Multifunctional automated semen analysis system of global SDF and DSBs provides new insights into varicocele diagnosis

Varicocele is the most common cause of reversible male infertility. Although abnormalities in basic semen parameters are frequently observed, they offer limited value for assessing fertility potential. Elevated sperm DNA fragmentation (SDF) has been consistently reported in varicocele patients, but conventional SDF methods are hindered by technical complexity and poor standardization, limiting clinical usability. This study evaluated an automated platform integrating basic semen analysis, global SDF assessment (SCD-X12), and double-strand break (DSB) detection (SDFR-X12) for varicocele fertility assessment. Seventy varicocele patients and thirty normozoospermic controls were enrolled at the Department of Urology, University of Foggia (Foggia, Italy), from July 2023 to January 2025. All semen samples were analyzed using the LensHooke® X12 system, with additional aliquots assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test. Inter-test agreement between SCD-X12 and TUNEL was good in healthy men (accuracy: 77.0%) but poor in varicocele patients (accuracy: 61.0%), indicating that global SDF assays perform reliably under normal conditions but lose interpretability when DNA damage becomes heterogeneous, as seen in varicocele patients. Although DSBs theoretically contribute to global DNA fragmentation, agreement between DSB levels (SDFR-X12) and global SDF was modest in healthy subjects (prevalence-adjusted bias-adjusted kappa [PABAK]: 0.4) and negligible in varicocele patients (PABAK: 0.06), indicating that DSB-related damage is distinct and varicocele-specific. In conclusion, global SDF assays (TUNEL and SCD-X12) are informative in non-varicocele men. In contrast, the SDFR-X12 module identifies elevated DSBs as a varicocele-specific signature, offering clearer characterization of varicocele-associated DNA damage. By integrating semen parameters, global SDF, and DSB assessment, the automated X12 system enhances scenario-specific testing and supports improved management of varicocele-related male infertility.