Table2_Altered mRNAs Profiles in the Testis of Patients With “Secondary Idiopathic Non-Obstructive Azoospermia”.XLSX

Background: Non-obstructive azoospermia (NOA) is the most severe form of male infertility. Currently, known causative factors, including congenital and several acquired causes only account for approximately 30% of NOA cases. The causes for NOA remain unclear for most patients, which is known as idiopathic (iNOA). However, whether iNOA is due to congenital defects or acquired abnormalities is a confusing problem due to the delayed diagnosis of this frustrating condition until the childbearing age. Therefore, we collected several cases with “secondary idiopathic NOA” and detected the altered mRNAs profiles in the testicular tissues to explore the possible molecular basis.
Materials and Methods: In this study, several patients with a previous history of natural pregnancy with their partners before, who were diagnosed as iNOA based on the outcomes of routine semen analysis and multiple testis biopsies now, were enrolled. Some known risk factors and genetic factors were excluded. Therefore, we defined this phenotype as “secondary idiopathic NOA.” To explore the possible molecular basis of this disease, we performed mRNA expression analysis through next-generation sequencing on three cases and other three patients with obstructive azoospermia as controls. Bioinformatics analyses were conducted to assess differentially expressed genes and possible biological mechanisms involved in the disease. Quantitative real-time reverse transcription polymerase chain reaction assays were applied to confirm the results in several selected mRNAs involved in stages and metabolism of Sertoli cells.
Results: A series of mRNAs were found to be altered in testicular tissues between patients with “secondary idiopathic NOA” and controls, including 6,028 downregulated and 3,402 upregulated mRNAs. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) analyses revealed a range of GO and KEGG terms, such as cellular process involved in reproduction, protein degradation, and absorption.
Conclusion: The present study introduces a novel classification called “secondary idiopathic NOA.” We provide a global view of the altered mRNAs involved in spermatogenetic failure in these cases. Regarding the limited samples, further studies should be taken to understand this new classification.

背景：非梗阻性无精子症（Non-obstructive azoospermia, NOA）是最严重的男性不育类型。目前已知的致病因素涵盖先天性因素与数种获得性因素，仅能解释约30%的NOA病例。绝大多数NOA患者的病因仍未明确，这类病例被称为特发性非梗阻性无精子症（idiopathic NOA, iNOA）。由于该疾病往往至生育年龄才获确诊，特发性NOA究竟源于先天性缺陷还是获得性异常，始终是一个令人困惑的问题。为此，本研究收集了若干“继发性特发性非梗阻性无精子症”病例，对其睾丸组织的mRNA表达谱改变进行检测，以探索其潜在分子机制。
材料与方法：本研究纳入的患者均曾与伴侣实现自然妊娠，后续经常规精液分析与多次睾丸活检确诊为iNOA，且已排除已知的致病风险因素与遗传因素，据此将该表型定义为“继发性特发性非梗阻性无精子症”。为探索该疾病的潜在分子基础，针对3例继发性iNOA患者与3例梗阻性无精子症（obstructive azoospermia）患者作为对照，采用下一代测序（next-generation sequencing）开展mRNA表达分析。通过生物信息学分析筛选差异表达基因，并解析该疾病涉及的潜在生物学机制。此外，选取与支持细胞（Sertoli cells）发育阶段及代谢相关的若干靶mRNA，采用实时定量逆转录聚合酶链反应（quantitative real-time reverse transcription polymerase chain reaction）对测序结果进行验证。
结果：与梗阻性无精子症对照组相比，继发性特发性非梗阻性无精子症患者的睾丸组织中存在一系列mRNA表达异常，其中6028个mRNA表达下调，3402个mRNA表达上调。基因本体论（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析共筛选出多个相关条目，包括生殖相关细胞过程、蛋白质降解与吸收等。
结论：本研究提出了一种全新的疾病分类——“继发性特发性非梗阻性无精子症”，并全面解析了该类病例中与精子发生失败相关的mRNA表达改变。鉴于本研究样本量有限，后续需开展进一步研究以深入阐释这一新的疾病分类。