Single-cell transcriptomic analysis reveals upregulation of EGR1, FOSB, and KLF4 in Leydig cells of idiopathic non-obstructive Azoospermia.

BACKGROUND: Idiopathic non-obstructive azoospermia (iNOA) is the most severe form of male infertility, yet its transcriptional regulatory mechanisms remain largely unknown. Leydig cells (LCs) are essential for androgen production and maintaining spermatogenesis, but the transcription factors driving LC dysfunction in iNOA have not been systematically characterized. METHODS: We integrated public single-cell RNA sequencing (scRNA-seq) data (GSE149512) from testicular tissues of three iNOA patients and three normal adults. A comprehensive bioinformatics analysis was performed, including UMAP clustering, differential gene expression, functional enrichment, RNA velocity (scVelo), cell-cell communication (CellChat), and pseudotime trajectory reconstruction (Monocle 2, Slingshot). Key findings were validated at the protein level by immunofluorescence co-staining of EGR1, FOSB, KLF4, and the LC marker CYP17A1. RESULTS: Unbiased clustering identified all major testicular cell types. In LCs, 144 differentially expressed genes (DEGs) were found, with the transcription factors EGR1, FOSB, and KLF4 identified as the most significantly upregulated. Functional analysis linked their upregulation to stress-response pathways. Pseudotime analysis revealed that iNOA LCs are arrested in an immature differentiation state, coinciding with high expression of these three factors. Virtual knockout predicted they act as transcriptional repressors of key LC functional genes. Critically, immunofluorescence staining confirmed their significant protein-level upregulation specifically within LCs of iNOA patients compared to controls. CONCLUSION: EGR1, FOSB, and KLF4 are upregulated in iNOA LCs and may impair LC maturation and androgen synthesis by repressing downstream target gene expression and disrupting spermatogenesis-supporting pathways. Targeting the EGR1/FOSB/KLF4 axis may offer a novel therapeutic strategy to restore Leydig cell maturation and steroidogenic capacity in iNOA.