SYBR-14/PI双染法流式细胞术检测精子质膜完整性的研究

目的:探讨应用荧光染料SYBR-14/碘化丙啶(SYBR-14/PI)双色标记法进行流式细胞术检测精子质膜完整性的可行性及其临床意义。方法:收集208例男性精液标本,按WHO精液分析标准分为正常组(n=31)与异常组(n=177)。通过计算机辅助精液分析系统进行精液常规分析。精液标本洗涤处理后经SYBR-14/PI双染后上流式细胞仪(FCM)分析,用发绿色荧光精子百分率(SYBR-14+/PI-%)表示质膜完整精子(PMI)的比例。结果:正常组与异常组SYBR-14+/PI-与SYBR-14-/PI+精子百分率均存在统计学差异(P均<0.05)。正常组精子SYBR-14+/PI-%为(55.66±20.64)%,显著高于异常组(39.71±19.21)%,P=0.000]。208例标本中,SYBR-14+/PI-%与精子活动率呈显著正相关(r=0.408,P=0.000),与(a+b)级精子百分率呈显著正相关(r=0.398,P=0.000),与d级精子百分率呈显著负相关(r=-0.413,P=0.000);SYBR-14-/PI+%与精子活动率呈显著负相关(r=-0.380,P=0.000),与(a+b)级精子百分率呈显著负相关(r=-0.397,P=0.000),与d级精子百分率呈显著正相关(r=0.385,P=0.000);SYBR-14+/PI+%与精子活动率呈正相关(r=0.172,P=0.013),与(a+b)级精子百分率呈正相关(r=0.177,P=0.011),与d级精子百分率呈负相关(r=-0.164,P=0.018)。结论:应用流式细胞术SYBR-14/PI双染法检测精子质膜完整性具有可行性,可用于评估男性生育力。

Evaluation of sperm plasma membrane integrity by SYBR-14/PI dual fluorescent staining and flow cytometry

Objective:To investigate the feasibility and clinical significance of detecting the plasma membrane integrity (PMI) of sperm by SYBR-14/PI fluorescent staining and flow cytometry. Methods:A total of 208 semen samples were divided into a normal (n = 31) and an abnormal group (n = 177),subjected to conventional computer-assisted semen analysis (CASA),and then evaluated for sperm PMI by flow cytometry after washed and SYBR-14/PI dual fluorescent staining. The percentage of sperm with PMI was indicated as that of the sperm emitting green fluorescence (SYBR-14+/PI-%). Results:Significant differences were detected in SYBR-14+/PI-and SYBR-14-/PI+ between the normal and abnormal groups (P < 0.05),with the SYBR-14+/PI-% significantly higher in the former (55.66 ± 20.64]%) than in the latter (39.71 ± 19.21]%) (P = 0.000). The SYBR-14+/PI-% showed significant positive correlations with sperm motility (r = 0.408,P = 0.000) and the percentage of grade a + b sperm (r = 0.398,P = 0.000),and a significant negative correlation with the percentage of grade d sperm (r=-0.413,P = 0.000); the SYBR-14-/PI+% exhibited significant negative correlations with sperm motility (r=-0.380,P = 0.000) and the percentage of grade a + b sperm (r=-0.397,P = 0.000),and a significant positive correlation with the percentage of grade d sperm (r=0.385,P = 0.000); the SYBR-14+/PI+% showed positive correlations with sperm motility (r = 0.172,P = 0.013) and the percentage of grade a + b sperm (r = 0.177,P = 0.011),and a negative correlation with grade d sperm (r =-0.164,P = 0.018). Conclusion:SYBR-14/PI dual fluorescent staining and flow cytometry could be readily used to detect sperm PMI and evaluate male reproductivity.