Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa

Appropriate semen processing and assessment are critical for successful infertility treatment. We investigated whether laboratory procedures including semen preparation and incubation affect sperm DNA integrity. A total of 153 infertile men were involved. Conventional semen parameters and sperm chromatin structure assay (SCSA) parameters, that is, DNA fragmentation index (%DFI) and high DNA stainability (%HDS), were assessed on the fresh ejaculated semen samples, which were treated and incubated under different conditions. Negative correlations were identified between the %DFI and sperm concentration, motility, progressive motility and morphology. A lower percentage of DFI was detected in spermatozoa when density gradient centrifugation (DGC) was followed by swim-up treatment in comparison with DGC alone (P < 0.01). Although the %DFI increased in a time-dependent manner with incubation both at room temperature (RT) and at 37°C in air, the %DFI after 24 h at RT was significantly lower than that at 37°C (P < 0.05). Incubation with 5% CO₂ was effective in maintaining sperm motility (P < 0.01); however, it induced further elevation of %DFI (P < 0.001). Thus, sperm DNA damage was associated with longer incubation periods. Interestingly, common culture conditions, such as maintaining pH and temperature, compromised the sperm DNA integrity.