Flow Cytometric Assessment of Sperm DNA Fragmentation by TUNEL and Acridine Orange: Methodological and Clinical Insights

Background/Objectives: Male infertility contributes to nearly half of global infertility cases, yet conventional semen analysis is insufficient to predict assisted reproductive technology (ART) outcomes such as intracytoplasmic sperm injection (ICSI). Sperm DNA fragmentation (SDF) is a promising biomarker of genomic integrity, but clinical implementation remains hindered by methodological heterogeneity. This study compared two SDF assays—TUNEL and Acridine Orange (AO)—regarding their correlations with semen parameters and ICSI outcomes. Methods: Sixty men undergoing ICSI were prospectively enrolled. SDF was analyzed using two flow cytometric assays: TUNEL (detecting DNA strand breaks) and AO (assessing chromatin instability). Semen quality and ICSI outcomes (fertilization, cleavage, blastulation, and embryo utilization rates) were evaluated. Statistical analyses included Spearman’s rank correlation and Mann–Whitney tests. Results: Median SDF levels were significantly higher by TUNEL than AO (17.2% vs. 10.15%; p = 0.0065). Inter-assay correlation was weak (r = 0.299, p = 0.01). AO-derived SDF correlated positively with age (r = 0.311, p = 0.02), while TUNEL showed no such trend. Neither assay correlated significantly with semen parameters or ICSI outcomes, although AO tended to associate with lower motility and slightly reduced top-quality embryo rates. Conclusions: TUNEL and AO capture distinct facets of sperm DNA damage. Their limited correlation and lack of predictive value for ICSI outcomes highlight the need for assay-specific interpretation and standardization. Integration of SDF with additional biomarkers and oocyte factors may enhance its clinical utility.