2.6. Assessment of DNA Fragmentation Index

The AO technique was used to evaluate the chromatin stability of sperm. A smear was collected from each specimen and placed on a transparent microscope slide on the same day of the inspection. Afterward, it was mixed with a freshly prepared Carnoy's solution. The next day, the slides were inserted into the AO solution for 5 min. Afterward, they were thoroughly washed with tap water before being stored in a cool and dark place until the evaluation was finished. The fraction of spermatozoa with normal DNA was determined by measuring at least 200 spermatozoa under a fluorescence microscope at a magnification of 400. Spermatozoa with normal, undamaged, and double-stranded DNA fluoresced green at a wavelength of 450-490 nm, while those with denatured DNA fluoresced red or orange. Genomic instability was measured using the DFI, which measures the amount of transient transfection as a percentage of total DNA ( 27 ).