ABSTRACT
Mechanisms of nuclear DNA damage in human spermatozoa are poorly understood. High levels of apoptosis were detected in spermatozoa from infertile men. The contribution of apoptosis and reactive oxygen species [ROS] in nuclear DNA damage of spermatozoa is not clear. The aim of this study was to elucidate the correlation between levels of apoptosis and ROS with percentage DNA damage in a group of infertile patients and normal donors. Our study included a randomly selected group of infertile men [n = 28] attending our fertility clinic for a history of infertility of more than one year. Semen samples were obtained after 2 to 3 days of sexual abstinence and were examined according to World health organization [WHO] guidelines to determine sperm concentration, motility and morphology. Samples from 22 normal donors with normal standard semen parameters were included as a control. Histologic features of apoptosis were studied using electron microscopy. Levels of apoptosis were detected using Annexin-V staining assay that detects externalization of phosphatidylserine to the outer surface of the plasma membrane of apoptotic spermatozoa. Propidium iodide stain was used to exclude necrotic spermatozoa. The percent apoptosis and necrosis were determined by epifluorescent microscopy. Two-hundred spermatozoa were randomly assessed and classified as normal [negative annexin- V and PI], apoptotic [positive annexin- V and negative PI] and necrotic [positive annexin- V and PI]. Levels of ROS were determined using a chemiluminescence assay and the results expressed as X 106 counted photons per minute [cpm] / 20 X 10[6] spermatozoa/mL. Sperm nuclear DNA damage was assessed by sperm chromatin structure assay [SCSA] to determine the percentage of cells outside the main population [%DFI] with abnormal chromatin structure. The percent apoptosis in spermatozoa from normal donors [6 +/- 2.2] were significantly lower than in patients [11.9 +/- 5.4] [P <0.001]. No significant difference was observed in percent apoptosis in patients with abnormal sperm parameters versus those with normal sperm parameters [P = 0.67]. The percent necrosis was significantly higher in the patients [54.2 +/- 13.6] versus donors [37,0 +/- 13.6] [P < 0.001]. A significant negative correlation was observed between apoptosis and sperm motility [r = -0.34, P = 0.04]. The correlation of apoptosis with sperm concentration and% normal sperm forms did not reach the standard significance [p = 0.07]. Levels of apoptosis were significantly correlated with levels of ROS [r = 0.3, P = 0.02]. A highly significant positive correlation has been demonstrated between levels of apoptosis and%DFI [r = 0.4, P = 0.02], Mean DFI [r = 0.5, P = 0.004], and SO DPI [r = 0.5, P = 0.002]. The range of% DFI was from 13.2% to 59% while that of percent apoptosis was from 1.5 to 25.5%. Our results indicate a significant correlation between levels of apoptosis, ROS and DNA damage in human spermatozoa. We conclude that apoptosis may play a significant role in DNA damage in human spermatozoa. However, apoptosis alone is not responsible for all DNA damage that could be encountered in spermatozoa. Other implicated factors such as ROS are possibly directly involved