诊断NOA患者睾丸精子形成的cfs-mRNAs鉴定及机制研究

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。

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.