基于血浆转录组学进行生精障碍无创诊断分子标志物筛选和临床评价的研究

Testis biopsy has been considered as the "gold standard" of judging spermatogenesis status in clinic. However, the invasiveness and spermatogenic function injury of testis biopsy are the obstacles in application. Also, the accuracy was damaged by focal and heterogeneity of spermatogenesis in testis. So, we need to develop more accurate and operable but non-invasive diagnosis techniques. Nowadays, noninvasive plasma cell-free mRNA diagnosis technology has been widely studied and applied to the field of tumor detection and prenatal diagnosis. In our previous study, specific expression of a variety spermatogenesis specific genes mRNAs had been detected in plasma and their expression patterns varied with the types of testicular pathology. On the basis of the results, plasma cell-free mRNA gene expression profiles in the infertile men will be detected by microarray tecnology with the classification of testicular pathology. Then the pathological type specific expression of the gene and/or gene clusters will be sieved out and the model for diagnosis be established by bioinformatics technology such as the differential gene expression, hierarchical cluster analysis and classification analysis etc. Afterwards, the technology for non-invasive diagnosis of spermatogenesis status by cell-free mRNA analysis in plasma shoud be validated to lay the foundation for clinical application. Furthermore,investigation for the molecular mechanism of normal spermatogenesis and/or spermatogenic dsfunction will be explored.

临床上对男性不育症患者生精功能的最终诊断依赖于睾丸组织病理检查。但睾丸活检的创伤性及损伤生精功能的缺点，且精子发生具有局灶性和双侧睾丸差异性，故活组织检查准确性尚不够，故亟待发展更为准确、无创性的检查技术。无创性的血浆 cell-free mRNA 检查技术已在肿瘤及产前诊断领域得到了广泛研究和应用。我们前期研究发现血浆内存在多种精子发生特异表达基因的 mRNAs，其表达组合模式随睾丸病理类型的不同而异,即血浆 cell-free mRNA 表达谱与生精功能发生具有相关性。本研究拟采用基因表达芯片检测各睾丸病理类型患者血浆 cell-free mRNA 基因表达谱，利用基因差异表达及聚类分析等生物信息学技术，筛出各病理学类型特异表达基因（组）并进行临床验证，奠定血浆 cell-free mRNA做为判断睾丸生精功能的无创性检测的分子标志物的基础，探索正常人类精子发生和精子发生障碍分子机制。

项目背景.临床上对男性不育症患者生精功能的诊断依赖于睾丸活组织病理检查。而且，准确诊断睾丸生精状态对男性不育患者具有指导性意义，但睾丸中精子发生具有局灶性和双侧睾丸差异性，故活组织检查准确性尚不够。加之睾丸活检的创伤性及损伤生精功能的缺点，亟待发展更为准确、可操作性强而又无创性的检查技术。.主要研究内容.在本研究中，我们根据前期的课题设计，首先对正常精子发生健康对照（normal spermatogenesis, NS）和非梗阻性无精症患者（Non-obstructive azoospermia, NOA）血浆中富集的small RNA进行了二代测序及表达差异性分析，并进行了靶基因的预测和初步验证。而在对同样两类人群精浆的mRNA及small RNA的二代测序及其后的联合分析结果表明：结合GTEx数据库数据，所筛选出的42个差异mRNA大部分都是在睾丸组织高表达（有两个基因无数据），其中37个最高表达组织为睾丸。其后，我们对部分基因（包括small RNA）进行了验证性检测和应用性分析。.主要研究结果.结果显示，目前我们所检测的基因中，① 血浆ZPBP（Zona pellucida-binding protein）蛋白可能为一无创诊断的候选靶分子。②精浆游离SPATA3 （spermatogenesis associated 3） mRNA，其cutoff值为0.27，敏感性0.91，特异性 0.73，AUC 0.794。③精浆游离SMCP （sperm mitochondria associated cysteine rich protein）mRNA，其cutoff值为0.23，敏感性1，特异性 0.65，AUC 0.756。④对正常精液和弱精症患者精浆游离mRNA的分析表明，HSPA2 （heat shock 70kDa protein 2）mRNA含量分别和精子活力负相关（R2 = 0.213，P = 0.004），与精子浓度负相关（R2 = 0.133，P = 0.026）。.科学意义.上述分子的筛选将有助于无创性诊断技术开发及在男性不育临床应用的推广，同时也有助于探索正常人类精子发生和精子发生障碍分子机制。