诊断NOA患者睾丸精子形成的cfs-mRNAs鉴定及机制研究

Non-obstructive azoospermia (NOA) is a common and severe condition in male infertility. Although no sperm can be obtained in the ejaculate, sperm can be retrieved from testis of many (43%-63%) patients with NOA, thus they have the opportunity to have their genetic offspring. The method for the prediction of successful sperm retrieval is thus desired. Some factors, such as the testis volume, hormones, body weight index, may contribute to the prediction of successful sperm retrieval, but many of them remain controversial. Biopsy, the present golden standard for testicular phenotype, is either not very reliable because of the heterogeneity of spermatogenesis. Our previous study of the presence and characterization of cell-free seminal mRNA (cfs-mRNA) in human seminal plasma, and the successful application of some seminal mRNAs for the diagnosis of obstructive azoospermia and Sertoli-Cell-Only syndrome indicated that cfs-mRNA should be a promising non-invasive biomarker for disorders of male reproductive system, with high sensitivity and specificity. We have obtained the cfs-mRNAs profiles of NOA with different testicular phenotypes, and found 28 cfs-mRNAs are significantly higher in NOA patients with hypospermatogenesis, which have sperm in testis, than in NOA men with maturation arrest. The present proposal is aimed to screen and identify the cfs-mRNAs from these 28 mRNAs for the diagnosis of the presence of sperm in NOA men. We will screen the candidate cfs-mRNAs by comparing their levels in seminal plasma of NOA men with sperm and without sperm revealed by microdissection testicular sperm extraction (micro-TESE). Candidate cfs-mRNAs then will be further identified in a prospective study to find the cfs-mRNAs with high specificity and sensitivity for the diagnosis of testicular sperm presence in NOA men. To elucidate the mechanism for the diagnosis, the dynamic changes of mRNA expression of these candidate genes will be evaluated during human spermatogenesis; and the main existing forms of these candidate cfs-mRNAs will be determined in the human semen. The objective of this project is to gain a solid foundation for the novel non-invasive prediction of successful sperm retrieval from NOA patients based on cfs-mRNA.

非梗阻性无精子症（NOA）是男性不育常见而严重的类型，50%左右患者可在睾丸找到精子用于辅助生殖技术而生育血缘关系后代；然而，临床缺乏无创性诊断NOA患者睾丸有无精子形成的可靠方法。我们前期发现了人精浆游离mRNA（cfs-mRNA），可无创性反映精浆分泌器官的基因表达；并建立了NOA不同睾丸病理类型对应的cfs-mRNAs谱，筛出28个在精子形成阶段显著上调表达的cfs-mRNAs，因而有诊断睾丸精子形成的潜能。本课题将这些cfs-mRNAs在睾丸切开显微取精术有、无精子的NOA患者中比较，筛选有诊断价值的指标；再通过前瞻性研究，鉴定出敏感性和特异性俱佳的cfs-mRNAs；并研究鉴定出的cfs-mRNAs基因在人精子发生中的转录动态变化及其分泌入精浆中的主要方式，为cfs-mRNAs诊断睾丸精子形成提供机制支持。本课题旨在为建立一种无创性诊断NOA患者睾丸精子形成的新途径奠定基础。

随着辅助生殖技术的发展，约半数非梗阻性无精子症（NOA）可以在睾丸中找到精子用于辅助生殖技术（ICSI）而生育血缘关系后代，然而，目前没有可靠的方法能预测哪些NOA患者睾丸中能找到精子。诊断NOA病理类型的金标准，即睾丸活检，也不能准确预测NOA患者精子形成情况。因此，找到一种能预测NOA患者睾丸精子形成的方法，尤其是无创性方法，有着很好的临床意义和需求。我们在前期研究精浆游离mRNAs（cfs-mRNAs）特点及用于唯支持细胞综合症的诊断基础上，本课题中首先筛选cfs-mRNAs用于诊断NOA患者减数分裂是否阻滞，由于减数分裂是精子发生中精子形成的最关键的前提，再进一步筛选cfs-mRNAs用于预测NOA患者睾丸精子形成。主要结果：先明确了影响精浆游离RNA的临床因素，表明禁欲天数和阴囊热激会影响cfs-mRNAs水平，也解决了NOA患者纳入的相关问题，表明近期受到关注的X染色体相关CNVs与我国NOA发生无关，因此在本课题执行中排除了一些临床混杂因素的影响；通过表达谱芯片比较减数分裂阻滞NOA患者与睾丸显微取精成功NOA患者cfs-mRNAs差异，从睾丸显微取精成功NOA患者上调差异表达的cfs-mRNAs中：①结合基因功能和表达模式，筛选出18个cfs-mRNAs；②通过比较梗阻性无精子症、精液参数正常男性及完全性唯支持细胞综合症患者这18个cfs-mRNAs水平，找出主要来源于睾丸的cfs-mRNAs（共11个）；③在减数分裂阻滞和生精功能低下的NOA患者中，用绝对定量RT-qPCR的方法，筛选出了用于诊断减数分裂阻滞的cfs-mRNAs，其中BOLL基因较为突出（ROC曲线下面积AUC=0.819，P<0.001，灵敏度为84.2%，特异度为78.3%）。通过qRT-PCR分析在睾丸显微取精成功与显微取精失败的NOA患者精浆中的水平，进一步分析筛选出的cfs-mRNAs预测睾丸显微取精的准确性，BOLL基因也显示较好结果。原位杂交及cfs-mRNA存在形式实验结果也支持其用于诊断减数分裂和预测精子形成的结果。