Hsa-circ_0081481-miR 3960-FBXO24 regulatory axis in non-obstructive azoospermia identifies potential biomarkers of spermatogenic failure

The etiology of non-obstructive azoospermia (NOA) remains largely idiopathic, emphasizing the urgent need for molecular biomarkers to predict sperm retrieval outcomes. Here, we integrate bioinformatic analyses of public transcriptomic data with experimental expression profiles to investigate a potential regulatory network involving hsa-circ_0081481, miR-3960 and FBXO24 in NOA. Seven GEO datasets were analyzed to identify differentially expressed genes, and FBXO24 was found to be a consistently downregulated gene in NOA. Upstream regulators and interacting non-coding RNAs were predicted, leading to the selection of miR-3960 (a microRNA targeting FBXO24) and hsa-circ_0081481 (a circular RNA predicted to sponge miR-3960). Subsequently, FBXO24, miR-3960 and hsa-circ_0081481 expression levels were measured in the plasma of 60 men with NOA (categorized by histopathology and sperm retrieval outcome) and 40 fertile controls. FBXO24 and hsa-circ_0081481 were significantly downregulated in NOA (especially in hypospermatogenesis and maturation arrest), whereas miR-3960 was significantly upregulated in these subgroups. Of note, hsa-circ_0081481 achieved excellent diagnostic performance (AUC 0.920) in distinguishing NOA cases with successful and failed sperm retrieval, respectively. miR-3960 also showed high accuracy (AUC 0.861) after its inverse expression pattern was taken into account, and FBXO24 showed moderate predictive value. Our findings suggest that the hsa-circ_0081481/miR-3960/FBXO24 axis is dysregulated in NOA and may serve as a novel trio of non-invasive biomarkers. This study provides new insights into the molecular mechanisms of spermatogenic failure in NOA and provides a basis for improved diagnosis and personalized fertility treatment.