Paraquat inhibits progesterone synthesis in human placental mitochondria

IntroductionHuman placenta mitochondria produces huge amounts of progesterone necessary for maintaining the pregnancy. Lipid peroxidation in human placental mitochondria inhibits progesterone synthesis and that inhibition can be reversed by superoxide dismutase and other antioxidants. Paraquat (PQ) a highly toxic herbicide generates superoxide radical inside cells and induces lipid peroxidation. Hence, it is supposed to stimulate lipid peroxidation in human placental mitochondria and in consequence to inhibit a placental mitochondrial steroidogenesis.MethodsPlacentas were obtained from normal pregnancies. All experiments were done using isolated human placental mitochondria. Mitochondrial lipid peroxidation was determined as tiobarbituric acid reactive substances (TBARS). A conversion of cholesterol to pregnenolone or pregnenolone to progesterone was measured using radiolabeled steroids and thin layer chromatography.ResultsPQ enhanced the iron-dependent lipid peroxidation as also PQ heightened the inhibitory action of this process on progesterone synthesis in isolated human placental mitochondria. Paradoxically, a superoxide dismutase (SOD) reversed the inhibition of progesterone synthesis only minimally although it strongly inhibited PQ stimulated iron-dependent lipid peroxidation. When iron was absent, PQ stimulated only negligible lipid peroxidation but strongly inhibited progesterone synthesis. SOD had no effect on inhibition of progesterone synthesis by PQ. PQ strongly inhibited of the conversion of cholesterol to pregnenolone but had not got any influence on the enzymatic activity of mitochondrial 3β-hydroxysteroid dehydrogenase. PQ strongly decreased the efficiency of NADPH-dependent cytochrome P450 reduction as well as it promoted the rapid oxidation of the pre-reduced mitochondrial cytochrome P450. However PQ has not inhibited combined activity of adrenodoxin reductase and adrenodoxin.DiscussionWe conclude that the most important reason of the inhibition of progesterone synthesis by PQ is the escape of electrons from cytochrome P450scc to that compound what leads to cytochrome oxidation and, in consequence the inhibition of the reaction catalyzed by it. The action of PQ described here should be considered as potentially harmful for pregnancy and fetal development.     

Human placental ultrastructure after in vitro dual perfusion

Unperfused term placental tissue maintained at 37 degrees C for two hours preserved a major part of its structure while displaying a number of signs of hypoxic damage. Most placentae perfused through maternal and fetal circulations with Krebs-Ringer medium retained their ultrastructural integrity after two or more hours of perfusion.     

Differential control of placental lactogen release and progesterone production by ovine placental tissue in vitro

The hypothesis that placental secretion of progesterone (P4) and ovine placental lactogen (oPL) are controlled through different mechanisms was tested. Placental tissue was obtained at days 133-138 of pregnancy, and explant incubations were established using 200 mg tissue per flask in 5 ml O2-saturated DMEM containing 24 mM HEPES and lacking phenol red (pH 7.4). Following a 30-min preincubation, and a 15-min control period, test substances were added and incubations continued, with periodic gassing, for 4 h at 37 degrees C in a shaking water bath. Dopamine (DA), norepinephrine (NE) and epinephrine significantly stimulated P4 production (P less than 0.05). The enhancement of placental P4 production was mimicked by the addition of 8-bromo-cyclic adenosine monophosphate and forskolin (P less than 0.05). The response to catecholamines was abolished by the addition of propranolol (P less than 0.05) but not by phentolamine (P greater than 0.05). Inclusion of a membrane-permeant substrate for P4 synthesis, 25-hydroxycholesterol, increased basal (P less than 0.05) but did not enhance agonist-induced P4 production (P greater than 0.05). High performance liquid chromatographic analysis of placental tissue demonstrated the presence of DA (80.8 +/- 7.07 pg/mg) and NE (48.8 +/- 5.77 pg/mg), as well as catecholamine metabolites. Addition of 1,2-dioctanoyl-sn-glycerol (DAG) or phorbol 12-myristate-13-acetate (PMA) enhanced oPL secretion (P less than 0.05) without affecting P4 production. The response to DAG and PMA, representing the release of considerably more oPL than can be detected by extracting the tissue, was not influenced by treatment with cycloheximide (P greater than 0.05) indicating that secretion of preformed oPL is regulated by the protein kinase C pathway. These results support the hypothesis that the secretion of oPL and the production of P4 are controlled by different mechanisms.     

Ethical aspects in placental perfusion studies: views of the researchers

Within the EU-project NewGeneris human placental perfusion has been used for predicting fetal exposure to food carcinogens. Within the work package of ethical aspects of the research, we studied opinions of the researchers (n = 23) who carried out perfusions of human placenta. Data were collected by focus group interviews (n = 12) and an open-ended questionnaire (n = 19 of which 8 were also attending the group session) from scientists representing 9 different nationalities. Both types of data were analysed together thematically and with data triangulation. Studied researchers considered communication between all stakeholders extremely important. Good communication was considered a prerequisite for the recruitment of mothers to donate the placenta, as well as for the process of getting the informed consent. Voluntariness, confidentiality and societal meaning were mentioned as important by all studied researchers. Educating the hospital personnel was regarded as essential in order to provide the best possible information to the mothers. The researchers also pointed out that cultural aspects should be respected, and that in Western thinking placenta is mostly considered as waste. Some researchers suggested that current guidelines and processes for obtaining informed consent should be reviewed also from a cultural perspective. With the development of biobanks, the use of human tissues, including placenta will most probably increase in the future, and the awareness of ethical considerations both in legislation and in practice need support. Thus, continuous effort for better research ethics is essential and requires research on research ethics.     

Inhibition of human placental progesterone synthesis by danazol in vitro

In vitro, danazol showed a slight dose-dependent inhibition of the mitochondrial cholesterol side chain cleavage enzyme isolated from early gestational (8th to 12th week of gestation) placenta. In the presence of 100 microM danazol, the enzyme activity was 65% of controls. Danazol inhibits dose-dependently the mitochondrial 3 beta-hydroxysteroid dehydrogenase (I50 = 3.1 microM; Ki = 1 microM) (noncompetitive inhibition) and the cytoplasmic 20 alpha-hydroxysteroid dehydrogenase (I50 = 1.4 microM; Ki = 2.6 microM) (competitive inhibition). The inhibition of human placental progesterone synthesis by danazol in vitro is a further example for the direct interference of danazol with steroidogenesis.     

Changes in placental progesterone receptors in term and preterm labour

Currently, preterm labour is associated with increased fetal mortality and morbidity and is often associated with elevated levels of inflammatory cytokines. However, the exact mechanisms that lead to this pathology are not fully elucidated. In the present study evidence was obtained using a specific membrane progesterone receptor (mPR) agonist, Org OD 02-0, that the progestin antagonism of apoptotic effects of a cytokine, IL-1β, on human placental BeWo cells is mediated through mPRs. Therefore the aim of this study was to determine whether the gene expression of mPRs and all other known progesterone receptors changes in human placentas at term and during labour. Quantitative PCR (qPCR) in clinical samples revealed a 2.8 fold decrease of mPRβ in labouring comparing to non-labouring tissues and 4.6 fold higher levels of mPRγ in preterm mPRγ compared to term placentas. The ratio of mPRα to PR-B was increased in term compared to preterm samples, whereas it was decreased in labour compared to non-labour placentas. There was also a high correlation between mPRα and PGRMC1 expression irrespective of pathologies. Collectively, our data indicates that changes in the ratios of progesterone receptors rather than individual fluctuations might affect progesterone signalling at the placental level.     

Labetalol does not decrease placental perfusion in the hypertensive term-pregnant rat

The acute effect of labetalol hydrochloride, a combined nonspecific beta-adrenergic and postsynaptic alpha 1-adrenergic blocker, on maternal hemodynamics and organ perfusion was investigated in 10 hypertensive, term-pregnant, spontaneously hypertensive rats with the use of the radioactive-labeled microsphere technique. The normal fall in blood pressure during pregnancy was prevented by the reduction of litter size to two conceptuses on day 7 of gestation. Labetalol (1 to 6 mg/kg) effectively lowered mean arterial pressure 22% by decreasing cardiac output 16%; total peripheral resistance was not significantly decreased. Thus, the blood pressure lowering effect of labetalol was due primarily to its beta-adrenergic blocking effect. Regional flows to the carcass and splanchnic circulation were decreased 19% and 15%, respectively, after labetalol administration. Uterine wall and ovarian perfusion were significantly reduced, but placental perfusion was not significantly altered. Because labetalol lowers blood pressure without reducing placental perfusion, it may be a useful alternative to hydralazine for the treatment of hypertensive emergencies in pregnancy.     

The effect of hydralazine on placental perfusion in the spontaneously hypertensive rat

Intravenous hydralazine was administered to 16 spontaneously hypertensive rats on day 21 of gestation. The radioactive labeled microsphere technique was used to assess the change in organ perfusion produced by the drug. Vascular resistance to most organs was decreased, except to the placentas, stomach, and cecum, where it increased by 43%, 104%, and 44%, respectively. Blood flow to the organs was redistributed, and although it was increased to the lungs, kidneys, liver, and adrenals, it was significantly reduced to the spleen, stomach, placentas, cecum, large intestine, and pancreas. The effect of hydralazine on placental perfusion was opposite to the effect on the uterus (myometrium). Patients with the highest blood pressures tend to have the poorest placental perfusion. Intravenous hydralazine should be used cautiously in these patients.     

Maternal-fetal transfer of saquinavir studied in the ex vivo placental perfusion model

OBJECTIVE: This study was undertaken to determine the placental transfer of the human immunodeficiency virus protease inhibitor saquinavir. STUDY DESIGN: An ex vivo perfused human placental cotyledon model was used. Ten placental perfusion studies were performed, with concentrations of saquinavir in the maternal compartment ranging from 322 to 2197 ng/mL (within reference therapeutic ranges). Drug concentrations were determined by high-performance liquid chromatography. RESULTS: The mean (+/- SD) fetal transfer rate of saquinavir was 1.8% +/- 1.6%, and the mean (+/- SD) clearance index was 0.05 +/- 0.05. A mean (+/- SD) of 1.6% +/- 3.1% of the perfused saquinavir was retained by the cotyledon. The small amount of saquinavir that crossed the placenta corresponded to the fraction not bound to human serum albumin. CONCLUSION: The low rate of placental transfer of saquinavir suggests that use of this antiretroviral drug by pregnant women may not lead to significant exposure of the fetus.     

Lack of short-term modulation of in vitro placental progesterone secretion in sheep

Experiments were performed in order to determine whether progesterone secretion in the ovine placenta can be short-term regulated. There was an increase in progesterone content per unit weight in ovine fetal cotyledons as gestation progressed: 17.0 +/- 4.7 ng/100 mg of wet tissue in ewes between 40 and 54 days of pregnancy (n = 7) and 70.7 +/- 18.8 (n = 9) between 100 and 118 days. At all stages of pregnancy, neither progesterone nor 20 alpha-dihydroprogesterone synthesis were significantly affected when fetal cotyledons were incubated for 3 h in the presence of LH, 8-Br-cAMP, GnRH agonist or GnRH antagonist. Addition of pregnenolone to the incubation medium increased progesterone secretion in a dose-dependent manner while addition of 25-hydroxycholesterol did not. These results suggest that the existent (basal) synthesis of progesterone reflects the maximal capacity of steroidogenesis through the cholesterol side-chain-cleavage system. In the presence of these precursors, LH, 8-Br-cAMP, the phorbol ester derivative PMA and calcium ionophore A23187 were not able to modify progesterone or 20 alpha-dihydroprogesterone synthesis. These results also suggest that LH or GnRH and the two signal mechanisms involved in their action, i.e. cAMP and Ca2+ sensitive-inositol phospholipid-dependent mechanisms are not implicated in the short-term regulation of progesterone synthesis in the ovine placenta.     

Ovine placental perfusion balance: effect of marijuana smoke

This study was designed to test the hypothesis that maternal marijuana smoking impairs placental oxygen transfer in late ovine pregnancy by disrupting perfusion balance between the maternal and fetal placental circulations. Placental hemodynamics were assessed in nine chronically prepared ewes 1 hour after exposure to smoke from either a marijuana (n = 5) or a placebo (n = 4) cigarette. When compared with placebo smoke, maternal marijuana smoke exposure resulted in a fall in both uterine and umbilical placental vascular resistance and a 30% improvement in placental perfusion balance at a cotyledonary level. We conclude that maternal marijuana smoking in late ovine pregnancy has a direct relaxant effect on both maternal and fetal placental vascular smooth muscles that decreases the normal heterogeneity of flow and improves macroscopic placental perfusion balance. However, the observed concurrent 6 torr decrease in fetal oxygen tension suggests that perfusion balance is actually disrupted at a microcirculatory level.     

Ultrastructure of human placental tissue after 6 h of normoxic and hypoxic dual in vitro placental perfusion

The dual in vitro perfusion model of human placental tissue allows the study of different aspects of placental function, such as metabolism, transport and secretion of proteohormones, cytokines and prostaglandins. The integrity of the perfused placental tissue is an important parameter to validate the perfusion system. Using light and electron microscopy, the morphology of villous tissue was examined before and after six hours of normoxic (n=10) vs. hypoxic (n=10) perfusion. An apical shift of the rough endoplasmic reticulum and occasional vacuoles were found in the syncytiotrophoblast of the terminal villi, the exchange area of the placenta. No unexpected pathological findings were seen before the perfusion experiments and only slight changes with moderate distension of the endoplasmic reticulum after 6 h of normoxic perfusion. After hypoxic perfusions, distinct ultrastructural alterations, such as oedematous villous stroma, swollen or completely destroyed cell organelles (e.g., mitochondria and endoplasmic reticulum), multiple vacuoles inside syncytio- and cytotrophoblasts as well as the microvilli were seen, which leads to an impairment of the placental barrier and other functions. The ultrastructural examination of placental tissue before and after dual in vitro perfusion broadens the knowledge of physiological and pathophysiological processes in the perfused placenta and may be a beneficial part of regular validation.     

Reduced placental perfusion causes an increase in maternal serum leptin

OBJECTIVE: We tested the hypothesis that the inadequately perfused placenta increases production of leptin, which can be detected in maternal serum. STUDY DESIGN: Sprague-Dawley rats (n=13), on day 14 of gestation, had placement of clips on the aorta and the ovarian arteries providing 35 per cent occlusion of the vessels. Eight rats had sham surgery and 14 rats served as non-surgical controls. All animals were sacrificed on day 19 of gestation. Maternal serum was obtained, and pups and placentae were weighed. RESULTS: Both placental weights and pup weights were reduced due to reduced uterine perfusion and were negatively correlated with maternal serum leptin (P=0.018 and 0.028, respectively). Maternal serum leptin was increased in the treatment group (2.21 ng/ml+/-64 ng/ml) compared to controls (1.66 ng/ml+/-38 ng/ml) (P=0.031). CONCLUSIONS: Our findings suggest that reduced placental perfusion results in an increase in maternal serum leptin. Further investigation is needed to determine if maternal serum leptin may be useful in identifying pregnancies with uteroplacental insufficiency.