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Objective: The aim of the article is to determine whether prior spontaneous abortion (SAB) or induced abortion (IAB), or the interpregnancy interval are associated with subsequent adverse pregnancy outcomes in nulliparous women., Methods: We performed a secondary analysis of data collected from nulliparous women enrolled in a completed trial of vitamins C and E or placebo for preeclampsia prevention. Adjusted odds ratios (ORs) for maternal and fetal outcomes were determined for nulliparous women with prior SABs and IABs as compared with primigravid participants., Results: Compared with primigravidas, women with one prior SAB were at increased risk for perinatal death (adj. OR, 1.5; 95% CI, 1.1-2.3) in subsequent pregnancies. Two or more SABs were associated with an increased risk for spontaneous preterm birth (PTB) (adj. OR, 2.6, 95% CI, 1.7-4.0), preterm premature rupture of membranes (PROM) (adj. OR, 2.9; 95% CI, 1.6-5.3), and perinatal death (adj. OR, 2.8; 95% CI, 1.5-5.3). Women with one previous IAB had higher rates of spontaneous PTB (adj. OR, 1.4; 95% CI, 1.0-1.9) and preterm PROM (OR, 2.0; 95% CI, 1.4-3.0). An interpregnancy interval less than 6 months after SAB was not associated with adverse outcomes., Conclusion: Nulliparous women with a history of SAB or IAB, especially multiple SABs, are at increased risk for adverse pregnancy outcomes., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

The placenta plays a key role in regulation of fetal growth and development and in mediating in utero developmental programming. Obesity, which is associated with chronic inflammation and mitochondrial dysfunction in many tissues, exerts a programming effect in pregnancy. We determined the effect of increasing maternal adiposity and of fetal sex on placental ATP generation, mitochondrial biogenesis, expression of electron transport chain subunits, and mitochondrial function in isolated trophoblasts. Placental tissue was collected from women with prepregnancy BMI ranging from 18.5 to 45 following C-section at term with no labor. Increasing maternal adiposity was associated with excessive production of reactive oxygen species and a significant reduction in placental ATP levels in placentae with male and female fetuses. To explore the potential mechanism of placental mitochondrial dysfunction, levels of transcription factors regulating the expression of genes involved in electron transport and mitochondrial biogenesis were measured. Our in vitro studies showed significant reduction in mitochondrial respiration in cultured primary trophoblasts with increasing maternal obesity along with an abnormal metabolic flexibility of these cells. This reduction in placental mitochondrial respiration in pregnancies complicated by maternal obesity could compromise placental function and potentially underlie the increased susceptibility of these pregnancies to fetal demise in late gestation and to developmental programming., (Copyright © 2014 the American Physiological Society.)

Abundant evidence indicates a pivotal role of prostaglandin F2α (PGF2α) in human parturition. Both the fetal and maternal sides of the fetal membranes synthesize PGF2α. In addition to the synthesis of PGF2α from PGH2 by PGF synthase (PGFS), PGF2α can also be converted from PGE2 by carbonyl reductase 1 (CBR1). Here, we showed that there was concurrent increased production of cortisol and PGF2α in association with the elevation of CBR1 in human amnion obtained at term with labor versus term without labor. In cultured primary human amnion fibroblasts, cortisol (0.01-1μM) increased PGF2α production in a concentration-dependent manner, in parallel with elevation of CBR1 levels. Either siRNA-mediated knockdown of glucocorticoid receptor (GR) expression or GR antagonist RU486 attenuated the induction of CBR1 by cortisol. Chromatin immunoprecipitation (ChIP) showed an increased enrichment of both GR and RNA polymerase II to CBR1 promoter. Knockdown of CBR1 expression with siRNA or inhibition of CBR1 activity with rutin decreased both basal and cortisol-stimulated PGF2α production in human amnion fibroblasts. In conclusion, CBR1 may play a critical role in PGF2α synthesis in human amnion fibroblasts, and cortisol promotes the conversion of PGE2 into PGF2α via GR-mediated induction of CBR1 in human amnion fibroblasts. This stimulatory effect of cortisol on CBR1 expression may partly explain the concurrent increases of cortisol and PGF2α in human amnion tissue with labor, and these findings may account for the increased production of PGF2α in the fetal membranes prior to the onset of labor.

Preeclampsia is a pregnancy-specific syndrome, defined by such clinical hallmarks as the onset of maternal hypertension and proteinuria after 20 weeks of gestation. The syndrome is also characterized by impaired blood flow through the utero-placental circulation and relative placental ischemia, which in turn, may generate feto-placental endothelial dysfunction. Endothelial dysfunction in offspring born from preeclamptic pregnancies has been associated with an increased risk of cardiovascular disease, including hypertension, later in life. Interestingly, diminished endothelial function, manifested by low angiogenic capacity, leads to hypertension in animal studies. Recently, we have shown that the adenosine receptor A2A/nitric oxide/vascular endothelial growth factor axis is reduced in human umbilical vein endothelial cells derived from preeclamptic pregnancies, an effect correlated with gestational age at onset of preeclampsia. We and others suggested that impaired vascular function might be associated with high cardiovascular risk in offspring exposed to pregnancy diseases. However, we are not aware of any studies that examine impaired adenosine-mediated angiogenesis as a possible link to hypertension in offspring born from preeclamptic pregnancies. In this review, we present evidence supporting the hypothesis that reduced adenosine-mediated angiogenesis during preeclamptic pregnancies might be associated with development of hypertension in the offspring.

Preeclampsia remains a major problem worldwide for mothers and babies. Despite intensive study, we have not been able to improve the management or early recognition of preeclampsia. At least part of this is because of failure to standardize the approach to studying this complex syndrome. It is possible that within the syndrome there may be different phenotypes with pathogenic pathways that differ between the subtypes. The capacity to recognize and to exploit different subtypes is of obvious importance for prediction, prevention, and treatment. We present a strategy for research to study preeclampsia, which will allow discrimination of such possible subtypes and also allow comparison and perhaps combinations of findings in different studies by standardized data and biosample collection. To make studies relevant to current clinical practice, the definition of preeclampsia can be that currently used and accepted. However, more importantly, sufficient data should be collected to allow other diagnostic criteria to be used and applied retrospectively. To that end, we present what we consider to be the minimum requirements for a data set in a study of preeclampsia that will facilitate comparisons. We also present a comprehensive or optimal data set for in-depth investigation of pathophysiology. As we approach the definition of phenotypes of preeclampsia by clinical and biochemical criteria, adherence to standardized protocols will hasten our understanding of the causes of preeclampsia and development of targeted treatment strategies.

Preterm birth involves the interaction of societal and environmental factors potentially modulating the length of gestation via the epigenome. An established form of epigenetic regulation is DNA methylation where promoter hypermethylation is associated with gene repression. We hypothesized we would find differences in DNA methylation in the myometrium of women with preterm labor of different phenotypes versus normal term labor. Myometrial tissue was obtained at cesarean section at term with or without labor, preterm without labor, idiopathic preterm labor, and twin gestations with labor. Genomic DNA was isolated, and samples in each group were combined and analyzed on a NimbleGen 2.1M human DNA methylation array. Differences in methylation from -8 to +3 kb of transcription start sites of 22 contraction-associated genes were determined. Cytosine methylation was not present in CpG islands of any gene but was present outside of CpG islands in shores and shelves in 19 genes. No differential methylation was found across the tissue groups for six genes (PTGES3L, PTGER2, PTGER4, PTGFRN, ESR2, and GJA1). For 13 genes, differential methylation occurred in several patterns between tissue groups. We find a correlation between hypomethylation and increased mRNA expression of PTGES/mPGES-1, indicating potential functional relevance of methylation, but no such correlation for PTGS2/COX-2, suggesting other regulatory mechanisms for PTGS2 at labor. The majority of differential DNA methylation of myometrial contraction-associated genes with different labor phenotypes occurs outside of CpG islands in gene promoters, suggesting that the entirety of DNA methylation across the genome should be considered.

The pivotal role of glucocorticoids in the initiation of parturition has been very well documented in several domestic mammalian animal species. However the role of glucocorticoids in human parturition remains controversial mainly because of the absence of effect of synthetic glucocorticoids, given to promote fetal organ maturation in pregnant women with threatened preterm delivery, on the length of gestation. This article will review studies of glucocorticoids in human parturition and provide evidence for an important role of glucocorticoids in human parturition as well but a simultaneous high concentration of estrogen within the intrauterine tissues may be necessary for GCs to initiate parturition. The synthetic GCs dexamethasone and betamethasone pass through the placenta intact resulting in potent negative feedback on the fetal HPA axis and diminished production of DHEA from fetal adrenal glands for estrogen synthesis by the placenta. This may negate the effect of systemic administration of GCs on the induction of labor, especially in cases where the myometrium is not yet fully primed by estrogen. Endogenous glucocorticoids are inactivated by the placental 11β-HSD2 thus limiting the negative feedback of maternal cortisol on the fetal HPA axis and allowing the simultaneous rise of cortisol and estrogen levels towards the end of gestation. Therefore, endogenous glucocorticoids, particularly glucocorticoids produced locally in the intrauterine tissues may play an important role in parturition in humans by enhancing prostaglandin production in the fetal membranes and stimulating estrogen and CRH production in the placenta., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Biobanks provide an important repository of samples for research purposes. However, for those samples to reflect the in vivo state, and for experimental reliability and reproducibility, careful attention to collection, processing and storage is essential. This is particularly true for the placenta, which is potentially subjected to stressful conditions during delivery, and sample collection may be delayed owing to routine postpartum inspection by clinical staff. In addition, standardisation of the collection procedure enables samples to be shared among research groups, allowing larger datasets to be established. Here, we provide an evidence-based and experts' review of the factors surrounding collection that may influence data obtained from the human placenta. We outline particular requirements for specific techniques, and propose a protocol for optimal sample collection. We recognise that the relevance of these factors, and of the sample types collected to a particular study will depend on the research questions being addressed. We therefore anticipate that researchers will select from the protocol to meet their needs and resources available. Wherever possible, we encourage researchers to extend their collection to include additional samples that can be shared on an international collaborative basis, with appropriate informed consent, to raise the quality, as well as quantity, of placental research., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In pregnancy fetal growth and development occur in a sexually dimorphic manner. Male and female fetuses respond differently to the intrauterine environment with males disproportionately suffering from perinatal morbidity and mortality. We have demonstrated placental dysfunction and sexually dimorphic responses in pregnancies complicated by severe preeclampsia. Production of cytokines and apoptosis in the male placenta is heightened relative to that of the female placenta. We also find increased expression and stabilization and a sexual dimorphism in expression of the transcription factor HIF-1α, but a defect in binding to the hypoxia response element with corresponding reduced expression of HIF-1α target genes including VEGF and Glut-1. HIF-1α is involved in crosstalk with the redox sensitive transcription factor NFκB in regulation by cytokines, reactive oxygen species and expression of inflammatory genes. We find increased placental expression and DNA binding of NFκB and a sexually dimorphic response suggesting a role for NFκB in placental dysfunction with preeclampsia. Placental mitochondrial complex III activity and complex I and IV expression are reduced and alterations in mitochondrial morphology are found in preeclampsia and are linked to the hypoxamir miR-210. We propose that with severe PE placental HIF-1α is stabilized by excessive ROS, inflammation and relative hypoxia. This increases the expression of miR-210 in the placenta causing repression of mitochondria-associated target genes, potentially leading to mitochondrial and placental dysfunction. This placental dysfunction may lead to a fetal programming effect that results in disease in later life.

Preeclampsia (PE) affects 5-8% of pregnancies and is responsible for 18% of maternal deaths in the US, and for long-term complications in mother and child. PE is an inflammatory state and may influence placental function in a sex-specific manner. We determined if there is a sexual dimorphism in the placental inflammatory and apoptotic responses in preeclamptic pregnancies. Placentas were collected from normotensive and preeclamptic pregnancies with either male or female fetuses (MPE and FPE respectively) after c-section at term with no labor. Expression patterns of markers of inflammation measured by ELISA, as well as hypoxia, apoptosis and angiogenesis markers measured by Western blotting were determined in the placenta. Consistent with previous studies, an increase in inflammation, hypoxia, and apoptotic cell death was observed in PE compared to normotensive pregnancies. Levels of TNFα, IL-6 and IL-8, and HIF-1α were significantly greater, whereas the angiogenic marker VEGF was significantly reduced in MPE vs. FPE. Sexual dimorphism was also observed in the activation of cell death: the number of TUNEL-positive cells, and the expression pro-apoptotic markers PUMA and Bax being higher in MPE vs. FPE. We also found an increase in the levels of protein and DNA-binding activity of NFκB p65 in MPE vs. FPE. In summary, we show here that in preeclamptic pregnancies the placentas of males were associated with significantly higher expression of inflammatory, hypoxia and apoptotic molecules but reduced expression of a pro-angiogenic marker compared to placentas of female fetuses. We propose that the transcription factor NFκB p65 might, at least partially, be involved in sexual dimorphism during PE., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human and animal studies show that suboptimal intrauterine environments lead to fetal programming, predisposing offspring to disease in later life. Maternal obesity has been shown to program offspring for cardiovascular disease (CVD), diabetes, and obesity. MicroRNAs (miRNAs) are small, noncoding RNA molecules that act as key regulators of numerous cellular processes. Compelling evidence links miRNAs to the control of cardiac development and etiology of cardiac pathology; however, little is known about their role in the fetal cardiac response to maternal obesity. Our aim was to sequence and profile the cardiac miRNAs that are dysregulated in the hearts of baboon fetuses born to high fat/high fructose-diet (HFD) fed mothers for comparison with fetal hearts from mothers eating a regular diet. Eighty miRNAs were differentially expressed. Of those, 55 miRNAs were upregulated and 25 downregulated with HFD. Twenty-two miRNAs were mapped to human; 14 of these miRNAs were previously reported to be dysregulated in experimental or human CVD. We used an Ingenuity Pathway Analysis to integrate miRNA profiling and bioinformatics predictions to determine miRNA-regulated processes and genes potentially involved in fetal programming. We found a correlation between miRNA expression and putative gene targets involved in developmental disorders and CVD. Cellular death, growth, and proliferation were the most affected cellular functions in response to maternal obesity. Thus, the current study reveals significant alterations in cardiac miRNA expression in the fetus of obese baboons. The epigenetic modifications caused by adverse prenatal environment may represent one of the mechanisms underlying fetal programming of CVD.

Objective: To determine if change in maternal angiogenic biomarkers between the first and second trimesters predicts pre-eclampsia in low-risk nulliparous women., Design: A nested case-control study of change in maternal plasma soluble Flt-1 (sFlt-1), soluble endoglin (sEng) and placenta growth factor (PlGF). We studied 158 pregnancies complicated by pre-eclampsia and 468 normotensive nonproteinuric controls., Setting: A multicentre study in 16 academic medical centres in the USA., Population: Low-risk nulliparous women., Methods: Luminex assays for PlGF, sFlt-1 and sEng performed on maternal EDTA plasma collected at 9-12, 15-18 and 23-26 weeks of gestation. Rate of change of analyte between first and either early or late second trimester was calculated with and without adjustment for baseline clinical characteristics., Main Outcome Measures: Change in PlGF, sFlt-1 and sEng., Results: Rates of change of PlGF, sEng and sFlt-1 between first and either early or late second trimesters were significantly different in women who developed pre-eclampsia, severe pre-eclampsia or early-onset pre-eclampsia compared with women who remained normotensive. Inclusion of clinical characteristics (race, body mass index and blood pressure at entry) increased sensitivity for detecting severe and particularly early-onset pre-eclampsia but not pre-eclampsia overall. Receiver operating characteristics curves for change from first to early second trimester in sEng, PlGF and sFlt-1 with clinical characteristics had areas under the curve of 0.88, 0.84 and 0.86, respectively, and for early-onset pre-eclampsia with sensitivities of 88% (95% CI 64-99), 77% (95% CI 50-93) and 77% (95% CI 50-93) for 80% specificity, respectively. Similar results were seen in the change from first to late second trimester., Conclusion: Change in angiogenic biomarkers between first and early second trimester combined with clinical characteristics has strong utility for predicting early-onset pre-eclampsia., (© Published 2013 This article is a U.S. Government work and is in the public domain in the USA © 2013 RCOG.)

Objective: To evaluate pregnancy outcomes according to 2009 Institute of Medicine (IOM) gestational weight gain guidelines., Methods: This study is a secondary analysis of a preeclampsia prevention trial among nulliparas carrying singletons. Odds ratios and 95% confidence intervals (adjusted for maternal age, race, smoking, and treatment group) were calculated based on total weight gain below or above the IOM guidelines stratified by prepregnancy body mass index (BMI). The referent group was weight gain within the guidelines., Results: Of 8,293 pregnancies, 9.5% had weight gain below, 17.5% within, and 73% above IOM guidelines. With excess weight gain, all BMI categories had an increased risk of hypertensive disorders; normal weight and overweight women also had increased risk of cesarean delivery and neonatal birth weight at or above the 90 centile but a decreased risk of weight below the 10 centile. There were no consistent associations with insufficient weight gain and adverse outcomes., Conclusion: Excess weight gain was prevalent and associated with an increased risk of hypertensive disorders, cesarean delivery, and large-for-gestational-age neonates.

Maternal nutrient reduction (MNR) during fetal development may predispose offspring to chronic disease later in life. Increased regeneration of active glucocorticoids by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in metabolic tissues is fundamental to the developmental programming of metabolic syndrome, but underlying mechanisms are unknown. Hexose-6-phosphate dehydrogenase (H6PD) generates NADPH, the cofactor for 11β-HSD1 reductase activity. CCAAT/enhancer binding proteins (C/EBPs) and the glucocorticoid receptor (GR) regulate 11β-HSD1 expression. We hypothesize that MNR increases expression of fetal C/EBPs, GR, and H6PD, thereby increasing expression of 11β-HSD1 and reductase activity in fetal liver and adipose tissues. Pregnant MNR baboons ate 70% of what controls ate from 0.16 to 0.9 gestation (term, 184 days). Cortisol levels in maternal and fetal circulations increased in MNR pregnancies at 0.9 gestation. MNR increased expression of 11β-HSD1; H6PD; C/EBPα, -β, -γ; and GR in female but not male perirenal adipose tissue and in male but not female liver at 0.9 gestation. Local cortisol level and its targets PEPCK1 and PPARγ increased correspondingly in adipose and liver tissues. C/EBPα and GR were found to be bound to the 11β-HSD1 promoter. In conclusion, sex- and tissue-specific increases of 11β-HSD1, H6PD, GR, and C/EBPs may contribute to sexual dimorphism in the programming of exaggerated cortisol regeneration in liver and adipose tissues and offsprings' susceptibility to metabolic syndrome.

Problem: Human labour is an inflammatory process with a heavy infiltration of immune cells into the myometrium and cervix induced by local chemokine production. Myometrial cells also express chemokine receptors, but there is little information about their behaviour or function during pregnancy and labour., Method of Study: We studied the behaviour of the receptors (CCR2, CXCR1 and CXCR2) for the CCL2 and CXCL8 in human myometrium, because both have been shown to be important in labour., Results: We found that there was a significant decline in the mRNA expression of all three receptors in the upper segment and a similar trend in the lower segment with the onset of term labour (TL). Chemokine receptor mRNA expression was increased by stretch, reduced by oxytocin and PGF(2α) acting via phospholipase C (PLC). CXCR2 declined with exposure to CXCL8, consistent with the negative relationship observed in labouring myometrial tissue. The mRNA changes were confirmed by western analysis and flow cytometry., Conclusion: These data show that myometrial chemokine receptor expression is reduced with the onset of term labour probably in response to the increased activity of chemokines, oxytocin and PGF(2α) ., (© 2012 John Wiley & Sons A/S.)

Haptoglobin's (Hp) antioxidant and pro-angiogenic properties differ between the 1-1, 2-1, and 2-2 phenotypes. Hp phenotype affects cardiovascular disease risk and treatment response to antioxidant vitamins in some non-pregnant populations. We previously demonstrated that preeclampsia risk was doubled in white Hp 2-1 women, compared to Hp 1-1 women. Our objectives were to determine whether we could reproduce this finding in a larger cohort, and to determine whether Hp phenotype influences lack of efficacy of antioxidant vitamins in preventing preeclampsia and serious complications of pregnancy-associated hypertension (PAH). This is a secondary analysis of a randomized controlled trial in which 10,154 low-risk women received daily vitamin C and E, or placebo, from 9-16 weeks gestation until delivery. Hp phenotype was determined in the study prediction cohort (n = 2,393) and a case-control cohort (703 cases, 1,406 controls). The primary outcome was severe PAH, or mild or severe PAH with elevated liver enzymes, elevated serum creatinine, thrombocytopenia, eclampsia, fetal growth restriction, medically indicated preterm birth or perinatal death. Preeclampsia was a secondary outcome. Odds ratios were estimated by logistic regression. Sampling weights were used to reduce bias from an overrepresentation of women with preeclampsia or the primary outcome. There was no relationship between Hp phenotype and the primary outcome or preeclampsia in Hispanic, white/other or black women. Vitamin supplementation did not reduce the risk of the primary outcome or preeclampsia in women of any phenotype. Supplementation increased preeclampsia risk (odds ratio 3.30; 95% confidence interval 1.61-6.82, p<0.01) in Hispanic Hp 2-2 women. Hp phenotype does not influence preeclampsia risk, or identify a subset of women who may benefit from vitamin C and E supplementation to prevent preeclampsia.

Human fetal membranes express 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which reduces biologically inert cortisone to active cortisol and may provide an extraadrenal source of cortisol mediating fetal development and parturition. The reductase activity of 11β-HSD1 depends on the availability of the cofactor reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) derived from the enzymatic activity of hexose-6-phosphodehydrogenase (H6PD). Based on the feed-forward induction of 11β-HSD1 by glucocorticoids in human fetal membranes, we hypothesize that glucocorticoids simultaneously induce H6PD in the fetal membranes. We found a parallel distribution of H6PD and 11β-HSD1 in the amnion, chorion, and decidua. In cultured human amnion fibroblasts, small interfering RNA-mediated knockdown of H6PD expression significantly attenuated the conversion of cortisone to cortisol. Cortisol (0.01-1 μm) induced H6PD expression in a concentration-dependent manner, which was attenuated by glucocorticoid receptor (GR) antagonist RU486. Cortisol induced the expression of p300, a histone acetyltransferase, whereas C646, an inhibitor of p300, attenuated the induction of H6PD by cortisol. Coimmunoprecipitation revealed GR and p300 in the same nuclear protein complex upon cortisol stimulation. Chromatin immunoprecipitation showed that cortisol increased the binding of p300 and GR to H6PD promoter and the acetylation of histone 3 lysine 9 on the promoters. In conclusion, the induction of H6PD by cortisol requires the participation of GR and p300 as well as the acetylation of H3K9 by p300. This may be a prerequisite for the parallel induction of reductase activity of 11β-HSD1 in human amnion fibroblasts in a feed-forward loop that may influence fetal development and the onset of parturition.

Preeclampsia (PE) affects 5-8% of all pregnancies and is associated with significant maternal and fetal morbidity and mortality. Placental mitochondrial dysfunction has been reported in PE. MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression through mRNA degradation and translational repression. MiR-210 has been previously shown to be upregulated in placentas from pregnancies complicated by PE. We hypothesized that placental mitochondrial dysfunction during PE can be mediated by miR-210. Placentas were collected at term from normotensive pregnancies (CTRL) and those complicated by severe PE (n = 6 each) following c-section (no labor). Villous tissue from PE showed significantly increased levels of HIF-1α compared to CTRL with no change in corresponding mRNA expression but with reduced DNA-binding activity. Mitochondrial complex III was significantly decreased in PE along with significantly reduced protein expression in complex I and IV during PE. Among the four miRNAs tested, miR-210 showed significant up regulation in PE and significant downregulation of its target, ISCU mRNA. To understand the role of miR-210 in PE, loss- and gain-of-function studies were performed using primary trophoblasts. Trophoblasts were transfected with miR-210 inhibitor or pre-miR-210 and mitochondrial function was measured using Seahorse Extracellular Flux Analyzer. Cells transfected with pre-miR-210 showed significant reduction in oxygen consumption. In contrast, transfection of trophoblast with AntagomiR-210 was sufficient to prevent the DFO-mediated respiratory deficiency. These data collectively suggest that miR-210 overexpression during PE could be responsible for placental mitochondria dysfunction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: The underlying pathophysiology of preeclampsia is thought to be abnormal trophoblast invasion of the spiral arteries leading to maldevelopment of uteroplacental perfusion. We estimated whether uterine artery Doppler measurements made in the early second trimester would predict the subsequent development of preeclampsia., Methods: Uterine artery Doppler measurements before 21 weeks of gestation (median 16.6 weeks) were correlated with subsequent development of preeclampsia in a cohort of 2,188 low-risk nulliparous women in a randomized control trial of antioxidant supplementation for prevention of preeclampsia. Preeclampsia developed in 165 (7.5%) women., Results: Development of preeclampsia overall was associated with increased resistance index, pulsatility index, a pulsatility index or resistance index multiple of the median at or above the 75th percentile but not the presence of a notch or a bilateral notch before 21 weeks of gestation. The sensitivity was 43% (95% confidence interval [CI] 35-51) and specificity 67% (95% CI 65-69) for prediction of preeclampsia overall. The presence of a notch or bilateral notch, resistance index, and pulsatility index multiple of the median was significantly associated with early onset (before 34 weeks of gestation) compared with late onset or no preeclampsia (odds ratio [OR] 6.9, 95% CI 2.3-20.9; sensitivity 78%, 95% CI 52-94; specificity 66%, 95% CI 64-68). The presence of a notch or resistance index multiple of the median at or above the 75th percentile increased the odds of developing severe compared with mild or no preeclampsia (OR 2.2, 95% CI 1.4-3.7; sensitivity 53%, 95% CI 40-65; specificity 66%, 95% CI 64-68)., Conclusion: Our data show poor sensitivity of second-trimester Doppler ultrasound measurements for prediction of preeclampsia overall in a well-characterized, low-risk, nulliparous population. The technique has utility in identifying poor trophoblast invasion of spiral arteries of a magnitude that severely compromises uteroplacental blood flow and gives early-onset disease., Level of Evidence: II.

Background: In utero exposure of the fetus to a stressor can lead to disease in later life. Epigenetic mechanisms are likely mediators of later-life expression of early-life events., Objectives: We examined the current state of understanding of later-life diseases resulting from early-life exposures in order to identify in utero and postnatal indicators of later-life diseases, develop an agenda for future research, and consider the risk assessment implications of this emerging knowledge., Methods: This review was developed based on our participation in a National Research Council workshop titled "Use of in Utero and Postnatal Indicators to Predict Health Outcomes Later in Life: State of the Science and Research Recommendations." We used a case study approach to highlight the later-life consequences of early-life malnutrition and arsenic exposure., Discussion: The environmental sensitivity of the epigenome is viewed as an adaptive mechanism by which the developing organism adjusts its metabolic and homeostatic systems to suit the anticipated extrauterine environment. Inappropriate adaptation may produce a mismatch resulting in subsequent increased susceptibility to disease. A nutritional mismatch between the prenatal and postnatal environments, or early-life obesogen exposure, may explain at least some of the recent rapid increases in the rates of obesity, type 2 diabetes, and cardiovascular diseases. Early-life arsenic exposure is also associated with later-life diseases, including cardiovascular disease and cancer., Conclusions: With mounting evidence connecting early-life exposures and later-life disease, new strategies are needed to incorporate this emerging knowledge into health protective practices.

Compelling evidence indicates a crucial role of prostaglandin F2α (PGF2α) in parturition. Both the maternal and fetal sides of the fetal membranes synthesize PGF2α, which exerts effects via the prostaglandin F2α receptor (FP) that is coupled to the activation of protein kinase C (PKC). Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step of the inducible synthesis of prostaglandin. Although activation of PKC is known to induce COX-2 expression, it is not clear whether PGF2α can induce COX-2 via FP receptor-coupled PKC activation. COX-2 promoter carries a cAMP-response element (CRE) and phosphorylation of CRE binding protein 1 (CREB1) is associated with COX-2 expression in human amnion fibroblasts. We demonstrated that human amnion fibroblasts produced PGF2α and expressed FP receptor. PGF2α increased COX-2 expression and CREB1 phosphorylation, which could be blocked by either the FP receptor antagonist AL8810 or PKC inhibitor Ro31-7549. The PKC activator, phorbol-12-myristate-13-acetate (PMA), could mimic the induction of COX-2 and CREB1 phosphorylation. The induction of COX-2 by PGF2α and PMA could be attenuated by the small interfering RNA-mediated knockdown of CREB1 expression or overexpressing dominant-negative CREB1. A chromatin immunoprecipitation assay showed that the binding of CREB1 to the COX-2 promoter was increased by PGF2α and PMA in amnion fibroblasts. In conclusion, we provide evidence that PGF2α induces COX-2 expression via the FP receptor and phosphorylates CREB1 by PKC, thus increasing CREB1 binding to the COX-2 promoter and the expression of COX-2 in human amnion fibroblasts. This feed-forward loop may be crucial for the production of prostaglandins in the fetal membranes prior to the onset of labor.

Objective: To identify clinical characteristics and biochemical markers in first-trimester samples that would possibly predict the subsequent development of preeclampsia., Methods: We conducted a multicenter observational study in 2,434 nulliparous women at low risk to identify biomarkers that possibly predict preeclampsia. Clinical history, complete blood count, and biochemical markers were assessed in the first trimester. The trophoblast and angiogenesis markers ADAM-12, pregnancy-associated plasma protein-A, placental protein 13, placental growth factor, soluble fms-like tyrosine kinase-1, and endoglin were measured in a case-control subset of 174 women with preeclampsia and 509 women in the control group., Results: Univariable analysis revealed maternal age, race, marital status, years of education, source of medical payment, prenatal caregiver, body mass index (BMI, calculated as weight (kg)/[height (m)]), and systolic blood pressure at enrollment were significantly associated with preeclampsia. Mean platelet volume was greater at enrollment in women who later had development of preeclampsia (median 9.4 compared with 9.0 femtoliter (fl); P=.02). First-trimester concentrations (multiples of the median) of ADAM-12 (1.14 compared with 1.04; P=.003), pregnancy-associated plasma protein-A (0.94 compared with 0.98; P=.04), and placental growth factor (0.83 compared with 1.04; P<.001) were significantly different in women who had development of preeclampsia compared with women in the control group. The optimal multivariable model included African American race, systolic blood pressure, BMI, education level, ADAM-12, pregnancy-associated plasma protein-A, and placental growth factor, and yielded an area under the curve of 0.73 (95% confidence interval 0.69-0.77) and a sensitivity of 46.1% (95% confidence interval 38.3-54.0) for 80% specificity., Conclusion: A multivariable analysis of clinical data and biochemical markers in the first trimester did not identify a model that had clinical utility for predicting preeclampsia in a nulliparous population at low risk., Level of Evidence: II.

Objective: To estimate whether there is an association between excessive early gestational weight gain and the development of gestational diabetes mellitus (GDM) and excessive fetal growth., Methods: This is a secondary analysis of a randomized controlled trial of vitamins C and E in nulliparous low-risk women. Maternal weight gain from prepregnancy (self-reported) to 15-18 weeks of gestation was measured, and expected gestational weight gain was determined using the Institute of Medicine 2009 guidelines for each prepregnancy body mass index category. Excessive early gestational weight gain was defined as gestational weight gain greater than the upper range of the Institute of Medicine guidelines. Rates of GDM, birth weight greater than 4,000 g, and large for gestational age (LGA, birth weight 90 percentile or higher) were calculated and compared between women with excessive early gestational weight gain and early nonexcessive gestational weight gain (within or below Institute of Medicine guidelines)., Results: A total of 7,985 women were studied. Excessive early gestational weight gain occurred in 47.5% of women. Ninety-three percent of women with excessive early gestational weight gain had total gestational weight gain greater than Institute of Medicine guidelines. In contrast, only 55% of women with nonexcessive early gestational weight gain had total gestational weight gain greater than Institute of Medicine guidelines (P<.001). Rates of GDM, LGA, and birth weight greater than 4,000 g were higher in women with excessive early gestational weight gain., Conclusion: In our population, excessive early gestational weight gain occurred in 93% of women who had total gestational weight gain greater than the Institute of Medicine guidelines. In low-risk nulliparous women, excessive early gestational weight gain is associated with the development of GDM and excessive fetal growth., Level of Evidence: II.

Pregnancy is a state of oxidative stress, which becomes exaggerated under pathological conditions, such as preeclampsia, IUGR, diabetes and obesity, where placental mitochondrial dysfunction is observed. The majority of investigations utilize isolated mitochondria when measuring mitochondrial activity in placenta. However, this does not provide a complete physiological readout of mitochondrial function. This technical note describes a method to measure respiratory function in intact primary syncytiotrophoblast from human term placenta., (Copyright © 2012. Published by Elsevier Ltd.)

Background: Preterm birth is a major societal and economic problem accounting for 80 to 90% of neonatal morbidity and mortality worldwide. It is recognized as a complex multifactorial condition comprising several distinct clinical phenotypes with different underlying etiologies. As animal models are expensive and fail to mimic the biology of spontaneous preterm birth in humans, understanding the pathophysiology requires detailed clinical studies. Meta-analyses and clinical translation of data, however, are limited by heterogeneity of study design and size, publication and reporting biases, definition of patient groups, and a lack of standard universal definitions. This article provides a harmonized open-source template for designing clinical studies addressing preterm birth., Methods: Recommendations are made for clinical definitions, choice and assignment to preterm birth phenotypes, selection of enriched populations and control pregnancies, and potential confounding factors. In addition, recommendations are made for study design, sample size and power calculations, the minimal data sets needed for any study of preterm birth, and the optimal data set of an ideal study., Results: Recommended patient phenotypes are infection, uterine overdistension, hemorrhage, stress (either maternal or fetal), and idiopathic. Confounding factors include medical conditions, obesity, antenatal glucocorticoids, multifetal pregnancies, and fetal sex. Guidelines regarding study design, sample size, and clinical data acquisition are provided to serve as a universal template for preterm birth studies., Conclusions: Adoption of a harmonized template will allow generation of protocols and studies with a basic degree of compatibility and will allow data to be compared, and samples and data sets to be combined for meaningful meta-analyses.

One of the dominant effects of glucocorticoids in triggering parturition in certain animal species is to drive the placental conversion of progesterone to estrogen. However, in the human placenta, estrogen is formed using dehydroepiandrosterone from the fetal adrenal glands rather than progesterone as precursor. Although aromatization of dehydroepiandrosterone is crucial in estrogen synthesis in human placenta, it is not known whether glucocorticoids affect aromatase expression. Human term placental syncytiotrophoblasts were used to examine the effect of cortisol on aromatase expression. The signaling pathway and transcription factors involved were identified in this study. Results showed that cortisol induced aromatase expression in a concentration-dependent manner, which was mediated indirectly by glucocorticoid receptor and required the participation of other proteins. The induction of aromatase by cortisol could be blocked by either specificity protein 1 (Sp1) antagonist mithramycin or knockdown of Sp1 expression. The induction of aromatase and Sp1 by cortisol could be prevented by inhibitors of the cAMP pathway, whereas activators of the cAMP pathway induced Sp1 and aromatase expression as well as Sp1 binding to aromatase promoter. Concomitantly, cortisol treatment and activation of the cAMP pathway led to increased acetylation and decreased methylation of histone 3 at the aromatase promoter. In conclusion, cortisol stimulates aromatase expression through the cAMP/Sp1 pathway in human placental syncytiotrophoblasts. These findings reveal a novel role of cortisol in increasing the local level of estrogen within the placenta that would help transform the myometrium to a contractile state, thereby contributing to a cascade of events leading to human parturition.

Both human preterm labor (PTL) and term labor are consistently associated with a chemokine-induced inflammatory infiltration of the myometrium. However, what regulates myometrial chemokine expression and whether the increase in expression precedes the onset of labor, and so may have a role in its causation, or occurs after, and is simply a consequence of labor, is uncertain. Therefore, we assessed 1) chemokine expression in nonlaboring and laboring myometrial samples obtained at and before term and 2) the factors that regulate myometrial chemokine expression. We found that term labor was characterized by an increase in CXCL8 and CCL2 in both upper and lower segments, whereas PTL was associated with a distinct pattern of chemokine expression, with increases in CCL5, CXCL5, and CCL20 in the lower segment myometrium only. Further, we found that chemokine expression in myometrial cell cultures was increased by stretch and inflammatory cytokines and reduced by prostglandins and oxytocin and that the primary mediator of stretch and cytokine effects was nuclear factor κB (NF-κB) and to a lesser extent MAPK. These data show that PTL appears to be associated with a distinct pattern of chemokine expression, that stretch and cytokines both drive myometrial chemokine expression primarily via activation of NF-κB. These data suggest that the modulation of NF-κB activity may be of potential benefit in the management of PTL.

Previously, we found that myometrial tumor necrosis factor-α (TNF-α) messenger RNA (mRNA) expression did not increase with preterm or term labor. To further investigate the role of TNF-α in human labor, we studied TNF-α receptor (TNFR1A and B) expression, regulation, and associated intracellular signaling pathways in human myometrial samples obtained both before and after the onset of labor and in primary cultures of uterine smooth muscle cells (USMCs). We found that the mRNA expression of both receptors increased with advancing gestation and labor and protein levels of TNFR1B were significantly higher in term laboring myometrial samples than in nonlabor controls. Tumor necrosis factor- treatment of USMCs activated all mitogen-activated protein kinase (MAPK) subtypes and nuclear factor κ-B (NF-κB). The TNF-α induced increases in the expression of TNFR1B and prostaglandin H synthase type 2 were reduced by inhibitors of NF-κB and MAPKs, respectively. The TNF-α induced increase in interleukin 8 (IL-8) appeared to be independent of MAPK and NF-κB pathway. These data suggest that the uterus may become more sensitive to the action of TNF-α with advancing gestation and labor and that TNF-α acts via MAPK and NF-κB to promote labor-associated gene expression.

Objective: We hypothesized that fetal folate serum concentrations are lower and placental folate transport is impaired in pregnancies of obese women., Study Design: Umbilical vein serum and placental tissue were collected from normal weight and obese pregnant women at term. Cellular localization (immunohistochemistry) of folate receptor-α (FR-α), proton coupled folate transporter (PCFT), and reduced folate carrier (RFC) was established. Protein expression (Western blot) and transporter activity (isotope labeled methyltetrahydrofolate) were determined in syncytiotrophoblast microvillus membranes (MVM)., Results: Fetal folate concentrations were similar in obese women as compared with normal weight women. Protein expression of FR-α in microvillus membranes was increased (+173%), in RFC was decreased (-41%), and in PCFT was unchanged. However, activity of FR-α, PCFT, and RFC was unaltered in obesity., Conclusion: Fetal serum folate concentrations and placental folate transport activity are not altered in obesity at term, which suggests that limited availability of folate does not contribute to abnormal gene methylation and developmental programming., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Context: Overexposure of the fetus to glucocorticoids early in gestation is detrimental to fetal development. Glucocorticoid concentrations in the fetal circulation are kept low by 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2, encoded by HSD11B2) in the placental syncytiotrophoblasts. However, cytotrophoblasts, the progenitors of syncytiotrophoblasts, express low levels of 11β-HSD2. Here we studied the molecular mechanisms underlying 11β-HSD2 induction upon syncytialization., Methods: Freshly isolated human term placental cytotrophoblasts and in vitro differentiated syncytiotrophoblasts were examined to determine the methylation status of HSD11B2 promoter. The transcription factor responsible for 11β-HSD2 induction was identified by observing its expression upon syncytialization, the effect of its attenuation, and its binding to the HSD11B2 promoter., Results: 11β-HSD2 expression was markedly increased upon syncytialization in vitro. No methylation differences of HSD11B2 promoter were found between cytotrophoblasts and syncytiotrophoblasts. Expression of the transcription factor Sp1 was markedly induced during syncytialization and further increased by activation of the cAMP pathway, which correlated with 11β-HSD2 expression. Importantly, small interfering RNA-mediated knockdown of Sp1 expression or inhibition of Sp1 activity with mithramycin A markedly attenuated not only basal but also cAMP pathway-stimulated expression of 11β-HSD2 in the syncytiotrophoblasts. Stimulation of the cAMP pathway also increased the binding of Sp1 and RNA polymerase II to HSD11B promoter in syncytiotrophoblasts. Concomitantly, acetylation at histone H3K9 was increased whereas methylation at histone H3K9 was decreased., Conclusions: 11β-HSD2 induction upon syncytialization is at least in part due to the increased expression of Sp1 upon activation of the cAMP pathway rather than the differential methylation of the HSD11B2 promoter.

Objective: The purpose of this study was to determine whether mid-trimester insulin resistance is associated with subsequent preeclampsia., Study Design: This was a secondary analysis of 10,154 nulliparous women who received vitamin C and E or placebo daily from 9-16 weeks gestation until delivery. Of these, 1187 women had fasting plasma glucose and insulin tested between 22 and 26 weeks gestation. Insulin resistance was calculated by the homeostasis model assessment of insulin resistance (HOMA-IR) and the quantitative insulin sensitivity check index., Results: Obese women were twice as likely to have a HOMA-IR result of ≥75th percentile. Hispanic and African American women had a higher percentage at ≥75th percentile for HOMA-IR than white women (42.2%, 27.2%, and 16.9%, respectively; P < .001). A HOMA-IR result of ≥75th percentile was higher among the 85 nulliparous women who subsequently had preeclampsia, compared with women who remained normotensive (40.5% vs 24.8%; adjusted odds ratio, 1.9; 95% confidence interval, 1.1-3.2). Quantitative insulin sensitivity check index results were similar to the HOMA-IR results., Conclusion: Midtrimester maternal insulin resistance is associated with subsequent preeclampsia., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Objective: To estimate whether maternally administered vitamins C and E lower the risk of spontaneous preterm birth., Methods: This is a secondary analysis of a randomized, double-masked, placebo-controlled trial in nulliparous women at low-risk administered 1,000 mg vitamin C and 400 international units vitamin E or placebo daily from 9 to 16 weeks of gestation until delivery. Outcomes include preterm birth attributable to premature rupture of membranes (PROM) and total spontaneous preterm births (spontaneous preterm birth attributable to PROM or spontaneous labor)., Results: Of the 10,154 women randomized, outcome data were available for 9,968 (4,992 vitamin group and 4,976 placebo group). A total of 1,038 women (10.4%) delivered preterm, of whom 698 (7.0%) had spontaneous preterm birth. A spontaneous preterm birth occurred in 356 women (7.1%) assigned to daily vitamin C and E supplementation and in 342 (6.9%) assigned to placebo. There were 253 women (2.5%) who delivered after preterm PROM and 445 (4.5%) after a spontaneous preterm labor. In women supplemented with vitamins C and E, births attributed to preterm PROM were similar at less than 37 and 35 weeks of gestation, but births were less frequent before 32 weeks of gestation (0.3% compared with 0.6%, adjusted odds ratio 0.3-0.9). However, total spontaneous preterm births across gestation in women supplemented with vitamins C and E or a placebo were similar., Conclusion: Maternal supplementation with vitamins C and E beginning at 9 to 16 weeks of gestation in nulliparous women at low risk did not reduce spontaneous preterm births., Clinical Trial Registration: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00135707., Level of Evidence: I.

Unlabelled: HYPOTHESIS AND STUDY OBJECTIVES: We hypothesized that maternal obesity is associated with increased placental amino acid transport and hyperleptinemia. Our objectives were to study placental amino acid transport and the effect of leptin on placental amino acid transport in vitro in the setting of maternal obesity., Materials and Methods: Seven lean, BMI at entry 22.4, and seven obese, BMI at entry 31.5 (p < 0.001), pregnant women were studied at 39 weeks. We measured baseline and leptin-stimulated placental system A sodium-dependent neutral amino acid transporter (SNAT) activity, placental immunoreactive protein expression of SNAT, leptin and leptin receptor, and maternal and fetal plasma leptin concentrations, with significance set at p

Cytosolic phospholipase A (cPLA(2alpha)) catalyzes the formation of arachidonic acid in prostaglandin synthesis. In contrast to the well-described down-regulation of cPLA(2alpha), up-regulation of cPLA(2alpha) by glucocorticoids has been reported in human amnion fibroblasts, which may play a key role in parturition. The mechanisms underlying this paradoxical induction of cPLA(2alpha) by glucocorticoids remain largely unknown. Using cultured human amnion fibroblasts, we found that the induction of cPLA(2alpha) by cortisol required ongoing transcription and synthesis of at least one other protein. The induction of cPLA(2alpha) by cortisol was abolished by mutagenesis of a glucocorticoid response element (GRE) in the promoter. The same GRE was found mediating the classical inhibition of cPLA(2alpha) expression by cortisol in human fetal lung fibroblasts (HFL-1). Cortisol increased Galpha(s) expression in amnion fibroblasts but not in HFL-1 cells. Inhibition of Galpha(s) with NF449 attenuated the phosphorylation of cAMP response element-binding protein-1 (CREB-1) and the induction of cPLA(2alpha) by cortisol in amnion fibroblasts. Both glucocorticoid receptor (GR) and CREB-1 were found bound to the GRE upon cortisol stimulation of amnion fibroblasts. The induction of cPLA(2alpha) by cortisol was blocked by GR antagonist RU486 or protein kinase A inhibitor H89 or dominant-negative CREB-1. In conclusion, cortisol activates the cAMP/protein kinase A/CREB-1 pathway via Galpha(s) induction, and the phosphorylated CREB-1 interacts with GR at the GRE to promote cPLA(2alpha) expression in amnion fibroblasts.

Background: Oxidative stress has been proposed as a mechanism linking the poor placental perfusion characteristic of preeclampsia with the clinical manifestations of the disorder. We assessed the effects of antioxidant supplementation with vitamins C and E, initiated early in pregnancy, on the risk of serious adverse maternal, fetal, and neonatal outcomes related to pregnancy-associated hypertension., Methods: We conducted a multicenter, randomized, double-blind trial involving nulliparous women who were at low risk for preeclampsia. Women were randomly assigned to begin daily supplementation with 1000 mg of vitamin C and 400 IU of vitamin E or matching placebo between the 9th and 16th weeks of pregnancy. The primary outcome was severe pregnancy-associated hypertension alone or severe or mild hypertension with elevated liver-enzyme levels, thrombocytopenia, elevated serum creatinine levels, eclamptic seizure, medically indicated preterm birth, fetal-growth restriction, or perinatal death., Results: A total of 10,154 women underwent randomization. The two groups were similar with respect to baseline characteristics and adherence to the study drug. Outcome data were available for 9969 women. There was no significant difference between the vitamin and placebo groups in the rates of the primary outcome (6.1% and 5.7%, respectively; relative risk in the vitamin group, 1.07; 95% confidence interval [CI], 0.91 to 1.25) or in the rates of preeclampsia (7.2% and 6.7%, respectively; relative risk, 1.07; 95% CI, 0.93 to 1.24). Rates of adverse perinatal outcomes did not differ significantly between the groups., Conclusions: Vitamin C and E supplementation initiated in the 9th to 16th week of pregnancy in an unselected cohort of low-risk, nulliparous women did not reduce the rate of adverse maternal or perinatal outcomes related to pregnancy-associated hypertension (ClinicalTrials.gov number, NCT00135707)., (2010 Massachusetts Medical Society)

The placenta regulates fetal growth and development via transport of nutrients and gases, and synthesis and secretion of steroid and peptide hormones. These functions are determined by vascular development and blood flow and by growth and differentiation of the trophoblast, which contains receptors, transporters and enzymes. The placenta generates reactive oxygen species which may contribute to the oxidative stress seen even in normal pregnancy but this is increased in pregnancies complicated by preeclampsia, IUGR and pregestational diabetes where oxidative and nitrative stress have been clearly documented. Nitrative stress is the covalent modification of proteins and DNA by peroxynitrite formed by the interaction of superoxide and nitric oxide. We have demonstrated nitrative stress by localizing nitrotyrosine residues in these placentas and found increased expression of NADPH oxidase (NOX) enzyme isoforms 1 and 5 as a potential source of superoxide generation. The presence of nitrative stress was associated with diminished vascular reactivity of the fetal placental circulation, a situation that could be reproduced by treatment with peroxynitrite in vitro. We find many nitrated proteins in the placenta, including p38 MAP kinase which has a role in development of the villous vasculature. Nitration of p38 MAPK was increased in the preeclamptic placenta and associated with loss of catalytic activity. We hypothesize that nitration of proteins in the placenta including receptors, transporters, enzymes and structural proteins can alter protein and placental function and this influences fetal growth and development. Increasing nitrative stress but a decrease in oxidative stress, measured as protein carbonylation, is found in the placenta with increasing BMI. Formation of peroxynitrite may then consume superoxide, decreasing nitrative stress. As protein carbonylation is a covalent modification at Lys, Arg, Pro and Thr residues the switch from carbonylation to nitration at tyrosine residues may alter protein function and hence placental function., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The renin-angiotensin system is upregulated in pregnant women and may play a role in myometrial hypertrophy during pregnancy. We examined whether angiotensin II could induce myometrial protein synthesis as determined by (3)H-leucine incorporation in an immortalized human myometrial smooth muscle cell line (ULTR cells). The effects of angiotensin II were mediated by NADPH oxidase because diphenylene iodonium abolished angiotensin II-induced protein synthesis. We investigated gene expression and cellular localization of NADPH oxidase isoforms in ULTR cells and confirmed expression of NOX1, NOX4, and NOX5 in myometrial tissue. Angiotensin II induced a cellular redistribution and upregulation of NOX5 protein without altering NOX1 and NOX4 expression. It seems the effect of angiotensin II relies on the type 1 receptor (AT1), because losartan significantly blocked angiotensin II-induced increase in (3)H-leucine incorporation. We conclude that NADPH oxidase mediates angiotensin II-stimulated protein synthesis downstream of AT1 in myometrium smooth muscle cells.

Prostaglandin E2 (PGE2) may regulate uterine activation and cervical ripening for labor through specific contractile and relaxatory receptors (EP1-4). The aim of this study was to determine the expression of PGE2 receptor isoforms in pregnant rat cervix during RU486-induced labor and progesterone supplementation to delay labor. Localization and expression of cervical PGE2 receptors were evaluated, and quantitative real-time polymerase chain reaction (PCR) for EP1-4 was performed. EP1-4 were found in both cervical epithelium and smooth muscle. RU486 treatment increased EP2 and EP4 messenger RNA (mRNA) and protein expression. Progesterone treatment had no effect on EP2 and EP4 mRNA expression but decreased EP4 protein. Hormonal manipulation resulted in differences in cellular localization of EP1 and EP3 in cervical epithelial cells, suggesting a specific role in that cell. Progesterone differentially regulates the expression of PGE2 receptor isoforms in the cervix. Elucidating the regulation of PGE2 receptors may facilitate improved approaches to the prevention and treatment of preterm labor.

The fetal membranes fulfill several functions during pregnancy. In addition to containing the products of conception and amniotic fluid, they presumably have barrier functions and fulfill paracrine signaling functions between the maternal (decidual) and fetal compartments. As the membranes are in an ideal place to receive both maternal and fetal signals and transmit signals to uterine myometrium, there has been a specific focus on the role of membranes in the initiation and maintenance of parturition. In this review, we summarize the data obtained in our laboratories as well as the data reported in the literature particularly with regard to the synthesis of steroids and prostaglandins in the fetal membranes, in signaling fetal maturation and in parturition. The fetal membranes are a major site both of prostaglandin synthesis and of prostaglandin metabolism. In addition, the abundant expression of 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1), which converts biologically inactive cortisone into active cortisol, in the fetal membranes may provide an extra-adrenal source of glucocorticoids for the fetal compartment during gestation. Accumulating evidence indicates that a positive feedback loop involving glucocorticoids, proinflammatory cytokines, prostaglandins, surfactant protein-A (SP-A) and 11beta-HSD1 is formed locally in human fetal membranes towards term or in preterm labor. This positive feedback loop would produce abundant biologically active glucocorticoids and prostaglandins in the fetal membranes or amniotic fluid, which would ultimately promote fetal organ maturation and initiate parturition.

Context: Human amnion fibroblasts produce abundant prostaglandins toward the end of gestation, which is believed to be one of the major events leading to parturition. Glucocorticoids have been shown to up-regulate cyclooxygenase-2 (COX-2) expression, the crucial enzyme catalyzing prostaglandin synthesis, in human amnion fibroblasts. Although a major propregnancy hormone, the effect of progesterone and the associated progesterone receptor subtypes in the regulation of both basal and glucocorticoid-induced COX-2 expression in human amnion fibroblasts have not been resolved., Methods and Results: Cultured human amnion fibroblasts prepared from the fetal membranes at term pregnancy without labor mainly expressed the progesterone receptor A form (PRA). Inhibition of endogenous progesterone production with trilostane or knockdown of PRA expression with small interfering RNA significantly enhanced the glucocorticoid receptor (GR)-mediated COX-2 induction by cortisol, whereas overexpression of PRA attenuated the induction by cortisol. Co-immunoprecipitation assay revealed PRA in the GR protein complex. Although exogenous progesterone did not alter COX-2 expression under basal conditions, it attenuated cortisol-induced COX-2 expression at concentrations about 10- to 50-fold higher, which might be achieved by competition with cortisol for GR., Conclusions: We demonstrated in this study that endogenous progesterone might counteract the induction of prostaglandin synthesis by cortisol via PRA transdominant repression of GR function, whereas high levels of progesterone might further inhibit the induction by cortisol via competitive binding to GR in human amnion fibroblasts. These inhibitory actions of progesterone and PRA on glucocorticoids and GR may partly explain the inconsistent effects of glucocorticoids on parturition in humans.

Overexpression of human telomerase reverse transcriptase (hTERT) has facilitated establishing in vitro model systems for biological research. The plasmid containing hTERT gene was stably transfected into ULTR cells, a retroviral transformed human uterine leiomyomatous smooth-muscle cell line. Cells that express hTERT, termed as ULTR-hT, shared the morphological characteristics of the parental proliferative ULTR cells. They expressed a set of smooth-muscle-specific genes and had increased proliferation rate and prolonged lifespan. Quantitative real-time polymerase chain reaction (PCR) analysis revealed a correlation of proliferation rates of ULTR-hT clonal cells with the level of hTERT expression. ULTRhT cells also preserved expression of estrogen, progesterone, and oxytocin receptor genes, confirming a myometrial phenotype. Expression of angiotensin II receptors and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoforms were also preserved. Our finding suggests that ULTR-hT cells can be a useful in vitro model for studying human myometrium differentiation both in pregnancy and pathological growth.

Human amnion fibroblasts produce abundant prostaglandins toward the end of gestation, which is one of the major events leading to parturition. In marked contrast to its well-described antiinflammatory effect, glucocorticoids have been shown to up-regulate cyclooxygenase-2 (COX-2) expression in human amnion fibroblasts. The mechanisms underlying this paradoxical induction of COX-2 by glucocorticoids have not been resolved. Using cultured human amnion fibroblasts, we found that the induction of COX-2 mRNA expression by cortisol was a glucocorticoid receptor (GR)-dependent process requiring ongoing transcription. Upon transfection of a COX-2 promoter-driven reporter gene into the amnion fibroblasts, cortisol stimulated the COX-2 promoter activity. This was abolished by mutagenesis of a cAMP response element (CRE) at -53 to approximately -59bp as well as by cotransfection of a plasmid expressing dominant-negative CRE-binding protein (CREB). The phosphorylation level of CREB-1 was significantly increased by cortisol treatment of the amnion fibroblasts, whereas the effect was attenuated either by the protein kinase A inhibitor H89 or the p38 -MAPK inhibitor SB203580. The induction of the COX-2 promoter activity and the phosphorylation of CREB-1 were also blocked by the GR antagonist RU486. Chromatin immunoprecipitation (ChIP) assay revealed that the binding of CREB-1 to the CRE of the COX-2 promoter was increased by cortisol treatment of the amnion fibroblasts. In conclusion, cortisol, via binding to GR, stimulated COX-2 expression by increasing phosphorylated CREB-1 binding to the CRE of the COX-2 gene. Cortisol may phosphorylate CREB-1 by activating either protein kinase A or p38-MAPK in the amnion fibroblasts.

Intrauterine growth restriction (IUGR) and fetal overgrowth occur in 15% of all pregnancies and lead to the delivery of a baby with an abnormally low or high birth weight, respectively. Both these conditions of pathological fetal growth increase the risk for perinatal complications and predispose the baby for the development of cardiovascular disease and diabetes in childhood and later in life. Fetal growth is closely related to the capacity of the placenta to transport nutrients and ions, which is dependent of the expression and activity of specific transporter proteins in the plasma membrane of the syncytiotrophoblast, the transporting epithelium of the human placenta. In human IUGR, some trophoblast nutrient and ion transporters are down regulated, whereas fetal overgrowth is associated with an up-regulation of transporters for amino acids and glucose in the placental barrier. Experimental studies have provided evidence to suggest that these changes in placental transport capacity constitute a direct cause of altered fetal growth. Therefore, regulation of placental nutrient transporters play a critical role in determining fetal growth and development, as well as the future health of the baby. This review is focused on the human and (i) summarizes the evidence that changes in the activity and expression of trophoblast nutrient and ion transporters play a central role in determining fetal growth, (ii) discusses the molecular mechanisms regulating trophoblast transporters, and (iii) highlights the implications of these findings for the development of pregnancy complications and fetal programming of cardiovascular and metabolic disease.

Objective: The aim of this study was to identify changes in protein expression in normal pregnancy compared with preterm labor by using 3 proteomic methods., Study Design: Serum was collected from 25 nonpregnant (n = 5) and pregnant women at 24-40 weeks' gestation (n = 20) who had preterm labor resulting in preterm delivery (n = 5), preterm labor with term delivery (n = 5), term labor resulting in delivery (n = 5), or at term with contractions (n = 5). Undepleted serum was used for surface-enhanced laser desorption ionization and immune-depleted serum for matrix-assisted laser desorption ionization and 2-dimensional electrophoresis., Results: Surface-enhanced laser desorption ionization identified significantly different peaks between preterm labor resulting in preterm delivery vs term labor resulting in delivery and preterm labor resulting in preterm delivery vs preterm labor with term delivery using 4 surfaces. In preterm labor resulting in preterm delivery vs preterm labor with term delivery, a peak of 7783.2 m/z was significantly up-regulated and at 3164 m/z down-regulated on 3 surfaces. By using 2-dimensional electrophoresis, protein 5364 was significantly different between preterm labor resulting in preterm delivery and term labor resulting in delivery. In preterm labor resulting in preterm delivery, 6 proteins showed decreasing trend and 1 showed increasing trend vs preterm labor with term delivery. Matrix-assisted laser desorption ionization showed a striking difference at 55,000 m/z between preterm labor resulting in preterm delivery and term labor resulting in delivery., Conclusion: Surface-enhanced laser desorption ionization identified 2 proteins fulfilling the criteria of putative biomarkers. Biomarker identification may aid in identifying women with preterm labor who will deliver preterm.

The fetus has a high requirement for cholesterol and synthesizes cholesterol at elevated rates. Recent studies suggest that fetal cholesterol also can be obtained from exogenous sources. The purpose of the current study was to examine the transport of maternal cholesterol to the fetus and determine the mechanism responsible for any cholesterol-driven changes in transport. Studies were completed in pregnant hamsters with normal and elevated plasma cholesterol concentrations. Cholesterol feeding resulted in a 3.1-fold increase in the amount of LDL-cholesterol taken up by the fetus and a 2.4-fold increase in the amount of HDL-cholesterol taken up. LDL-cholesterol was transported to the fetus primarily by the placenta, and HDL-cholesterol was transported by the yolk sac and placenta. Several proteins associated with sterol transport and efflux, including those induced by activated liver X receptor, were expressed in hamster and human placentas: NPC1, NPC1L1, ABCA2, SCP-x, and ABCG1, but not ABCG8. NPC1L1 was the only protein increased in hypercholesterolemic placentas. Thus, increasing maternal lipoprotein-cholesterol concentrations can enhance transport of maternal cholesterol to the fetus, leading to 1) increased movement of cholesterol down a concentration gradient in the placenta, 2) increased lipoprotein secretion from the yolk sac (shown previously), and possibly 3) increased placental NPC1L1 expression.

In a search for genes involved in regulation of uterine contractility, we cloned a novel calcium-activated chloride channel gene, named rat Clca4, from pregnant rat uterus. The gene shares approximately 83% and 70% nucleotide homology with mouse Clca6 and human CLCA4, respectively, and was expressed primarily in rat uterus. The transcripts were upregulated at Gestational Day 22 (prior to parturition), implying a functional involvement in parturition. Western blot analysis showed that rat CLCA4 protein was present in uterus, lung, and heart, but not in any other tissues examined. Confocal microscopy revealed that rat CLCA4 is localized in cell membrane and could not be removed by alkaline or PBS washing. Transient transfection of rat CLCA4-enhanced green fluorescent protein in Chinese hamster ovary cells resulted in production of characteristic Cl(-) currents that could be activated by Ca(2+) and ionomycin but inhibited by niflumic acid, a CLCA-channel blocker. The identification and characterization of rat Clca4 help decipher the contribution of Ca(2+)-activated Cl(-) conductance in myometrial contractility.

Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema, resolves on delivery of the placenta. Normal pregnancy is itself characterized by systemic inflammation, oxidative stress and alterations in levels of angiogenic factors and vascular reactivity. This is exacerbated in preeclampsia with an associated breakdown of compensatory mechanisms, eventually leading to placental and vascular dysfunction. The underlying pathology of preeclampsia is thought to be a relatively hypoxic or ischemic placenta. Both the placenta and maternal vasculatures are major sources of reactive oxygen and nitrogen species which can interact to produce peroxynitrite a powerful prooxidant that covalently modifies proteins by nitration of tyrosine residues, to possibly alter vascular function in preeclampsia. The linkage between placental hypoxia and maternal vascular dysfunction has been proposed to be via placental syncytiotrophoblast basement membranes shed by the placenta or via angiogenic factors which include soluble flt1 and endoglin secreted by the placenta that bind vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) in the maternal circulation. There is also abundant evidence of altered reactivity of the maternal and placental vasculature and of the altered production of autocoids in preeclampsia. The occurrence of preeclampsia is increased in women with preexisting vascular disease and confers a long-term risk for development of cardiovascular disease. The vascular stress test of pregnancy thus identifies those women with a previously unrecognized at risk vascular system and promotes the development of preeclampsia. Preexisting maternal vascular dysfunction intensified by placental factors is possibly responsible for the individual pathologies of preeclampsia.

Maternal obesity is an increasing problem in obstetrics associated with adverse pregnancy outcomes and delivery complications. As an inflammatory state, where elevated levels of pro-inflammatory cytokines are found, obesity can lead to the increased incidence of oxidative and nitrative stress. These stresses may result in protein oxidation and protein nitration respectively, which are post- translational covalent modifications that can modify the structure and subsequently alter the function of a protein. The objective of this study was to examine whether placental oxidative and nitrative stress increase with increasing maternal body mass index. Placental tissue was collected from three groups of patients categorized as lean, overweight and obese. The presence of nitrotyrosine residues, a marker of nitrative stress, and antioxidant enzymes, as markers of oxidative stress, were assessed by immunohistochemistry, Western blot and ELISA. Protein carbonyl formation, a specific measure of protein oxidation, was measured by OxyBlot kit. Nitrotyrosine residues were increased in obese compared to lean and overweight groups although localization was unaltered across the three groups. Superoxide dismutase enzyme expression, localization and activity was unaltered between the groups. Protein carbonyl formation was greater in the lean compared to the overweight individuals. This study demonstrates that with increasing maternal body mass index there is an increase in placental nitrative stress. There does not appear to be a corresponding increase in oxidative stress and indeed we demonstrate some evidence of a decrease in oxidative effects in these placenta samples. Potentially the formation of peroxynitrite may be consuming reactive oxygen species and reducing oxidative stress. There may be a shift in the balance between nitrative and oxidative stress, which may be a protective mechanism for the placenta.