Microbial infection causes inflammatory process which is usually accompanied by oxidative stress. Peroxidation of the membrane structures is one of the most important biological effects connected with overproduction of reactive oxygen intermediates (ROI) and, owing to their high content of polyunsaturated fatty acids, spermatozoa are particularly susceptible to this process. The in vitro model of semen infection created in this study was intended to assess the influence of selected bacterial strains commonly isolated from semen of infertile patients (Escherichia coli, Streptococcus oralis, Staphylococcus haemolyticus, Bacteroides ureolyticus, and Ureaplasma urealyticum) on oxidative stress and consequently its influence on sperm membrane status. Human white blood cells (WBC) were isolated from the whole heparinized blood using a density gradient centrifugation technique (Histopaque-1.077). Spermatozoa were separated from semen samples with normal sperm quality parameters using swim-up technique (swim-up fraction) as well as a double-step discontinuous Percoll gradient (90% and 47% Percoll fractions). The ROI generation in co-incubated mixtures was measured by a chemiluminescent assay in the presence of luminol. Lipid peroxidation was determined by a high-performance liquid chromatography (HPLC) technique on the basis of the amount of malondialdehyde (MDA) produced in all sperm fractions incubated with bacteria and/or WBC. In general, the presence of bacteria in the co-incubated mixtures of leukocytes and sperm was found to decrease effective neutralization of leukocyte- derived ROI by applied spermatozoal fractions, particularly by those isolated by the swim-up technique. Most of the studied bacterial strains increased MDA concentrations in sperm membranes, which was associated with the high degree of chemiluminescent signal. The damage of sperm membrane lipids caused by bacteria was higher in the presence of leukocytes, irrespectively of the bacterial strains used. The increase in MDA content was highly significant with S. haemolyticus and B. ureolyticus (p<0.05 and p<0.01, respectively) compared with sperm incubated with leukocytes only. We can conclude that oxidative stress induced in vitro by bacteria greatly affects sperm function. We were able to demonstrate a cooperation between an infectious factor and leukocytes in deepening the harmful effect of oxidative stress on sperm membrane lipids. This constitutes a further evidence for a correlation between bacterial semen infection and male infertility.

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