Effect of seminal redox status on lipid peroxidation, apoptosis and DNA fragmentation in spermatozoa of infertile Saudi males

Abstract

Objectives: To assess the effect of seminal redox status on lipid peroxidation (LPO), apoptosis and integrity of sperm DNA in infertile males. Methods: In this case-control study, the total antioxidant status (TAS) and reactive oxygen species (ROS) levels were analyzed within the seminal plasma of fertile normozoospermic, n=40 and infertile (asthenozoospermic, n=30; oligoasthenoteratozoospermic, n=30) males. Additionally, the level of 4-hydroxynonenal (4-HNE), DNA fragmentation, and caspase-3 activity were estimated in the spermatozoa.

Results: Significantly (p less than 0.001) increased seminal ROS level with decreased TAS scores was observed in the infertile groups compared to normozoospermics. The infertile males showed marked elevated (p less than 0.001) levels of 4-HNE, DNA fragmentation and caspase-3 activity compared to normozoospermics, which was positively correlated to increased seminal ROS levels and negatively to the TAS score in the studied groups. Seminal ROS level was significantly inverse correlated to the semen parameters. Additionally, a strong negative correlation between DNA fragmentation, LPO, caspase-3activity and seminal parameters were observed. Conclusion: Seminal oxidative stress is a potential risk factor for LPO, DNA damage, and apoptosis in spermatozoa, which can affect semen quality and male fertility. Thus, in addition to conventional seminological parameters, measurement of seminal oxidative stress and sperm DNA integrity may also be employed to investigate the functional integrity of spermatozoa at the molecular level.

Figure 1

Comparison of seminal A) reactive oxygen species (ROS) levels and B) total antioxidant status (TAS) between the fertile normozoospermic (control) and asthenozoospermic (AST) and oligoasthenoteratozoospermic (OAT) groups. The level of ROS was significantly higher whereas, TAS score in both infertile groups was significantly lower compared to normozoospermic group. Values represent means ± SD. *p<0.001, AST group versus the control group; **p<0.001, OAT group versus the control group.

Figure 2

Comparison of the levels of 4-hydroxynonenal (4-HNE) in sperm cell lysates derived from the fertile normozoospermic (control) and asthenozoospermic (AST) and oligoasthenoteratozoospermic (OAT) males. The mean 4-HNE level in both infertile groups was significantly higher than that in the control group. Values represent means ± SD. *p=0.003, AST group vs the control group; **p<0.001, OAT group versus the control group.

Figure 3

Comparison of the caspase-3 activity in sperm cell lysates derived from the fertile normozoospermic (control) and asthenozoospermic (AST) and oligoasthenoteratozoospermic (OAT) groups. The mean caspase-3 activity in both infertile groups was significantly higher than that in the control group. Values represent means ± SD. *p<0.005, AST group versus the control group; **p<0.001, OAT group versus the control group.

Figure 4

DNA fragmentation in human sperm as determined by TUNEL assay. A) Fluorescence microscopy images of sperm derived from men in (a) the fertile normozoospermic group (control), (b) asthenozoospermic (AST) group, and (c) oligoasthenoteratozoospermic (OAT) group. White arrows indicate TUNEL-positive nuclei (bright green), while DAPI (blue) staining indicates the total number of nuclei (scale bar: 50 µm). B) Quantitative determination (percentage of apoptotic cells versus the total number of cells) of DNA fragmentation in sperm of males in the control, AST, and OAT groups. A greater number of TUNEL-positive nuclei were observed in the OAT and AST groups than in the control group. Data are expressed as means ± SD. *p<0.001 control versus AST; **p<0.001 control versus OAT.