A novel flow cytometry approach resolves testicular cell heterogeneity and enables diagnostic profiling in male infertility.

Male infertility, particularly non-obstructive azoospermia (NOA), remains a major clinical challenge. Although the testis is one of the most transcriptionally complex organs, stage-specific changes in total RNA abundance remain poorly characterized. Through total RNA staining, we identified distinct RNA content profiles across spermatogenic stages, supporting its value as a discriminative marker. Based on this, we developed RNA-DNA dual-parameter flow cytometry (RD-Cyto), a novel method that simultaneously measures RNA and DNA content at single-cell resolution for high-precision classification of testicular cells. Compared to conventional Hoechst-based cytometry, RD-Cyto offers superior resolution, enabling clear separation of pachytene (P) and diplotene (D) spermatocytes, first-time identification of metaphase I spermatocytes (MI), and subdivision of metaphase II spermatocytes (MII) into two subsets. To assess its utility in mechanistic research, we applied RD-Cyto to investigate the RNA-binding protein (RBP) DEAD-Box Helicase 17 (DDX17) and found that its deletion causes RNA accumulation in preleptotene spermatocytes and meiotic failure-demonstrating RD-Cyto's sensitivity to stage-specific regulatory disruptions. We further applied RD-Cyto to the clinical diagnosis of azoospermia. Combined with testicular fine-needle aspiration cytology (FNAC), RD-Cyto accurately evaluated spermatogenesis in all enrolled patients, showing 100 % concordance with testicular sperm retrieval outcomes. It successfully identified Sertoli cell-only syndrome (SCOS) in NOA, predicted sperm retrieval in rare NOA cases with residual spermatogenesis, and revealed spermatogenic and morphological abnormalities linked to pathogenic mutations in oligoasthenospermia (OAS). These findings establish RD-Cyto as a robust platform for both mechanistic investigation and diagnostic stratification in male infertility.