[Peroxidation components of sperm lipid membranes in male infertility]. / Peroksydacja komponenty lipidowej blon biologicznych plemników w nieplodnosci meskiej.

We have studied the lipid peroxidation product (malondiadehyde-MDA) levels after thiobarbituric acid reaction, spectrophotometrically and by a high-performance liquid chromatography (HPLC) with UV detection in seminal plasma as well as in the cell fraction. Semen samples were obtained from healthy volunteers and infertile males. Ejaculates were previously classified into studied subgroups according to standard andrological criteria (sperm number, motility, morphology) and defined as: normozoospermia (K), azoospermia (Az), teratozoospermia (T), asthenoteratozoospermia (AT), oligoasthenoteratozoospermia (OAT) and idiopathic infertility (NIF). MDA is a good marker for lipid peroxidation. There were found elevated MDA concentrations (determined by HPLC) in seminal plasma of the all analysed pathological semen samples, especially in patients with OAT. The more pronounced differences between healthy controls and NIF patients were observed in intracellular compartment. The lipid peroxidation of rich in unsaturated fatty acids sperm membranes is considered to be one of the most important effects from ROS-induced cell damage what may lead to persistent infertility.

Severe antioxidase deficiency in human semen samples with pathological spermiogram parameters.

Spermatozoa of 103 ejaculates from infertile patients and fertile healthy individuals were separated from seminal plasma and purified on Percoll gradient to determine the activities of superoxide dismutase (SOD) and catalase (CAT) in seminal plasma as well as in spermatozoal supernatants after hypotonic disintegration of the sperm plasma membrane. Out of collected specimens, a subgroup of ejaculates from 40 individuals was examined whose female partners had developed malignant processes in the cervix uteri (oncological subgroup). All sperm samples were classified into normal and pathological semen samples according to WHO criteria. While no significant differences of SOD levels were detected in seminal plasma of patients with primary infertility, a catalase deficiency seemed to be associated with combined sperm pathology-oligoasthenoteratozoospermia (OAT). Liberated concentrations of both SOD and catalase were diminished by 10-70% in the oncological subgroup compared to normozoospermia. In four OAT samples obtained from infertile males of the oncological subgroup, total depletion from both antioxidases was observed. A lack of sufficient antioxidase protection in cases of severe sperm pathology (OAT) may also lead to cervical dysplasia.

Effect of reactive oxygen species and the activity of antioxidant systems on human semen; association with male infertility.

A range of compounds with a role in oxidative stress were measured in ejaculates from 40 normozoospermic individuals and 93 infertile males. Ejaculates were classified according to WHO criteria. Seminal plasma and the sperm cell fraction were assessed separately for superoxide dismutase (SOD), catalase, xanthine oxidase, capability for singlet oxygen trapping and content of thiobarbituric acid-reactive substances (TBARS). Pathological cases defined as oligozoospermia, asthenozoospermia, or teratozoospermia revealed different backgrounds of oxidative stress as reflected by different levels of tested substances in every type of sperm pathology. In the majority of abnormal ejaculates, a significant increase in intracellular activity of SOD, decreased intracellular levels of catalase, elevated levels of xanthine oxidase and TBARS, and severely impaired singlet oxygen trapping were observed when compared to normozoospermic ejaculates. Interrelationships between SOD and TBARS, and between xanthine oxidase and catalase, appeared to be of key importance when analysed separately in seminal plasma and in spermatozoa or in a combination of both. Elevated xanthine oxidase levels and low capacity for singlet oxygen trapping are statistically significant factors for the evaluation of male infertility which can develop as a result of persistent oxidative stress.

Oxidative stress and male infertility.

We have studied the activity of substances (superoxide dismutase [SOD], catalase [Cat], malonaldehyde, xanthine oxidase [XO], nitric oxide [NOx]) participating in oxidative stress. Seminal plasma samples of 147 ejaculates obtained from normal and from infertile males were examined. Activities of SOD, Cat, and XO were measured chemiluminometrically while malonaldehydes and NOx were measured by spectrophotometer in seminal plasma samples. Ejaculates were previously characterized according to World Health Organization andrological criteria (sperm number, motility, and morphology). Procedures were performed in a university laboratory. Statistically significant changes (in comparison to normozoospermic samples) were noted in activities of SOD, XO, and malonaldehyde levels. The SOD activity exceeded values obtained for normozoospermic samples only in oligozoospermia. Otherwise low SOD levels in analyzed infertile subgroups inversely related to elevated malonaldehydes. Because diminished activity of SOD in seminal plasma was associated with increased levels of malonaldehydes and XO, we could postulate some significance of these monitored substances in evaluation of the cause of male infertility.

Seminal plasma can be a predictive factor for male infertility.

Seminal plasma from ejaculates of 10 healthy, fertile volunteers and 63 infertile males was analysed for superoxide dismutase (SOD) and xanthine oxidase (XO) activities using a chemiluminometer. There was not statistically significant difference in the activity of either enzyme between control and infertile populations (113 +/- 74 IU/ml for SOD and 1.17 +/- 0.52 IU/ml for XO) in samples from normozoospermic ejaculates. Sperm progressive motility was positively correlated with SOD activity in seminal plasma of corresponding ejaculates (P < 0.05) and negatively with XO activity (P < 0.001). An 'oxido-sensitive' index was defined as the SOD/XO ratio and was found to be inversely related to sperm progressive motility samples (P < 0.01). Analysing this index among all tested samples of semen including those with pathological spermiograms, as well as normospermic (N) samples we found statistically significant (elevated) differences in oligoasthenoteratospermia (OAT) in comparison with N (P <0.05); OAT samples were also significantly different from oligospermic (O) and oligoteratospermic (OT) samples (P < 0.05). This suggests that the 'oxido-sensitive' index of seminal plasma may be a simple diagnostic factor, useful in the determination of male infertility.

Antiinflammatory effects of NADPH oxidase inhibitors.

Proinflammatory cytokines prime the membrane-bound NADPH oxidase of neutrophils and monocytes of mice suffering from experimental arthritis so as to attain an activated state, which, upon a second stimulus, releases 6-fold increased levels of reactive oxygen species (ROS) than do unprimed phagocytes. Enhanced NADPH oxidase activity deregulates ROS-dependent signal transduction pathways of inflammation, which play a crucial role in the pathogenesis of arthritis. The antiarthritic reactivity of two inhibitors of NADPH oxidase, diphenylene iodoniumchloride (DPI) and staurosporine, was tested in male DBA/1 x B10A(4R) hybrid mice suffering from potassium peroxochromate arthritis. Daily doses of 2.8 mumol/kg of DPI or 30 nmol/kg of staurosporine sufficed to inhibit the arthritis by 50%. A complete inhibition was obtained with 10 mumol/kg of DPI, and 100 nmol/kg of staurosporine suppressed the arthritis by 85%. The onset, progression, and remission of arthritis correlated to both the activity of phagocytic NADPH oxidase (r = 0.750) and to overt disease symptoms as judged by the arthritis index. Our data support the hypothesis that oxidative stress plays a pivotal role in the pathology of arthritis, which can be therapeutically targeted by NADPH oxidase inhibitors.

Effects of allopurinol on in vivo suppression of arthritis in mice and ex vivo modulation of phagocytic production of oxygen radicals in whole human blood.

Recently, we demonstrated elevated levels of xanthine oxidase in serum of patients with various inflammatory and autoimmune rheumatic diseases. The present study reports the antiarthritic efficacy of the xanthine oxidase inhibitor and immunosuppressant allopurinol in DBA/1xB10A(4r) mice suffering from peroxochromate-induced arthritis. A profound dose-dependent suppression of arthritis was noted (P < 0.001). The ED50 was 80 +/- 14 mumol/kg/day. The arthritis index correlated positively to the phagocytic production of oxygen radicals (r2 > 0.672) and negatively to the concentrations of allopurinol (r2 = 0.915). Ex vivo, allopurinol and various conventional antirheumatic drugs were screened for the inhibition of 12-O-tetradecanoylphorbol-13-acetate-stimulated whole human blood chemiluminescence. The concentrations of antirheumatic drugs required to inhibit the chemiluminescence by 50% were compared to the therapeutic doses administered to rheumatic patients. While D-penicillamine and cis-platinum(II) increased the phagocytic generation of superoxide, nonsteroidal antiinflammatory drugs (NSAIDs), steroids, and slow-acting antirheumatic drugs (SAARDs) inhibited the whole blood chemiluminescence in a dose-dependent manner. Therapeutic doses of NSAIDs, SAARDs, or steroids inhibited the phagocytic generation of reactive oxygen species by 10-50%. In addition to well-known mechanisms of action of NSAIDs and SAARDs, our results support the hypothesis that most common anti-rheumatic drugs act also by modulating the levels of reactive oxygen species, which serve important mediator and signal transduction functions in inflammatory and autoimmune diseases. Pharmacologically safe antioxidants like allopurinol, which simultaneously modify the oxidative burst of phagocytes, inhibit xanthine oxidase, and display immunosuppressive effects may well be suited to control the consequences of chronic phagocytic hyperreactivity in rheumatic patients.