论文部分内容阅读
目的研究精浆游离miR-34b在体外不同条件下的稳定性及其与男性不育症患者精液参数的关系。方法选取男性不育症患者80例和同期因女方因素不孕前来检查的生育力评估正常的健康男性20例为正常对照组,将8例正常的精液标本分别在室温和-80℃下放置,以不同时间点检测精浆miR-34b的稳定性。采用实时荧光定量PCR检测精浆游离miR-34b的相对含量,精子浓度及精子活力用计算机辅助精子分析系统,精子存活率采用低盐膨胀实验检测,正常精子形态百分率采用Diff-Quick染色法。分析男性不育症组和正常对照组精液参数及miR-34b表达量的差异,对精浆游离rniR-34b在男性不育组精浆中的诊断价值进行ROC曲线分析,并采用Pearson直线相关分析miR-34b与男性不育组精液各参数的相关性。结果精浆游离miR-34b在室温和低温下不同时间点表达量无明显变化(F=0.13,P=0.97;F=0.35,P=0.84)。与正常组比较发现,男性不育组精浆游离miR-34b表达下调(P<0.01)。两组的精子浓度、精子存活率、前向运动精子百分率(PR)和正常形态精子百分率均有差异(P<0.01)。ROC曲线显示miR-34b能够较好的区分不育组与健康对照组,曲线下面积分比(AUC)为0.85(0.79-0.95,95%)。Pearson直线相关分析发现男性不育患者精浆miR-34b表达量与精子密度(r=0.28,P<0.05)、精子活率(r=0.56,P<0.01)、前向运动精子百分率(r=0.41,P<0.01)正相关,与精子形态无相关性(r=O.15,P>0.05)。结论精浆miR-34b在室温下稳定,并能在-80℃条件下长期保存,在男性不育患者精浆中表达下调,并与精子浓度、精子存活率、前向运动精子百分率呈正相关,与精子形态无相关性。
Objective To study the stability of seminal plasma free miR-34b under different conditions in vitro and its relationship with sperm parameters in male infertility patients. Methods 80 cases of male infertility and 20 cases of fertility normal women who were fertile due to female infertility were selected as normal control group. Eight normal semen specimens were placed at room temperature and -80 ℃ respectively, The stability of seminal plasma miR-34b was measured at different time points. Real-time fluorescence quantitative PCR was used to detect the relative content of seminal plasma free miR-34b. The sperm concentration and sperm motility were determined by computer-aided sperm analysis system. The sperm survival rate was measured by low-salt swollen test. The percentage of normal sperm morphology was calculated by Diff-Quick staining. The difference of semen parameters and expression of miR-34b between male infertility group and normal control group was analyzed. The diagnostic value of seminal plasma free rniR-34b in male infertility group was analyzed by ROC curve and Pearson’s linear correlation analysis Correlation of various parameters of sperm between miR-34b and male infertility group. Results There was no significant change in the expression of seminal plasma free miR-34b at different time points (F = 0.13, P = 0.97; F = 0.35, P = 0.84) at room temperature and low temperature. Compared with normal group, the expression of free miR-34b in male infertility group was down-regulated (P <0.01). The sperm concentration, sperm survival rate, percentage of motile sperm motility (PR) and percentage of normal spermatozoa in both groups were significantly different (P <0.01). The ROC curve showed that miR-34b was able to distinguish the infertile group from the healthy control group well. The AUC of the curve was 0.85 (0.79-0.95, 95%). Pearson’s linear correlation analysis showed that the positive rate of sperm motility (r = 0.56, P <0.01) and the percentage of motile spermatozoa (r = 0.41, P <0.01), but no correlation with sperm morphology (r = 0.15, P> 0.05). CONCLUSIONS: Seminal plasma miR-34b is stable at room temperature and can be stored at -80 ℃ for a long time. It is down-regulated in seminal plasma of male infertility patients and positively correlated with sperm concentration, sperm motility and percentage of forward motility sperm. No correlation with sperm morphology.