Background: DNA damage in human spermatozoa is known to be associated with a variety of adverse clinical outcomes affecting both reproductive efficiency and the health and wellbeing of the offspring. However, the origin of this damage,its biochemical nature and strategies for its amelioration, still await resolution. Methods: Using novel methods to simultaneously assess DNA fragmentation (modified TUNEL assay), DNA-base adduct formation (8-hydroxy-2′-deoxyguanosine [8OHdG]) and cell vitality, spermatozoa from a cohort of 50 assisted conception patients were examined and compared with a group of donors. Receiver operating characteristic (ROC) curve analysis was then used to examine the frequency distribution of the data and to determine optimized thresholds for identifying patients exhibiting abnormally high levels of DNA damage. Results: 8OHdG formation and DNA fragmentation were highly correlated with each other and frequently associated with cell death. Percoll centrifugation improved sperm quality but, unexpectedly, increased 8OHdG formation in live cells, as did sperm fractionation using Puresperm® gradients. ROC analysis indicated that the frequency distribution of 8OHdG and DNA fragmentation data were significantly different between patients and donors (P < 0.001), permitting the development of thresholds that would allow the accurate diagnosis of DNA damage in the male germ line. Conclusion: The aetiology of DNA damage in spermatozoa involves a cascade of changes that progress from the induction of oxidative stress and oxidized DNA base adduct formation to DNA fragmentation and cell death. Preparation of spermatozoa on discontinuous density gradients aggravates the problem by stimulating the formation of 8OHdG in live cells. However, the development of novel methods and optimized thresholds for diagnosing oxidative DNA damage in human spermatozoa should assist in the clinical management of this pathology.
Human Reproduction Vol. 25, Issue 10, p. 2415-2426