Refining NOA diagnosis: a dual approach through immunostaining subclassification and metabolic markers in Sertoli cells

Background Non-obstructive azoospermia (NOA) is a leading cause of male infertility, affecting approximately 1% of men worldwide. Accurate pathological diagnosis and subclassification of NOA remain challenging due to the lack of reliable markers for germ cells (GCs) and Sertoli cells (SCs) within seminiferous tubules. Current histopathological evaluation relies on Hematoxylin and Eosin (H/E) staining, which lacks molecular specificity and cannot precisely quantify GCs or identify the stage of spermatogenic arrest. This limits both prognostic accuracy and the development of targeted therapeutic strategies. The identification of robust cellular and molecular markers is therefore essential to improve diagnosis, subclassification, and clinical management of NOA. Methods We performed immunofluorescence staining for SOX9, DAZL, and SYCP3 on 146 testicular samples to refine NOA subtyping. Single-cell RNA sequencing (scRNA-seq) was conducted on samples from 5 NOA patients, integrated with data from healthy and obstructive azoospermia (OA) controls. Candidate markers identified from SC-specific transcriptional profiles were validated via immunofluorescence in human and mouse testicular tissues. Findings Immunostaining enabled a refined classification of NOA into three subtypes: Maturation Arrest-Meiotic Arrested (MA-MA), Maturation Arrest-Premeiotic Arrested (MA-PA), and Sertoli cell-only (SCO). The scRNA-seq revealed that SCs in adult NOA patients exhibit an immature transcriptional state resembling prepubertal SCs, characterized by a distinct metabolic profile. From this profile, we identified LDHB and PKM2 as significantly upregulated markers in NOA SCs. Immunofluorescence confirmed their elevated expression in human NOA samples and in immature mouse testes. Interpretation Our integrated approach establishes a novel immunostaining-based classification for NOA and identifies an immature SC state as a key pathological feature. The overexpression of LDHB and PKM2 in SCs provides two new candidate biomarkers for improving the pathological diagnosis and subclassification of NOA, with potential implications for future diagnostic and therapeutic strategies.