Comparison of Sperm DNA Analysis Outcomes Using Different Gating Strategies in Flow Cytometry

Sperm DNA integrity is a key factor in ensuring successful fertilization, embryo development, and ongoing pregnancy. Sperm chromatin structure assessment (SCSA) using flow cytometry after DNA denaturation and acridine orange staining is considered the gold-standard method for evaluating sperm DNA. Two gating strategies for flow cytometry-based assessment of the sperm DNA fragmentation index (DFI) have been reported in the literature: polygonal gating (PGG) and cruciform gating (CFG). The aim of the present study was to compare the relationships of assay results between PGG and CFG, and to analyze their correlations with parameters of basic semen analysis. Data obtained independently by the two methods were compared using statistical analysis, including Bland-Altman plots and regression analysis. A total of 121 male outpatients undergoing fertility assessment at our hospital's reproductive center were selected via a completely random sampling method. Sperm DFI and related indicators were detected by flow cytometry using PGG and CFG, respectively. The results demonstrated that DFI values obtained from the two methods showed good agreement. The correlation coefficients between DFIp from the PGG method and DFIm, DFIs, and DFIc from the CFG method were 0.6497, 0.9404, and 0.9667, respectively (All p < 0.01). High DNA staining index (HDS) showed a slight negative correlation with DFIs (r = -0.3042, p < 0.05). The percentage of progressively motile sperm was negatively correlated with DFIp, DFIs, and DFIc (r = -0.3824, -0.3794, -0.3574, respectively, all p < 0.05). No significant correlation was observed between HDS and semen volume, sperm concentration, total sperm count, or PR (%). These findings indicate that DFIs are more closely associated with male fertility, suggesting that the CFG method, which provides this parameter, may be more suitable for clinical assessment of sperm DNA damage.