P-837: The effect of oxidative stress on human sperm morphology

Abstract

Oxidative stress plays a major role in induction of cell structural damage and can result in cellular dysfunction, loss of cell viability, and decreased motility. Correlations between oxidative stress-induced lipid membrane damage and altered cellular morphology have been reported for several tissue types. Although oxidative stress is widely accepted as a cause of male infertility, little is known about the effects of oxidative stress-induced lipid peroxidation on sperm morphology. This study 1) evaluated whether ROS-induced lipid peroxidation is a causative agent of human sperm morphological changes, 2) determined the ROS concentrations at which lipid peroxidation and morphological damage occurs, and 3) examined the potential protective effects of the antioxidant Trolox on ROS-induced morphological changes. In vitro dose response study. A semen sample was obtained from a fertile male and analyzed according to World Health Organization criteria. Sperm were isolated from the seminal plasma using Percoll discontinuous gradients. The sperm concentration in the 80% gradient was determined, divided into 6 equivalent sperm concentration aliquots, and treated with 0,10, 50, and 100 μM H2O2 +/- 40 μM Trolox in PBS. Following an incubation period, sperm were evaluated for: (A) lipid peroxidation using a spectrophotometric assay and (B) sperm morphology via bright field microscopy. 100 sperm were scored for each morphological parameter using WHO 1999 criteria Linear regression analysis (r= 0.8401 p<0.001) indicated that lipid peroxidation was H2O2-dose dependent. ANOVA results demonstrated that trolox treatments significantly decreased lipid peroxidation when compared to the control (p< 0.0082). The analysis of images indicated that sperm exposed to 50 and 100 μM H2O2 showed that 1) the number of sperm with severe morphological changes such as nuclear fragmentation, chromatin dispersion, and cellular agglutination increased, 2) the number of sperm with abnormal heads is concentration dependent, 3) trolox has a protective effect on sperm exposed to 50 μM H2O2. Conversely, the number of sperm with abnormal heads did not change, regardless of trolox treatment, at the 100 H2O2 concentration. Thus, trolox did not seem to protect sperm exposed to the highest H2O2 treatment used in this study. Human sperm exposed to 50 and 100 μM H2O2 are vulnerable to oxidative damage as evidenced by severe morphological alterations. Lipid peroxidation is an important factor that may induce detrimental morphological changes in the spermatozoa, which may be associated with male infertility. Trolox was effective in ameliorating damage in sperm exposed to 50 μM H2O2. At this concentration, trolox prevented abnormal sperm responses such as nuclear deformations, morphological modifications, and aggregation when sperm were exposed to oxidative stress.