流式细胞术检测精子线粒体膜电位影响因素分析

Analysis of influencing factors in detection of sperm mitochondrial membrane potential by flow cytometry

目的对运用流式细胞术检测精子线粒体膜电位(MMP)中的影响因素进行初步研究.方法对精子MMP的流式细胞检测技术进行重复性分析后,随机选取符合MMP检测要求的精液样本,观察样本放置时间、温度、样本保存液及冷冻等因素对MMP检查结果的影响.结果高、中、低MMP的精液样本使用流式细胞术重复检测10次,所得CV均低于7%;随着样本放置时间延长,精子MMP逐渐下降,与放置30 min(43.31±13.96)相比,放置2 h后MMP显著降低(35.44±17.23,P<0.05).不同样本的个体差异导致MMP的下降幅度存在差异;样本存放的温度:精液样本于2~8℃冷藏放置相较室温避光保存样本MMP下降幅度少,但是无统计学差异(P>0.05);精液样本添加自制样本保护液于2~8℃冷藏后,精子MMP显著高于无保护剂添加的样本(P<0.05),MMP下降速度明显减缓.精液样本1∶1添加冷冻保护剂后置于-20℃冷冻会对精子MMP造成显著损伤(P<0.01).结论MMP检测中,样本放置时间对MMP的检测结果有着较大的影响,最好在精液液化后半小时内完成检测.为了提高实验室的质量控制及工作效能,可将样本置于样本保存液中冷藏并在2小时内完成检测.冷冻精液样本无法作为精子MMP检测的室内质控品.

Objective To analyze the influencing factors in detection of sperm mitochondrial membrane potential(MMP)by flow cytometry.Methods After reproducibility analysis of sperm MMP using flow cytometry,semen samples that met the requirements of MMP detection were randomly collected,The effects of sample standing time,temperature,sample preservation solution,freezing and other factors on the results of MMP were analyzed.Results The semen samples with high,medium and low MMP levels were repeatedly tested by flow cytometry for 10 times,and the CV of the results was all less than 7%.With the extension of the sample storage time at room temperature,the level of sperm MMP was gradually decreased.Compared with the result of the placed sample for 30 min(43.31±13.96),the level of sperm MMP was significantly decreased in the placed sample for 2 hours(35.44±17.23).The decrease of MMP of semen samples stored at 2-8℃was less than that of samples stored at room temperature,but there was no significant difference(P>0.05).The level of sperm MMP in the samples with self-made sample protective solution store at 2-8℃was significantly higher than that of the samples without protective agent(P<0.05),and the decline rate of MMP level was significantly slower(P<0.05).The temperature of semen stored at 2-8℃was less than that of samples stored at room temperature,but there was no statistical difference.Semen samples with cryoprotectant at 1:1 were frozen at-20℃,which caused a significant damage of sperm MMP(P<0.01).Conclusion For sperm MMP detection using FCM,the sample standing time has a great influence on the test result.It is recommended to finish the test within half an hour after semen liquefaction.In order to improve the quality control and working efficiency of the laboratory,the samples is suggested to be refrigerated in the sample preservation solution and the testing can be completed within 2 hours.Frozen semen samples can not be used as internal quality control for sperm MMP detection.