cryopreservation）. The sperm motility was examined by Marker and CASC. The copy number and integrity of sperm mtDNA were detected by real time fluorescence quantitative （Q PCR）and long polymerase chain reaction（L PCR） respectively. Results： A total of22 sperm samples were collected. The age of the donors was（27. 8±3. 0）years,the number of abstinence days（6. 1 ± 0. 9）days, the volume of semen（5.0 ± 1.5）ml, the sperm concentration （75. 8±15. 8））×10^6/ml,the percentage of the progressive motility sperm（68±6）% ,and the recovery rate of motile sperm（72±8）%. After cryopreservation, sperm motility including the percentage of RPE（49.0± 6.5） G vs. （68.0 ± 6.1） G, P〈0. 051 and sperm motility parameters VAPE （35.8 ± 6.8） vs. （46.8 ± 9.5）, P〈0.051,VSL（27.3±3.3）vs. （35.1±8.3）,P〈0.05] and VCL[（57.6±6.9）vs. （91.8±10.2）,P〈 0. 051 was decreased significantly. The copy number of mtDNA of sperm with cryopreservation was increased significantly]（10.12±8.41）vs. （5. 66±5. 53） ,P〈0. 051. However,the sperm mtDNA integrity was not changed after cryopreservation[ （29.69±15.04）vs. （32.78±16.0）,P=0.077]. Conclusions： Sperm cryopreservation technology decreases the sperm motility and increases the copy number of human sperm mtDNA,but does not change the sperm mtDNA integrity,in the eligible sperm donors.

关 键 词：精子冷冻 生育力保存 线粒体DNA 

分 类 号：R714.8[医药卫生—妇产科学]