The role of oxidative stress in female infertility and in vitro fertilization.

Infertility problem involves many couples of reproductive age. It has been estimated that in Poland 0.7-1.0 million pairs require treatment, while for more than half of them assisted reproduction is the only recommended and effective method. Infertility affects 13 to 15% of the world's population. A major concern is the age-related decline in female fertility even more that often a decision about pregnancy is taken at later age. Recent studies show that increased production of reactive oxygen species is an important factor in etiopathogenesis of pregnancy and affects female reproduction. It was found that oxidative stress may damage the oocytes and may impair their fertilization capacity. Oxidative stress may also lead to embryo fragmentation and formation of numerous developmental abnormalities, and is regarded to be one of the important reasons of spontaneous and recurrent miscarriage. Moreover, overproduction of reactive oxygen species has a significant impact on the success of in vitro fertilization (IVF).

Aronia melanocarpa as a protector against nitration of fibrinogen.

Fibrinogen (Fg) also known as coagulation factor I represents about 4% of the total human plasma proteins. The main function of Fg is its involvement in last phase of blood coagulation cascade, when thrombin-induced conversion of dissolved plasma fibrinogen into an insoluble fibrin clot occurs. The reaction of fibrinogen with peroxynitrite causes both structural modifications and changes of the biological properties of this plasma glycoprotein. Recently, there is an increased interest in the screening of natural products present in fruits, vegetables and herbs for their possible antioxidative activities. Therefore, the aim of our study was to estimate the effect of extract from berries of Aronia melanocarpa against nitrative and oxidative damage induced by peroxynitrite. The extract from A. melanocarpa (0.5-50 µg/ml) added to Fg 10 min before peroxynitrite (100 µM) significantly inhibited both the formation of the high molecular weight protein aggregates and nitration of Fg molecule. The extract also abolished peroxynitrite-induced inhibition of fibrinogen polymerization (by 95% at 50 µg/ml). The obtained results indicate that natural extract from berries of A. melanocarpa has protective effects against peroxynitrite-induced nitrative damage of plasma fibrinogen, and therefore may contribute in the prevention of peroxynitrite-related cardiovascular or inflammatory diseases.

Coagulation and fibrynolitic parameters in women and the effects of hormone therapy; comparison of transdermal and oral administration.

It is established that hormone therapy (HT) is related with significant increased prothrombotic risk factor. The aim of our study was to assess the effects of oral hormone therapy (o-HT) and transdermal hormone therapy (t-HT) on hemostasis parameters: fibrinogen (Fg) concentration, the maximum velocity of polymerization of clot formation, fibrin half-time lysis, plasma level of thrombin inhibitor of fibrinolysis (TAFI) and activity of generated thrombin and plasmin amidolytic activity. We observed that values of initial velocity of polymerization in o-HT group were increased (94.64 mOD/min vs. 131.50 mOD/min, p < 0.001) compared to control group. Fibrin lysis half-time increased in both groups with HT (controls - 18.26 min vs. 32.43 min (o-HT); 23.34 min transdermal hormone therapy (t-HT) p < 0.001) compared to controls. The activity of thrombin was statistically higher in plasma of women after o-HT (72.6 ± 8.5 mOD/min) than in patients with t-HT (53.7 ± 10.1 mOD/min) and controls (51.2 ± 10 mOD/min. Plasmin activity was the highest in controls (84.5 ± 10.2 mOD/min). The highest level of TAFI we observed in patients after oral hormones (80.38 ± 8.23%); women on transdermal HT had 61.58 ± 9.81% and the lowest concentration of TAFI we noted in controls 44.70 ± 10.16). The results of our study show that HT may partly explain the increase in venous thrombosis (VTE) and cardiovascular events reported after the use of it, especially the oral form of treatment.

Oxidative stress markers in follicular fluid of women undergoing in vitro fertilization and embryo transfer.

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.

Oxidative stress measured by carbonyl groups level in postmenopausal women after oral and transdermal hormone therapy.

AIM: Menopause is associated with an increased risk of cardiovascular disorders, which are accompanied by oxidative stress. Our study was undertaken to determine whether oxidative stress in menopausal women could be reduced after six months of oral or transdermal hormonal therapy. MATERIAL AND METHODS: Carbonyl groups of proteins in blood plasma were estimated by sensitive ELISA method with anti-DNP antibodies. In this method, protein samples diluted in phosphate-buffered saline were adsorbed to wells of an ELISA plate and then reacted with dinitrophenylhydrazine (DNPH). RESULTS: Plasma protein carbonyl levels of postmenopausal women treated with o-HT and t-HT for six months (o-HT: 1.785 ± 0.31 nmol/mg; t-HT: 1.838 ± 0.33 nmol/mg) were lower when compared with the control group (2.232 ± 0.28 nmol/mg). There was no statistically significant difference in carbonyl levels between women after oral and transdermal HT (P = 0.149). CONCLUSION: Hormonal therapy reduces the level of carbonyl protein, a marker of oxidative stress, suggesting potential protective effect.

Protective effects of (-)-epicatechin against nitrative modifications of fibrinogen.

Fibrinogen appears to be particularly sensitive to toxic action of peroxynitrite; a potent oxidizing and nitrating species. An increased nitration of fibrinogen has been reported in cardiovascular diseases. The defense mechanisms against PN are crucial for complex hemostasis process. Flavonoids have antioxidative properties and could protect biomolecules against action of peroxynitrite. The aim of our studies was to establish, if (-)-epicatechin may in vitro protect fibrinogen molecule against peroxynitrite-induced nitration of tyrosines and change its thrombin-catalyzed polymerization. The exposure of purified fibrinogen (6 µM) to peroxynitrite (1-100 µM) resulted in both structural modifications and clotting ability of this glycoprotein. Peroxynitrite at the concentration of 1 µM increased maximum velocity of Fg polymerization, whereas exposure to 100 µM PN resulted in a significant decrease of Vmax. (-)-Epicatechin (1-100 µM) caused a dose-dependent inhibition of 3-nitrotyrosine formation in fibrinogen treated with peroxynitrite (100 µM) in both Western blot assays and C-ELISA assays. At the highest concentration of (-)-epicatechin (100 µM) the level of 3-NT in fibrinogen reached the control values. At lower doses (-)-epicatechin reduced tyrosine nitration by approx. 23% and 40% at the concentration of 1 µM and 10 µM, respectively. (-)-Epicatechin also abolished the pro-thrombotic effect of peroxynitrite on fibrinogen clotting. The presented in vitro results demonstrated for the first time that (-)-epicatechin might have protective effects against the impairment of structure and properties of Fg, caused by action of the strong biologic oxidant/nitration and inflammatory mediators.

Anticoagulant effect of polyphenols-rich extracts from black chokeberry and grape seeds.

Blood coagulation consists of a series of zymogens that can be converted by limited proteolysis to active enzymes leading to the generation of thrombin. Fresh plasma and human thrombin was incubated with extracts from berries of Aronia melanocarpa or seeds of Vitis vinifera (0.5; 5; 50 µg/ml). The in vitro experiments showed that both extracts prolonged clotting time and decreased the maximal velocity of fibrin polymerization in human plasma. Moreover thrombin incubation with both extracts results in the inhibition of amidolytic activity of this enzyme. It gives hopes for development of diet supplements, which may be preventing thrombosis in pathological states.

Changes in hemostatic parameters after oral and transdermal hormone therapy in postmenopausal women.

Hormone therapy (HT) can be prothrombotic risk factor. We compared the effects of oral HT (o-HT) and transdermal HT (t-HT) on the kinetic of clot formation and fibrinolysis in postmenopausal women after 6 months HT using a multiparameter test. We observed that after HT, the level of fibrinogen was higher than in controls (Fg 3.12 g/l vs. 4.24 g/l (o-HT); 3,7 g/l (t-HT); p < 0.001) and values of velocity of polymerization in o-HT group were increased (95.84 mOD/min vs. 146.50 mOD/min, p < 0.001) compared to controls. Maximum absorbance of formed clots was higher in o-HT group (0.279 vs. 0.312, p < 0.001) than in controls, but in t-HT group was lowest (0.268). Fibrin lysis half-time increased in both HT groups (controls 17.16 min vs. 31.43 min (o-HT); 23.34 min (t-HT) p < 0.001) compared to values in controls. The results of our study show that o-HT caused the changes in clot formation and fibrinolysis than t-HT in postmenopausal women. The increased level of fibrinogen and its accelerated kinetics of polymerization as well as a lower rate of clot lysis may partly explain the increase in venous thrombosis and cardiovascular events reported after the use of HT, especially the oral form of that.

Changes in hemostatic parameters after oral hormone therapy in postmenopausal women.

OBJECTIVE: Oral hormone therapy (HT) and menopausal age are both prothrombotic risk factors. The aim of our study was to compare the hemostatic parameters in plasma of postmenopausal women after 6 months of oral HT with parameters of control (without treatment) postmenopausal women. METHODS: Twenty-seven postmenopausal women were treated with 17ß-estradiol (1 mg) and dydrogesterone (5 mg) daily for 6 months. The control group (27 women) did not receive any HT. Hemostatic factors, such as fibrinogen (FG) concentration, activated partial thromboplastin time (APTT), platelet (PLT) count, maximum velocity of clot formation, and fibrin lysis half-time were estimated. RESULTS: The hemostatic parameters in both groups differ significantly. After 6 months oral HT, APTT and the level of FG were higher than in the control group (APTT 30.08 seconds vs. 28.18 seconds, p = 0.02; FG 4.14 g/L vs. 3.03 g/L, p < 0.001). However, the higher values of maximal velocity of FG polymerization (153.53 mOD/min vs. 92.87 mOD/min, p < 0.001), maximum absorbance values (0.306 vs. 0.275, p < 0.001), and fibrin lysis half-time (32.33 minutes vs. 18.11 minutes, p < 0.001) compared with values in the control group also were observed. There was no statistically significant difference in PLT counts between control and women treated with oral HT. CONCLUSIONS: Six months of oral combined HT (17ß-estradiol and dydrogesterone) caused increased initial velocity of clot formation and inhibition of fibrinolysis. The increased level of FG and its higher polymerization may help explain the increase in venous thrombosis and cardiovascular events reported after the use of oral HT.