Table3_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.

研究背景：非梗阻性无精子症（Non-obstructive azoospermia, NOA）是最为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多种获得性因素，仅能解释约30%的NOA病例。多数患者的NOA病因仍不明确，此类病例被称为特发性非梗阻性无精子症（idiopathic NOA, iNOA）。然而，由于该病症往往延迟至育龄期才得以确诊，特发性非梗阻性无精子症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，检测其睾丸组织中的mRNA表达谱变化，以探索其潜在的分子机制。
材料与方法：本研究纳入的受试者均为既往曾与伴侣实现自然妊娠，后经常规精液分析及多次睾丸活检确诊为iNOA的患者，同时排除了已知风险因素与遗传因素，因此将该表型定义为“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机制，本研究对3例患者与3例梗阻性无精子症（obstructive azoospermia）对照患者的样本开展下一代测序，以分析mRNA表达情况。通过生物信息学分析评估差异表达基因，并解析该疾病涉及的潜在生物学机制。采用实时定量逆转录聚合酶链反应（quantitative real-time reverse transcription polymerase chain reaction, qRT-PCR）对筛选出的、与支持细胞（Sertoli cells）发育阶段及代谢相关的若干mRNA进行验证，以确认测序结果。
研究结果：在“继发性特发性非梗阻性无精子症”患者与对照者的睾丸组织中，共发现一系列差异表达的mRNA，其中下调mRNA共6028条，上调mRNA共3402条。基因本体（Gene Ontology, GO）分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）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。
研究结论：本研究提出了一种新型分类——“继发性特发性非梗阻性无精子症”，并全面展示了此类病例中与精子发生失败相关的mRNA表达变化情况。鉴于本研究样本量有限，未来需开展进一步研究以深入阐明这一新分类的相关机制。