男性不育患者精浆miR-135a-5p和miR-135b-5p水平检测的价值

Clinical value of detecting seminal plasma levels of miR⁃135a⁃5p and miR⁃135b⁃5p in male patients with infertility

目的探讨精浆miR-135a-5p和miR-135b-5p表达特征及其在男性不育患者精浆中变化和价值。方法选取2019年1月至2021年12月于江苏省中医院检验科留存的血清、晨尿、胸水、脑脊液和精浆标本各30例;进一步选取2010年至2019年江苏省中医院和南京大学医学院附属金陵医院冻存的非梗阻性无精子症患者精浆标本、弱精子症患者精浆标本及正常对照精浆标本各54例。实时荧光定量PCR(RT-qPCR)检测不同体液、不育患者精浆和正常对照精浆中miR-135a-5p和miR-135b-5p表达。ROC曲线分析精浆miR-135a-5p、miR-135b-5p水平区分男性不育患者及对照的ROC曲线下面积(AUC^(ROC));相关性分析精浆miR-135a-5p、miR-135b-5p水平与精液参数的相关性。结果5种不同体液间miR-135a-5p和miR-135b-5p水平差异具有统计学意义(H=64.88,P<0.01和H=64.7,P<0.01),二者在血清和精浆中水平均最高,在尿液中水平均最低。无精子症组、弱精子症组及正常对照组精浆miR-135a-5p和miR-135b-5p水平差异具有统计学意义(H=32.14,P<0.01;H=21.37,P<0.01)。与正常对照组相比,无精子症组2种miRNA水平均显著降低(U=687,P<0.01;U=843,P<0.01);弱精子症组与正常对照组差异无统计学意义(P均>0.05);无精子症组与弱精子症组精浆miR-135a-5p和miR-135b-5p水平具有显著差异(U=635,P<0.01;U=779,P<0.01)。精浆miR-135a-5p和miR-135b-5p区分无精子症与正常对照及弱精子症患者AUCROC分别为0.764(95%CI:0.675~0.854),0.711(95%CI:0.612~0.810)和0.782(95%CI:0.695~0.869),0.733(95%CI:0.637~0.829)。相关性分析结果显示,精浆miR-135a-5p和miR-135b-5p水平与精子浓度、精子活动率、前向运动精子百分率和非前向运动精子百分率均呈正相关(P均<0.01)。结论miR-135a-5p和miR-135b-5p为精浆高表达miRNA,在无精子症患者中显著降低,二者与男性不育发生关系密切。

Objective To examine the expression pattern of miR135a5p and miR135b5p in seminal plasma as well as the clinical value of their variation in the male patients with infertility.Methods The clinical samples of 5 kinds,including serum,morning urine,pleural effusion,cerebrospinal fluid and seminal plasma,were collected from 30 patients respectively in the Department of Clinical Laboratory,Jiangsu Province Hospital of Chinese Medicine from January 2019 to December 2021.In addition,the frozen seminal plasma samples from the patients with nonobstructive azoospermia,asthenozoospermia of 54 cases for each illness statuses,and 54 healthy controls with fertile ability from 2010 to 2019 in Jiangsu Province Hospital of Chinese Medicine and Jinling Hospital were also selected.Fluorescent quantitative RTqPCR analyses were applied to measure the expression levels of miR135a5p and miR135b5p in seminal plasma.Receiver operating characteristic curve(ROC)was used to construct the area under the curve(AUC^(ROC))to evaluate the ability for discriminating infertility males from healthy controls.Correlation analyses were performed to explore the correlations between the levels of miR135a5p and miR135b5p in seminal plasma and other semen parameters.Results The concentrations of miR135a5p and miR135b5p in the samples from five different kinds of body fluid showed significantly difference(H=64.88,P<0.01 and H=64.7,P<0.01,respectively).The highest levels were found in serum and seminal plasma,and the lowest levels were in urine.The concentrations of miR135a5p and miR135b5p in seminal plasma were also showed marked difference among the azoospermia group,asthenozoospermia group and healthy fertile control group(H=32.14,P<0.01 and H=21.37,P<0.01,respectively).Compared to the fertile control group,the levels of miR135a5p and miR135b5p in seminal plasma were significantly downregulated in azoospermia patients(U=687,P<0.01 and U=843,P<0.01,respectively).The contents of the two miRNAs showed no statistically difference between asthenozoospermia patients and healthy fertile controls,but showed markedly difference between the two patients groups(U=635,P<0.01 and U=779,P<0.01,respectively).Receiver operating characteristic curve(ROC)analyses results revealed that levels of miR135a5p and miR135b5p in seminal plasma could successfully discriminate the azoospermia patients from fertile controls and asthenozoospermia patients with the AUCROC of 0.764(95%CI:0.675 to 0.854),0.711(95%CI:0.612 to 0.810)and 0.782(95%CI:0.695 to 0.869),and 0.733(95%CI:0.637 to 0.829),respectively.Correlation analyses demonstrated that the concentrations of miR135a5p and miR135b5p in seminal plasma were significantly associated with sperm concentration,total sperm motility,and percentages of progressive sperm and nonprogressive sperm(all with the Pvalue<0.01).Conclusion miR135a5p and miR135b5p are the highly expressed miRNA in seminal plasma,and significantly decreased in azoospermia patients.Both of them may play pivot roles in male infertility.