Development of a specific method to evaluate 8-hydroxy, 2-deoxyguanosine in sperm nuclei: relationship with semen quality in a cohort of 94 subjects.

Oxidative stress is involved in many diseases including male infertility. Human spermatozoa are very sensitive targets of reactive oxygen species (ROS) and most of sperm functions are impaired in case of oxidative stress. In addition, unbalanced production of ROS is considered one of the most important causes of sperm DNA fragmentation, a semen trait of infertile men. The relationship between oxidative damage and semen quality is partially controversial, likely due to the different methods and/or targets used to reveal the oxidative stress. In this study, by fluorescence microscopy and flow cytometry, we compared two methods to reveal 8-hydroxy,2-deoxyguanosine, the hallmark of oxidative DNA damage: an immunofluorescence method and the commercial OxyDNA kit. We found that although both methods localized the labelling in sperm nuclei, they yielded different measures and only with the immunofluorescence method the labelling resulted specific for sperm 8-hydroxy,2-deoxyguanosine. The immunofluorescence method, coupled to flow cytometry, was thus selected to analyse the 8-hydroxy,2-deoxyguanosine content in semen samples from 94 sub fertile patients and to investigate the relationship with semen quality. We found that the percentages of spermatozoa with 8-hydroxy,2-deoxyguanosine (mean±SD: 11.4±6.9%) were related to sperm count (r=-0.27, p=0.04), motility (progressive: r=-0.22, p=0.04; non progressive: r=0.25, 0.01) and normal morphology (r=-0.27, p=0.01). In conclusion, we demonstrate that immunofluorescence/flow cytometry is a reliable and specific method to detect 8-hydroxy,2-deoxyguanosine at single cell level and show that oxidative damage only partially overlaps poor semen quality, suggesting that it could provide additional information on the male fertility respect to routine semen analysis.