YBX1 downregulation links to impaired spermatogonial stem cells function potentially through inhibiting miRNAs production.

MicroRNAs are dysregulated in patients with idiopathic nonobstructive azoospermia (iNOA), but the underlying mechanisms remain unclear. This study investigated miRNA-processing regulators in iNOA pathogenesis using bioinformatics and experimental validation in 41 obstructive azoospermia (OA) and 51 iNOA patients. Primary human spermatogonial stem cells (SSCs) were isolated for functional studies. YBX1, a miRNA-binding protein, was identified to be reduced in testes and SSCs of patients with iNOA. Knockdown of YBX1 in SSCs inhibited the processing of pre-miR-17 and the transcription of primary miR-221/222 (pri-miR-221/222), reducing mature miR-17 and miR-221/222 levels. This led to upregulated KIT and PTEN, inhibited PI3K/AKT signaling, and induced apoptosis. Additionally, YBX1 depletion reduced oxygen consumption and glycolysis, increased reactive oxygen species (ROS) production, and elevated the Bax/Bcl2 ratio, which was partially reversed by miR-17 and miR-221/222 supplementation. YBX1 loss disrupts miRNA biogenesis, leading to spermatogenic failure via apoptotic and metabolic dysfunction.