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.