男性不育患者精浆exosome中miR-184水平变化及临床意义

The miR-184 level in the seminal plasma exosome of male infertility patients and its clinical significance

目的:探讨男性不育患者精浆外泌体(exosome)中miR-184的水平变化及临床意义。方法:收集2015~2019年在江苏省中医院、东部战区总医院及温州医科大学附属第一医院就诊的97例无精子症患者、96例弱精子症患者及同期募集的92例正常生育男性精浆标本,分离精浆exosome后运用透射电镜、Nanosight和Western印迹对exosome进行鉴定;运用实时荧光定量PCR(qRT-PCR)检测exosome miR-184的水平,统计分析miR-184变化、临床价值及与精液参数的相关性,进一步用生物信息学方法预测和分析miR-184靶基因、相关信号通路及与男性不育关系。结果:透射电镜、Nanosight和Western印迹结果显示,精浆中含有大量exosome。qRT-PCR结果显示,无精子症患者精浆exosome miR-184水平显著低于正常对照[0.227 (0.092, 0.790)vs 0.650 (0.408, 1.061)](P<0.01),弱精子症患者精浆exosome中miR-184水平显著高于正常对照[1.176 (0.661, 1.946)vs 0.650 (0.408, 1.061)](P<0.01)。受试者工作特征曲线(ROC)分析结果显示,精浆exosome miR-184无精子症、弱精子症患者的曲线下面积(AUC)为0.866(95%CI 0.815~0.916)和0.724(95%CI 0.653~0.795);区分弱精子症和无精子症患者的AUC为0.964(95%CI 0.943~0.985)。相关性分析显示,弱精子症患者精浆exosome miR-184水平与精子浓度呈显著正相关(r=0.243,P=0.017),与前向运动精子百分率呈显著负相关(r=-0.407,P=0.006)。生物信息学分析发现miR-184的靶基因显著富集于男性生殖和精子发生密切相关的蛋白调控通路。结论:男性不育患者精浆exosome miR-184水平与正常生育男性具有统计学差异,具备作为男性不育辅助诊断标志物潜能并可能参与男性不育的发生发展。

Objective: To study the miR-184 level in the seminal plasma exosome of male infertility patients and its clinical significance. Methods: Between 2015 and 2019, we collected 285 seminal plasma samples from 97 azoospermia(AS) and 96 asthenospermia(AZS) patients and 92 age-matched normal fertile controls in Jiangsu Provincial Hospital of Traditional Chinese Medicine, General Hospital of Eastern Theater Command and the First Hospital Affiliated to Wenzhou Medical University, identified the isolated seminal plasma exosomes by nanoparticle tracking analysis(NTA), transmission electron microscopy(TEM) and Western blot, and detected the miR-184 level in the seminal plasma exosomes by quantitative real-time PCR(qRT-PCR). We determined the clinical value of the miR-184 level and its correlation with semen parameters by multiple statistics, predicted the target genes and involved pathways of miR-184 by bioinformatic algorithms, and analyzed their relationship with male infertility. Results: NTA, TEM and Western blot exhibited plenty of exosomes in the seminal plasma of the patients. The results of qRT-PCR showed that the miR-184 level in the seminal plasma exosome was dramatically decreased in the AS patients compared with that in the normal fertile controls(0.227 [0.092, 0.790] vs 0.650 [0.408, 1.061], P < 0.01), but increased in AZS males in comparison with that in the control group(1.176 [0.661, 1.946] vs 0.650 [0.408, 1.061], P < 0.01). The areas under the ROC curve(AUC) for differentiating the AS and AZS patients from the controls were 0.866(95% CI: 0.815-0.916) and 0.724(95% CI: 0.653-0.795), respectively, and that for differentiating the AS from the AZS group was 0.964(95% CI: 0.943-0.985). The miR-184 level in the seminal plasma exosome of the AZS patients was correlated positively with the sperm count(r = 0.243, P = 0.017) but negatively with the percentage of progressively motile sperm(r =-0.407, P = 0.006). Bioinformatics analysis indicated that the downstream target genes of miR-184 were significantly enriched in the protein regulatory pathways closely related to male reproduction and spermatogenesis. Conclusion: The miR-184 level in the seminal plasma exosome of infertility patients is significantly different from that of normal fertile males, which may serve as a potential auxiliary marker for the diagnosis of and participate in the development and progression of male infertility.