Apoptosis Will be Increased after 2h Incubation of Human Sperm at 37°C

The main goal was to evaluate the impact of different incubation time intervals on human sperm DNA status using terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) test. This prospective study involved 21 normozoospermic specimens. After direct swim-up، sperm cells were incubated at 37°C and DNA damage was evaluated at different time intervals (0، 1، 2 and 3 h). After slide fixation with methanol 100% for 4 minutes and rinse in phosphate-buffered solution (PBS)، TUNEL was added to each slide and incubated for 60 minute in dark. Eosin-Nigrosin and Papanicolaou staining protocols were applied in order to assess sperm viability and morphology، respectively. Sperm viability and normal morphology was improved after sperm processing (100%، 72. 3% respectively، p<0. 0001). The rate of DNA damage was significantly higher after 2h compared to 0h (9. 19±0. 8% Vs 4. 9±0. 9%، respectively، p=0. 008). Also there was significant difference in abnormal sperm DNA between 3h and 1h (10. 95±0. 7% Vs 7. 1±1%، respectively، p=0. 020). Incubation of prepared normozoospermic samples at 37°C more than 2 h may be associated with sperm DNA fragmentation. Therefore، it seems that incubation of human spermatozoa at 37°C should be limited up to 2h prior to use in ART clinics.