H.P. Zahradnik

Twin pregnancies with single fetal death

Objective. Analysis of the fetal outcome of the surviving twin and the cause of fetal death. Patients. Between January 1979 and December 1992, 43 twin pregnancies with single fetal death were observed: in 11 cases (group I) before 16 weeks of gestation, in 11 cases (group II) between 17 and 24 weeks, and in 21 cases (group III) after 24 weeks of gestation.

Arachidonate metabolism in human placenta, fetal membranes, decidua and myometrium: Lipoxygenase and cytochrome P450 metabolites as main products in HPLC profiles

Eicosanoids play a key role in pregnancy maintenance and parturition. We investigated the metabolism of arachidonic acid (AA) in short-term tissue cultures of placenta, fetal membranes, decidua and myometrium. Tissues were obtained from caesarean sections before the onset of labour after uncomplicated pregnancies. The released metabolites were analysed by high performance liquid chromatography (HPLC) and specific immunoassays. In radiotracer experiments tissues were labelled with [3H]-AA and metabolites released after incubation with calcium ionophore A23187 were profiled by HPLC. Decidua was more active in metabolizing AA (turnover 34 per cent) than myometrium (28 per cent), placenta (21 per cent) and fetal membranes (17 per cent). Main product in placenta, decidua and myometrium was 12-hydroxyeicosatetraeinoic (12-HETE) (decidua: 19 per cent of released radioactivity, myometrium 14 per cent, placenta 7 per cent). Fetal membranes formed 5-HETE as main product. Another major metabolite in placenta, fetal membranes and decidua was characterized by HPLC as 5(6)-epoxyeicosatrienoic acid. Only myometrium released appreciable amounts of prostaglandins in form of 6-keto-prostaglandin F1 alpha. In non-radioactive experiments formation of eicosanoids from endogenous AA was investigated by HPLC (fluorescence- and UV-detection) and immunoassays. These experiments confirmed the high production of 12-HETE and the low formation of prostaglandins. Our results suggest that the biological role of AA-metabolites, other than prostaglandins, have as yet been underestimated.

Eicosanoid production by intrauterine tissues before and after labor in short-term tissue culture

Prostanoid production by intrauterine tissues from pregnant and non-pregnant women has been studied intensively over the last decade. Little is known about the lipoxygenase metabolites of arachidonic acid (AA). The production of prostaglandins and HETEs by pregnancy specific human tissues was investigated in a short-term culture system. Tissue samples were obtained after uncomplicated pregnancies from placenta, fetal membranes and decidua of deliveries before (n = 6) and after the onset of labor (n = 8) and incubated for 1 hour in oxygenated HBSS. In the supernatant, PGE2, PGF2 alpha, 6-keto-PGF1 alpha and TXB2 were measured with RIA and 15-, 12- and 5-HETE with HPLC and UV-detection. The main AA-metabolite in all tissue incubations was 12-HETE. Decidua produced 12 to 28 times more prostaglandins than placenta and fetal membranes with 6-keto-PGF1 alpha as the main metabolite. The main cyclooxygenase derivative measured from placenta and fetal membrane incubations was TXB2. After labor, fetal membranes showed an increase in total prostaglandin (significant for PGE2) and a decrease in HETE synthesis. The physiologic significance of 12-HETE in reproduction is still poorly understood, but a shift in AA metabolism from HETEs to prostaglandins may be involved in the initiation of labor. Furthermore, these results point to different roles of the tissue compartments within the pregnant uterus for the parturition process.

The effect of acetylsalicylic acid and indomethacin on the catecholamine- and oxytocin-induced cotractility and prostaglandin (6-keto-PGF1a, PGF2a)-production of human pregnant myometrial strips

The effects of acetylsalicylic acid (ASA) and indomethacin (IND) on the epinephrine and oxytocin stimulated contractility and prostaglandin (6-keto-PGF1 alpha, PGF2 alpha) production of superfused myometrial strips from the pregnant human uterus at term are reported. Without preincubation in ASA or IND epinephrine dose-dependently (10 ng/ml to 1 microgram/ml) stimulated the contractility and significantly increased the PG-release of the myometrial strips. The epinephrine induced increase in contractility was correlated to a higher increase in PGF2a production and a decreased 6-keto-PGF1 alpha/PGF2 alpha ratio (5.4 to 1.8). Superfusion of oxytocin increased myometrial contractions and PGF2 alpha release according to dose (3-12 microU/ml). However, 6-keto-PGF1 alpha production was not affected by oxytocin. Myometrial strips preincubated with ASA (100 micrograms/ml) or IND (10 micrograms/ml) demonstrated little spontaneous activity and the PG production was below the detection limit of the RIA. The stimulating effect of epinephrine and oxytocin on the contractility and PGF2 alpha release of the myometrial strips was inhibited significantly. During continuous superfusion of the ASA and IND preincubated myometrial strips with Tyrodes solution the inhibitory effect on spontaneous, epinephrine-, and oxytocin-stimulated contractility and PGF2 alpha release gradually declined over a period of 2 hours. This decrease of the inhibitory effect was more significant in ASA preincubated specimens. Our results demonstrate that spontaneous, epinephrine-, and oxytocin-stimulated contractility and PG release of human myometrial strips can be inhibited by ASA and IND and that this inhibitory effect is reversible. Furthermore our results suggest that in pregnant human myometrium the inhibition of PGF2 alpha production by ASA and IND is more pronounced than that of 6-keto-PGF1 alpha (PGI2).

Effects of nitric oxide donors on the contractility and prostaglandin synthesis of myometrial strips from pregnant and non-pregnant women.

Nitric oxide (NO) is a potent relaxant of smooth muscle and possibly plays a role in maintaining uterine quiescence during pregnancy. Clinical studies have shown beneficial effects of the stable NO donor glyceryl trinitrate (GTN) for the inhibition of pathological myometrial contractility that occurs in preterm labor or dysmenorrhea. Since there are contradictory results regarding the mediation of the relaxing effect of NO ,the myometrial prostaglandin synthesis during superfusion with NO donors was studied. Human myometrial strips obtained either at term Cesarean sections before the onset of labor or after hysterectomies in premenopausal women were studied in a superfusion system. After the manifestation of spontaneous contractions ,GTN was added in low doses comparable with in vivo levels (0.4-40 nM) and the effect on myometrial activity ,intracellular cGMP and prostaglandin production was analyzed. Additionally ,the effect of sodium nitroprusside (SNP) - which releases NO spontaneously - was compared with that of GTN. GTN caused a significant decrease in the contraction frequency of myometrial strips from both pregnant and non-pregnant women similar to that of SNP. There was no significant change in the myometrial synthesis of PGI2 ,PGF2α and PGE2 ,whereas the intracellular cGMP content was increased. In conclusion ,GTN showed a significant inhibitory effect on human myometrium in vitro in very low doses and therefore represents an interesting therapeutic alternative for the treatment of preterm labor and dysmenorrhea. GTN in low doses did not alter the prostaglandin synthesis of human myometrium.

Formation of prostaglandins by ovarian carcinomas

Tissue contents of prostaglandins (PG) PGE2, PGE2a and 6-keto-PGF1a (degradation product of PGI2) were determined in specimens of advanced human ovarian cancer (n = 11). The PG levels (ng/mg tissue protein) varied widley: PGE2 17-515; PGF2a 2-43 and 6-keto-PGF1a 5-105. Tumors of patients without response to chemotherapy contained more PGE2, PGF2a and 6-keto-PGF1a than did tumors responding to chemotherapy. PG production was investigated in two ovarian carcinoma-derived cell lines. The ability of these cells to synthesize PG varied depending on the cell density. An increase of cell number was associated with a decrease of PG yield. PG formation was inhibited by indomethacin in a concentration-dependent manner. The present study suggests that ovarian carcinoma cells form PG in vivo and vitro.

Efficacy, safety and sustainability of treatment continuation and results of an oral contraceptive containing 30 mcg ethinyl estradiol and 2 mg chlormadinone acetate, in long-term usage (up to 45 cycles) an open-label, prospective, noncontrolled, office-based Phase III study

BACKGROUND This open-label, noncontrolled study assessed the long-term efficacy and tolerability of the monophasic combined low-dose oral contraceptive (OC) ethinyl estradiol (EE) 30 mcg+chlormadinone acetate (CMA) 2 mg (Belara). STUDY DESIGN In total, 781 women who had already taken EE 30 mcg+CMA 2 mg for 24 cycles in a previous Phase III study were assessed for up to 45 cycles. RESULTS Over 23,033 cycles, the Pearl Index was 0.16 (95% confidence interval, 0.04-0.42). Approximately 86% of women had regular withdrawal bleeding in each cycle, while incidence of intracyclic bleedings (1.6-6.4%) and proportion of women with amenorrhea (4%) were low. The incidence of acne decreased from 13.8% to 5.7%, while rates of hirsutism, alopecia and seborrhea remained low (< or =4%) throughout this study. The most frequent adverse events were consistent with OC treatment, and no unexpected events occurred. No changes in mean blood pressure and pulse rate were observed during the study, and there were no clinically relevant changes in liver or hematological parameters, hemostasis or carbohydrate metabolism. The incidence of pathological findings in gynecological examination was low and decreased over time. CONCLUSION EE 30 mcg+CMA 2 mg was an effective and well-tolerated OC, with beneficial effects on cycle stability, intracyclic bleeding, amenorrhea and signs of androgenization that were maintained during long-term treatment for up to 5 years. There was no evidence of an increased risk of thromboembolic events, atherogenic disease or cervical cancer, suggesting that 30 EE mcg+CMA 2 mg is highly suitable for long-term use.

Effects of estradiol-17β and progesterone on the synthesis of prostaglandin F2α, prostaglandin E2 and prostaglandin I2 by fibroblasts from human endometrium in vitro

Abstract Estradiol-17β increases the production of prostaglandin F2α (PGF2α) in long term monolayer cell cultures of the human endometrium in a dose dependent manner. Progesterone in pharmacological dosage stimulates the syntheses of PGF2α and prostaglandin E2 (PGE2). The synthesis of prostaglandin I2 (PGI2) is not influenced by sex steroids in long term monolayer cell cultures of the human endoterium.

CYTOCHROME P450 METABOLITES OF ARACHIDONIC ACID IN HUMAN PLACENTA

Little is known about the epoxygenase pathway of the arachidonic acid cascade in uterine tissues. In this paper, we describe the formation of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs) in human term placenta after uncomplicated pregnancies. Metabolism of [3H]-arachidonic acid was analyzed in short term tissue cultures of placenta by reverse phase HPLC. Major metabolites coeluted with authentic EETs and DHETs. The formation of EETs was inhibited by carbon monoxide. In non-radioactive incubations with biopsies from seven different placentas, sufficient material for GC/MS analysis was obtained. The combined media were purified by solid phase extraction and reverse phase HPLC. The fraction coeluting with DHETs was derivatized with pentafluorobenzylbromide (PFB) and bis-(trimethylsilyl)-trifluoroacetylacetamide (BSTFA) and analyzed by GC/NICI/MS/MS. 11, 12-DHET and 14, 15-DHET were identified by their mass spectra displaying specific fragments at m/z 149 and m/z 189, respectively. Our results suggest that the epoxygenase pathway is active in human term placenta.

Expression of prostacyclin and thromboxane synthases in placenta and placental bed after pre-eclamptic pregnancies.

Prostacyclin and thromboxane are potent antagonistic regulators of vascular tone and platelet aggregation. In pre-eclampsia, the ratio of their metabolites is decreased. Little is known about the local regulation of intrauterine prostacyclin and thromboxane production in this condition. Placenta and placental bed biopsies were obtained from uncomplicated and pre-eclamptic pregnancies. Prostacyclin synthase (PCS) and thromboxane synthase (TXS) and their mRNAs were localized by immunohistochemistry using monoclonal antibodies and in situ hybridization. Protein and mRNA levels were quantified by immunoblot and RNase protection assay. PCS-like immunoreactivity was found in endothelial cells and leiomyocytes, whereas fetal and maternal macrophages showed positive staining for TXS. Their mRNA was localized to trophoblast and endothelium, and TXS mRNA could also be detected in macrophages. Quantitative analysis showed no significant difference in intrauterine protein or mRNA expression after pre-eclampsia. The prostacyclin and thromboxane production seems to be compartmentalized within the uteroplacental unit. The expression of their synthesizing enzymes might be regulated post-transcriptionally. Additional regulation of prostaglandin production could be metabolically or on the substrate level and requires further elucidation.