Department of Urology, Tianjin Medical University General Hospital, Tianjin, China.
Front Endocrinol (Lausanne). 2024 Sep 26;15:1356959. doi: 10.3389/fendo.2024.1356959. eCollection 2024.
Non-obstructive azoospermia (NOA) is a major contributor of male infertility. Herein, we used existing datasets to identify novel biomarkers for the diagnosis and prognosis of NOA, which could have great significance in the field of male infertility.
NOA datasets were obtained from the Gene Expression Omnibus (GEO) database. CIBERSORT was utilized to analyze the distributions of 22 immune cell populations. Hub genes were identified by applying weighted gene co-expression network analysis (WGCNA), machine learning methods, and protein-protein interaction (PPI) network analysis. The expression of hub genes was verified in external datasets and was assessed by receiver operating characteristic (ROC) curve analysis. Gene set enrichment analysis (GSEA) was applied to explore the important functions and pathways of hub genes. The mRNA-microRNA (miRNA)-transcription factors (TFs) regulatory network and potential drugs were predicted based on hub genes. Single-cell RNA sequencing data from the testes of patients with NOA were applied for analyzing the distribution of hub genes in single-cell clusters. Furthermore, testis tissue samples were obtained from patients with NOA and obstructive azoospermia (OA) who underwent testicular biopsy. RT-PCR and Western blot were used to validate hub gene expression.
Two immune-related oxidative stress hub genes ( and ) were identified. Both hub genes were highly expressed in NOA samples compared to control samples. ROC curve analysis showed a remarkable prediction ability (AUCs > 0.8). GSEA revealed that hub genes were predominantly enriched in toll-like receptor and Wnt signaling pathways. A total of 24 TFs, 82 miRNAs, and 111 potential drugs were predicted based on two hub genes. Single-cell RNA sequencing data in NOA patients indicated that and were highly expressed in endothelial cells and Leydig cells, respectively. RT-PCR and Western blot results showed that mRNA and protein levels of both hub genes were significantly upregulated in NOA testis tissue samples, which agree with the findings from analysis of the microarray data.
It appears that and could be significant immune-related oxidative stress biomarkers for detecting and managing patients with NOA. Our findings provide a novel viewpoint for illustrating potential pathogenesis in men suffering from infertility.
非阻塞性无精子症（NOA）是男性不育的主要原因之一。在此，我们利用现有的数据集来鉴定用于诊断和预测 NOA 的新型生物标志物，这在男性不育领域可能具有重要意义。
从基因表达综合数据库（GEO）中获取 NOA 数据集。使用 CIBERSORT 分析 22 种免疫细胞群体的分布。通过应用加权基因共表达网络分析（WGCNA）、机器学习方法和蛋白质-蛋白质相互作用（PPI）网络分析来鉴定枢纽基因。在外部数据集验证枢纽基因的表达，并通过接受者操作特征（ROC）曲线分析进行评估。应用基因集富集分析（GSEA）来探索枢纽基因的重要功能和途径。基于枢纽基因预测 mRNA-微小 RNA（miRNA）-转录因子（TFs）调控网络和潜在药物。应用睾丸内患者的单细胞 RNA 测序数据来分析枢纽基因在单细胞簇中的分布。此外，从接受睾丸活检的 NOA 和阻塞性无精子症（OA）患者中获取睾丸组织样本。使用 RT-PCR 和 Western blot 验证枢纽基因的表达。
鉴定出两个与免疫相关的氧化应激枢纽基因（和）。与对照样本相比，两个枢纽基因在 NOA 样本中的表达均较高。ROC 曲线分析显示出出色的预测能力（AUCs > 0.8）。GSEA 表明枢纽基因主要富集于 Toll 样受体和 Wnt 信号通路。基于两个枢纽基因共预测到 24 个 TFs、82 个 miRNAs 和 111 种潜在药物。NOA 患者的单细胞 RNA 测序数据表明，和分别在血管内皮细胞和睾丸间质细胞中高表达。RT-PCR 和 Western blot 结果显示，两个枢纽基因在 NOA 睾丸组织样本中的 mRNA 和蛋白水平均显著上调，与微阵列数据分析结果一致。
和可能是用于检测和管理 NOA 患者的重要免疫相关氧化应激生物标志物。我们的研究结果为阐明男性不育潜在发病机制提供了新的视角。
