Levels of Key Enzymes of Methionine-Homocysteine Metabolism in Preeclampsia

Objective. To evaluate the role of key enzymes in the methionine-homocysteine metabolism (MHM) in the physiopathology of preeclampsia (PE). Methods. Plasma and placenta from pregnant women (32 controls and 16 PE patients) were analyzed after informed consent. Protein was quantified by western blot. RNA was obtained with RNA purification kit and was quantified by reverse transcritase followed by real-time PCR (RT-qPCR). Identification of the C677T and A1298C methylenetetrahydrofolate reductase (MTHFR) single-nucleotide polymorphisms (SNPs) and A2756G methionine synthase (MTR) SNP was performed using PCR followed by a high-resolution melting (HRM) analysis. S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) were measured in plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). The SNP association analysis was carried out using Fisher’s exact test. Statistical analysis was performed using a Mann-Whitney test. Results. RNA expression of MTHFR and MTR was significantly higher in patients with PE as compared with controls. Protein, SAM, and SAH levels showed no significant difference between preeclamptic patients and controls. No statistical differences between controls and PE patients were observed with the different SNPs studied. Conclusion. The RNA expression of MTHFR and MTR is elevated in placentas of PE patients, highlighting a potential compensation mechanism of the methionine-homocysteine metabolism in the physiopathology of this disease

NFAT5 is up-regulated by hypoxia: possible implications in preeclampsia and intrauterine growth restriction

During gestation, low oxygen environment is a major determinant of early placentation process, while persistent placental hypoxia leads to pregnancy-related complications such as preeclampsia (PE) and intrauterine growth restriction (IUGR). PE affects 5%-8% of all pregnancies worldwide and is a cause of maternal and fetal morbidity and mortality. During placental development, persistent hypoxia due to poor trophoblast invasion and reduced uteroplacental perfusion leads to maternal endothelial dysfunction and clinical manifestation of PE. Here we hypothesized that nuclear factor of activated T cells-5 (NFAT5), a well-known osmosensitive renal factor and recently characterized hypoxia-inducible protein, is also activated in vivo in placentas of PE and IUGR complications as well as in the in vitro model of trophoblast hypoxia. In JAR cells, low oxygen tension (1% O2) induced NFAT5 mRNA and increased its nuclear abundance, peaking at 16 h. This increase did not occur in parallel with the earlier HIF1A induction. Real-time PCR and Western blot analysis confirmed up-regulation of NFAT5 mRNA and NFAT5 nuclear content in human preeclamptic placentas and in rabbit placentas of an experimentally induced IUGR model, as compared with the control groups. In vitro lambda protein phosphatase (lambda PPase) treatment revealed that increased abundance of NFAT5 protein in nuclei of either JAR cells (16 h of hypoxia) or PE and IUGR placentas is at least partially due to NFAT5 phosphorylation. NFAT5 downstream targets aldose reductase (AR) and sodium-myo-inositol cotransporter (SMIT; official symbol SLC5A3) were not significantly upregulated either in JAR cells exposed to hypoxia or in placentas of PE- and IUGR-complicated pregnancies, suggesting that hypoxia-dependent activation of NFAT5 serves as a separate function to its tonicity-dependent stimulation. In conclusion, we propose that NFAT5 may serve as a novel marker of placental hypoxia and ischemia independently of HIF1A

First trimester prediction of early onset preeclampsia using demographic, clinical, and sonographic data: a cohort study

Objective The aim of this research was to evaluate the performance of a predictive model for early onset preeclampsia (PE) during early gestation.Method Prospective multicenter cohort study was performed in women attending 11-14 weeks ultrasound. Medical history and biometrical variables were recorded and uterine artery Doppler was performed. All patients were followed until postpartum period. Constructed predictive models were compared using the area under the associated receiver operating characteristic curve. Sensitivity, specificity, and likelihood ratios were estimated for each outcome.Results A total of 627 patients were enrolled. Sixty-five (10.4%) developed gestational hypertension, of which 29 developed PE (4.6% of the total sample) and nine occurred before 34 weeks (1.5% of total sample). Prediction model generated for early onset PE (ePE) with 5% false positive achieve sensitivity of 62.5% and specificity of 95.5%. The positive and negative likelihood ratios for ePE were 13.9 and 0.39, respectively. Development of ePE was significantly associated with history of preterm labor (p = 0.002) and diabetes mellitus (p = 0.02).Conclusions This study confirms the advantage of combining multiple variables for prediction of ePE. (C) 2013 John Wiley & Sons, Ltd

Placental Aromatase Is Deficient in Placental Ischemia and Preeclampsia.

Preeclampsia is a maternal hypertensive disorder with uncertain etiology and a leading cause of maternal and fetal mortality worldwide, causing nearly 40% of premature births delivered before 35 weeks of gestation. The first stage of preeclampsia is characterized by reduction of utero-placental blood flow which is reflected in high blood pressure and proteinuria during the second half of pregnancy. In human placenta androgens derived from the maternal and fetal adrenal glands are converted into estrogens by the enzymatic action of placental aromatase. This implies that alterations in placental steroidogenesis and, subsequently, in the functionality or bioavailability of placental aromatase may be mechanistically involved in the pathophysiology of PE.Serum samples were collected at 32-36 weeks of gestation and placenta biopsies were collected at time of delivery from PE patients (n = 16) and pregnant controls (n = 32). The effect of oxygen tension on placental cells was assessed by incubation JEG-3 cells under 1% and 8% O2 for different time periods, Timed-mated, pregnant New Zealand white rabbits (n = 6) were used to establish an in vivo model of placental ischemia (achieved by ligature of uteroplacental vessels). Aromatase content and estrogens and androgens concentrations were measured.The protein and mRNA content of placental aromatase significantly diminished in placentae obtained from preeclamptic patients compared to controls. Similarly, the circulating concentrations of 17-β-estradiol/testosterone and estrone/androstenedione were reduced in preeclamptic patients vs. controls. These data are consistent with a concomitant decrease in aromatase activity. Aromatase content was reduced in response to low oxygen tension in the choriocarcinoma JEG-3 cell line and in rabbit placentae in response to partial ligation of uterine spiral arteries, suggesting that reduced placental aromatase activity in preeclamptic patients may be associated with chronic placental ischemia and hypoxia later in gestation.Placental aromatase expression and functionality are diminished in pregnancies complicated by preeclampsia in comparison with healthy pregnant controls

Aromatase is downregulated in JEG–3 cell line in response to hypoxia.

<p>JEG–3 cell line was exposed either to 8% or to 1% O₂ in an hypoxic chamber for 0, 4, 8, 16 and 24 h. <b>A</b>. Cells were collected at indicated times and protein lysates analyzed by western blot to determine the protein expression levels of aromatase, HIF–1α and β-Tubulin. Upper panel shows representative western blots and lower panels show aromatase and HIF–1α proteins densitometry data normalized to β-Tubulin loading control from n = 3 experiments. Data are shown in arbitrary units (A.U.) ±SEM. <b>B</b>. Cells were collected at indicated times and analyzed by qRT–PCR to determine aromatase mRNA transcript levels. Statistical analyses were performed using Student’s <i>t</i>-test and compared with the correspondent time point in the control, 8% O₂, cells. Columns are the mean of four independent experiments in duplicate; Data are reported as mean±SEM. *<i>P</i>≤0.05; **<i>P</i>≤0.01; *** <i>P</i>≤0.001, significant; n.s., non-significant.</p

Aromatase metabolite levels are dysregulated in PE patients.

<p>Aromatase functionality was measured in PE and normotensive pregnancies. The levels of aromatase precursors and metabolites including, <b>A</b>. testosterone, <b>B</b>. androstenedione, <b>C</b>. 17-β-estradiol, and <b>D</b>. estrone were measured by RIA in maternal serum samples collected at 32–36 weeks of gestation. Also shown are <b>E</b>. 17-β-estradiol/testosterone and <b>F</b>. estrone/androstenedione ratios. Data are reported as mean±SEM from 32 controls and 16 PE patients. Statistical analysis was performed using Mann-Whitney test. *<i>P</i>≤0.05; **<i>P</i>≤0.01, significant; n.s., non-significant.</p

Clinical characteristics of controls and preeclampsia patients at different times of gestation.

<p>Values are given as Mean±SEM. Statistical significance was assessed using Mann Whitney test and Fisher's exact test.</p><p>*p≤0.05</p><p>**p≤0.01</p><p>***p≤0.001</p><p>Clinical characteristics of controls and preeclampsia patients at different times of gestation.</p

Effect of hypoxia on placental aromatase expression <i>in vivo</i>.

<p><b>A</b>. Pups of timed-pregnant rabbits derived either from one uterine horn which placental spiral arteries had been ligated or from the non-ligated placentas on the contralateral horn (control horn, n = 17) were weighted <b>B</b>. and RNA was extracted from the correspondent placentas. Aromatase mRNA levels were analyzed by qRT-PCR in the control (n = 17) and hypoxic horn (n = 10). At least one hypoxic and one no-hypoxic placenta were collected from each of the 6 pregnant rabbits. Data are reported as mean±SEM. Statistical analysis was conducted using Mann-Whitney test. *<i>P</i>≤0.05; **<i>P</i>≤0.01, significant.</p