Single-cell RNA sequencing reveals immune-peritubular myoid cell crosstalk driving testicular interstitial fibrosis in idiopathic non-obstructive azoospermia

Infertility is a major global health challenge, and non-obstructive azoospermia (NOA) represents the most severe form of male infertility. While a small proportion of NOA cases are caused by congenital factors, the majority arise from unknown causes and are therefore classified as idiopathic non-obstructive azoospermia (iNOA). Understanding the pathogenesis of iNOA is essential for developing effective diagnostic and therapeutic strategies. In this study, we analyzed single-cell RNA sequencing (scRNA-seq) data from testicular tissues of patients with iNOA and healthy controls, and validated the findings using hematoxylin and eosin (H&E) staining, Masson's trichrome (MT) staining, and Multiplex Immunofluorescence of patient testicular sections. Our results identified peritubular myoid cells (PTMs) as the primary contributors to testicular interstitial fibrosis. Cell-cell communication analysis revealed that macrophage-derived PDGF signaling is significantly upregulated in iNOA and promotes PTMs fibrosis. Fibrotic PTMs were associated with changes in the immune microenvironment, including pro-inflammatory activation of macrophages, T cells, and mast cells, consistent with a potential fibrosis-inflammation interaction. Furthermore, through integrative network pharmacology and molecular docking analyses, we identified regorafenib as a potential therapeutic agent that targets PTM fibrosis. These findings offer new insights into the diagnosis and treatment of iNOA.