Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation

The origin and the meaning of DNA fragmentation in ejaculated human spermatozoa are not yet clear, although some hypotheses have been proposed. In the present study, we used investigated sperm DNA fragmentation by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL)-coupled flow cytometry to investigate DNA fragmentation in spermatozoa that were selected by the swim-up procedure and incubated long-term. In addition, using flow cytometry we detected annexin V binding assay and propidium iodide staining, and we also studied membrane phosphatidylserine translocation and the loss of membrane integrity in the same sperm populations that we used in the TUNEL investigation. We found that in vitro sperm DNA fragmentation 1) occurs after ejaculation under experimental conditions without the involvement of any external factor, 2) is not affected by treatment with the nuclease inhibitor aurintricarboxylic acid, 3) is increased by treatment with the glutathione peroxidase inhibitor mercaptosuccinate, 4) correlates with basal values (ie, just after swim-up selection) of DNA fragmentation in teratozoospermic but not in normospermic semen samples, 5) develops in a sharply associated manner with the in vitro occurrence of sperm necrosis, and 6) is predicted by the basal value of annexin V binding in viable spermatozoa. These findings suggest an involvement of endogenously produced reactive oxygen species as the possible cause of in vitro sperm DNA fragmentation.