The generation of superoxide anion radicals (O2*-) and the other reactive oxygen species (ROS) was estimated by means of cytochrome c reduction and chemiluminescence, as well in resting blood platelets and in platelets stimulated by thrombin in the presence or absence of some inhibitors of pathways involved in platelet activation. We used allopurinol (xanthine oxidase inhibitor), wortmannin (PI 3-kinase inhibitor) and staurosporine (protein kinase C inhibitor). To determine the involvement of the glutathione in ROS generation, we used L-buthionine sulfoximine (BSO) which blocks GSH synthesis. Our results confirmed that thrombin stimulates the production of ROS concomitant with metabolism of arachidonate and production of malonyldialdehyde (MDA) in blood platelets (P < 0.05) and showed that, in the presence of inhibitors, the generation of ROS in platelets (resting and stimulated) was reduced. This indicates that xanthine oxidase, PI 3-kinase or protein kinase C take part in the formation of ROS in blood platelets. Moreover, adhesion of platelets to fibrinogen and secretion of adenine nucleotides from platelets after wortmannin and staurosporine action was also inhibited. BSO not only decreased GSH level, but also reduced the amount of ROS; a correlation between the depletion of GSH and the decrease of ROS was observed (R = -0.987; P < 0.02). It is concluded that in blood platelets, ROS are produced in the receptor-mediated signaling pathways and platelet activation (arachidonic acid metabolism, the glutathione cycle, metabolism of phosphoinositoides and due to xanthine oxidase). Our results support the importance of ROS in platelet function.

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The aim of the study was to evaluate the levels of lipid and protein peroxidation markers, in the follicular fluids (FF) of 82 patients undergoing in vitro fertilization (IVF). This included, thiobarbituric acid-reactive substances (TBARS), protein carbonyl, and thiol groups. The oxidative stress markers were compared between the pregnancy positive and pregnancy negative patient groups. The two patient groups were compared in terms of their age, body mass index (BMI), cause of infertility, and the plasma hormone levels (AMH, E(2), peak E(2)). Protein carbonyl and thiol groups were estimated using an ELISA assay and with Ellman's reagent (5, 5'-dithiobis-2-nitrobenzoic acid, DTNB), respectively. The mean FF TBARS level of 29 pregnant women was 0.954 ± 0.420 µmol/l, whereas it was twice as high (1.961 ± 0.796 µmol/l) in a group of 53 non-pregnant patients (p < 0.0001). In non-pregnant patients, we observed 2-fold elevated levels of protein carbonyl groups when compared to pregnant women (2.969 ± 0.723 vs. 1.523 ± 0.254; p < 0.0001). The mean age of women and BMI were significantly higher in the pregnancy negative vs. pregnancy positive group. There were no significant differences in protein thiols and in the levels of the hormones tested between patient groups. Our results demonstrate that elevated FF lipid and protein peroxidation level may have a negative impact on IVF outcome. The findings support the idea that increased level of oxidative stress markers in follicular fluid may play an important role in fertility.

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