浮游板优化精液与密度梯度离心结合上游法的比较  

Comparison of the effects between optimization of semen with the floating plate and density gradient centrifugation with swim-up in split semen samples

作　　者：张玲[1,2] 辜秀丽 袁周 金其贝 张衷源 叶臻 孟天庆 周慧 李红钢[1] 熊承良 ZHANG Ling;GU Xiu-li;YUAN Zhou;JIN Qi-bei;ZHANG Zhong-yuan;YE Zhen;MENG Tian-qing;ZHOU Hui;LI Hong-gang;XIONG Cheng-liang(Institute of Reproductive Health,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030;Wuhan Huake Reproductive Specialist Hospital,Wuhan 430010)

机构地区：[1]华中科技大学同济医学院生殖健康研究所,武汉430030 [2]武汉华科生殖专科医院,武汉430010

出　　处：《生殖医学杂志》2024年第8期1080-1086,共7页Journal of Reproductive Medicine

基　　金：国家重点研发计划项目(2022YFC2703004)。

摘　　要：目的应用浮游板和密度梯度离心结合上游法优化人类精液,明确优化效果并比较两种方法优化后精子参数的差异。方法收集正常精液样本85例和轻中度少弱精子症精液样本59例,每例样本分为原液组、密梯组和浮游组各1 ml进行后续处理。原液组在精液液化后未做任何处理;密梯组精液液化后用密度梯度离心法结合上游法进行处理,浮游组则在精液液化后应用精子浮游板进行处理。比较原液组、密梯组、浮游组3个亚组间的精子浓度、前向运动精子百分率、正常形态精子百分率;应用流式细胞仪检测精子线粒体膜电位和精子DNA碎片指数,使用单精子活性氧分析仪检测单精子活性氧水平,使用电子显微镜观察精子超微结构并进行组间比较。结果无论是正常精液还是轻中度少弱精子症精液,与原液组比较,密梯组与浮游组的前向运动精子百分率、正常形态精子百分率及精子线粒体膜电位均显著升高(P<0.05),精子DNA碎片指数显著降低(P<0.05);密梯组和浮游组各指标无显著性差异(P>0.05)。正常精液中,浮游组的精子浓度显著高于密梯组(P=0.029),单精子活性氧水平显著低于原液组(P=0.038)。轻中度少弱精子症精液中3亚组间的单精子活性氧水平均无显著性差异(P>0.05)。电子显微镜观察发现正常精液中3个亚组的精子均未见明显异常;轻中度少弱精子症精液中密梯组部分精子可观察到精子细胞膜断裂和线粒体肿胀等异常,而浮游组精子超微结构未见明显异常。结论密度梯度离心结合上游法和浮游板法用于精液的处理和优化,均可有效回收活力、形态和功能更好的精子。精子浮游板法的体外干预小,操作简便,可以获得活性氧更少、损伤更小的精子。Objective:To optimize human semen with floating plate and density gradient centrifugation combined with upstream method in order to clarify the optimization effects and compare the difference of sperm quality between the two methods.Methods:Eighty-five normal semen samples and 59 oligoasthenozoospermia semen samples were obtained,and each sample was divided into the unprocessed semen group(1 ml),density-gradient centrifugation and swim-up group(DGU group,1 ml)as well as floating plate group(FP group,1 ml).The unprocessed semen group did not undergo any treatment after semen liquefaction,the DGU group was treated with density-gradient centrifugation with swim-up,while the FP group was treated with a microfluidic device called floating plate method.The sperm concentration,forward motility sperm percentage,and normal morphology sperm percentage were compared among the three subgroups of the unprocessed semen group,DGU group and FP group.Sperm mitochondrial membrane potential and sperm DNA fragmentation index were detected with flow cytometry,and sperm reactive oxygen species levels were evaluated with a single sperm reactive oxygen species analyzer,and the electron microscopy was used for the observation of sperm ultrastructure.The different indicators were compared among three groups.Results:Compared with the unprocessed semen group,the percentage of progressive motile sperm,normal morphology sperm,and mitochondrial membrane potential of sperm in the DGU group and FP group were significantly increased(P<0.05),while the DNA fragmentation index of sperm was significantly reduced(P<0.05)for both normal semen and oligoasthenozoospermia semen.There were no significant differences in these indicators between the FP and DGU groups.For normal semen,the sperm concentration of FP group was significantly higher than that of DGU group(P=0.029),and the level of reactive oxygen species in single sperm was significantly lower than that in the unprocessed semen group(P=0.038).For the oligoasthenozoospermia semen,there were no sign

关 键 词：密度梯度离心法 浮游板法 单精子活性氧 精子DNA碎片指数 精子线粒体膜电位 

分 类 号：R698.2[医药卫生—泌尿科学]