流式细胞术检测吸烟对精子质膜完整性的影响

Cigarette smoking affects sperm plasma membrane integrity

目的:应用荧光染料SYBR-14/PI双色标记法进行流式细胞术检测不育患者精子质膜完整性,分析吸烟对精子质膜完整性的影响并探讨其临床意义。方法:收集202例男性精液标本,其中132例为本院就诊男性不育患者,分为大烟量组(n=68)与小烟量组(n=64),正常生育男性为正常对照组(n=70)。通过计算机辅助精液分析系统进行精液常规分析。精液标本经PBS洗涤处理后用荧光染料SYBR-14/碘化丙锭(PI)双染后上流式细胞仪分析,用SYBR-14+/PI-(绿)表示质膜完整精子(PMI);SYBR-14-/PI+(红)表示质膜破损而坏死的精子;SYBR-14+/PI+(绿,红)表示正处于由活到死过度状态的精子。检测精子质膜完整性,并对精子质膜完整性与部分精液参数做相关性分析。结果:大烟量组与小烟量组质膜破损的坏死精子(SYBR-14-/PI+%)、过渡态的精子(SYBR-14+/PI-%)与正常对照组存在显著差异(P<0.01或0.05)。大烟量组SYBR-14+/PI-%明显低于小烟量组,SYBR-14-/PI+%明显高于小烟量组。所有202例标本SYBR14+/PI-%与精子活动率呈显著正相关(r=0.938,P=0.000)。结论:应用流式细胞术SYBR-14/PI双染法能快速、准确地检测精子质膜完整性。吸烟会导致精子质膜完整性下降,引起精子活力下降,可能是导致男性不育的重要原因之一。

Objective： To detect sperm plasma membrane integrity （PMI） of cigarette smoking infertile males using SYBR-14/ PI fluorescent staining and flow cytometry and investigate its clinical significance. Methods： We collected semen samples from 132 cigarette smoking infertile men and 70 normal fertile controls, the former divided into a heavy-smoker group （ 〉 20 cigarettes a clay, n = 68） and a light-smoker group （ ≤20 cigarettes a day, n = 64）. We performed computer-assisted semen analysis of the semen sam- ples, and determined sperm PMI by flow cytometry after rinsing with PBS and staining by SYBR-14/PI, the sperm with normal PMI in- dicated as the percentage of those emitting green fluorescence （ SYBR-14 ＋/PI - % ）, dead sperm as the percentage of those emitting red （SYBR-14-/PI ＋ ） , and moribund sperm as the percentage of those emitting both green and red （SYBR-14 -/PI ＋ ）. Results： Both the heavy- and light-smoker groups showed significant differences in SYBR-14 -/PI ＋ % and SYBR-14 ＋/PI - % from the normal controls （P 〈 0.01 or P 〈0.05 ）. SYBR-14 ＋/PI-% was remarkably lower, while SYBR-14-/PI＋% markedly higher in the heavy- smoker than in the light-smoker group （P 〈 0.05 ）. There was a significant correlation between SYBR-14 -/PI-% and sperm motility （ r = 0. 938, P = 0. 000）. Conclusion ： SYBR-14/PI fluorescent staining and flow cytometry analysis could quickly and exactly detectsperm PMI. Cigarette smoking reduces sperm PMI and consequently sperm motility, which might be an important factor of male infertil- ity.