Placental Global DNA Methylation Research Articles

Prenatal vitamin intake in first month of pregnancy and DNA methylation in cord blood and placenta in two prospective cohorts

BackgroundPrenatal vitamin use is recommended before and during pregnancies for normal fetal development. Prenatal vitamins do not have a standard formulation, but many contain calcium, folic acid, iodine, iron, omega-3 fatty acids, zinc, and vitamins A, B6, B12, and D, and usually they contain higher concentrations of folic acid and iron than regular multivitamins in the US Nutrient levels can impact epigenetic factors such as DNA methylation, but relationships between maternal prenatal vitamin use and DNA methylation have been relatively understudied. We examined use of prenatal vitamins in the first month of pregnancy in relation to cord blood and placenta DNA methylation in two prospective pregnancy cohorts: the Early Autism Risk Longitudinal Investigation (EARLI) and Markers of Autism Risk Learning Early Signs (MARBLES) studies.ResultsIn placenta, prenatal vitamin intake was marginally associated with −0.52% (95% CI −1.04, 0.01) lower mean array-wide DNA methylation in EARLI, and associated with −0.60% (−1.08, −0.13) lower mean array-wide DNA methylation in MARBLES. There was little consistency in the associations between prenatal vitamin intake and single DNA methylation site effect estimates across cohorts and tissues, with only a few overlapping sites with correlated effect estimates. However, the single DNA methylation sites with p-value < 0.01 (EARLI cord nCpGs = 4068, EARLI placenta nCpGs = 3647, MARBLES cord nCpGs = 4068, MARBLES placenta nCpGs = 9563) were consistently enriched in neuronal developmental pathways.ConclusionsTogether, our findings suggest that prenatal vitamin intake in the first month of pregnancy may be related to lower placental global DNA methylation and related to DNA methylation in brain-related pathways in both placenta and cord blood.

Association between placental global DNA methylation and blood pressure during human pregnancy.

Gene-specific placental DNA methylation patterns differ between normal pregnancies and pregnancies complicated by hypertension. However, whether global placental DNA methylation is associated with maternal blood pressure remains controversial. Using multiple linear regression models, we analysed the association between maternal mean arterial pressure (MAP) at the third trimester of pregnancy and global DNA methylation in the placenta in 922 mothers using LC-ESI-MS/MS. To better characterize the contribution of genetic or epigenetic mechanisms, we performed isolated analyses in mothers with and without a family history of hypertension. Mean placental global DNA methylation was 3.00 ± 0.46%. A significant negative correlation between placental global DNA methylation and mean arterial blood pressure (MAP) in the third trimester could be observed (P = 0.023, r = -0.075). This association remained significant after adjusting for confounders. In placenta samples from mothers with a family history of hypertension, mean maternal MAP was higher (86.1 ± 8.1 vs. 84.6 ± 7.5, P < 0.01) and placental global DNA methylation was lower (2.94 ± 0.43 vs. 3.04 ± 0.47, P < 0.01) compared with samples without a family history of hypertension. Furthermore, the significant independent negative correlation between global placental DNA methylation and MAP was only found in mothers without a family history of hypertension. This study showed an independent negative correlation between placental global DNA methylation and maternal MAP in mothers without a family history of hypertension.

Placental DNA methylation in caesarean sections – a pilot study

IntroductionCaesarean section (CS) is the most common medical procedure performed in women all over the world. There is a hypothesis that caesarean section changes DNA methylation, and it has been linked to immune-mediated diseases such as diabetes in late infant health outcomes. The aim of the study was to evaluate the relationship between caesarean section and DNA methylation patterning in the placenta by measurement of global DNA methylation.Material and methodsWe included in the study 111 healthy, pregnant women in singular pregnancy at term of delivery (gestational age at least 37 weeks). The study involved global methylation in placental tissue from 46 pregnant women, delivered by vaginal route, and 49 pregnant women, delivered by elective caesarean section (ECS) before the start of labour. In 16 of the elective caesarean section cases, regular uterine contractions were declared. An ELISA-based kit was used for an assessment of the global DNA methylation.ResultsGlobal DNA methylation in the placenta in elective caesarean sections (3.02 on average) was significantly lower compared to intrapartum caesarean sections (3.71 on average) and vaginal deliveries (3.64 on average), but did not significantly differ between intrapartum caesarean sections and vaginal deliveries. Global DNA methylation in the placenta in male newborns was significantly higher (4.86 on average) than in female newborns (3.31 on average) in vaginal deliveries but not in either elective or intrapartum caesarean sections.ConclusionsCaesarean sections performed without uterine contractions significantly influenced global DNA methylation in the placenta. Moreover, a sexual dimorphism at the level of placental global DNA methylation was demonstrated. Further studies investigating different panels of genes may help to identify genes with aberrant methylation in newborn infants delivered by caesarean section compared to vaginal delivery, as well as demonstrate sexual dimorphism.

Being Born Large for Gestational Age is Associated with Increased Global Placental DNA Methylation

Being born small (SGA) or large for gestational age (LGA) is associated with adverse birth outcomes and metabolic diseases in later life of the offspring. It is known that aberrations in growth during gestation are related to altered placental function. Placental function is regulated by epigenetic mechanisms such as DNA methylation. Several studies in recent years have demonstrated associations between altered patterns of DNA methylation and adverse birth outcomes. However, larger studies that reliably investigated global DNA methylation are lacking. The aim of this study was to characterize global placental DNA methylation in relationship to size for gestational age. Global DNA methylation was assessed in 1023 placental samples by LC-MS/MS. LGA offspring displayed significantly higher global placental DNA methylation compared to appropriate for gestational age (AGA; p < 0.001). ANCOVA analyses adjusted for known factors impacting on DNA methylation demonstrated an independent association between placental global DNA methylation and LGA births (p < 0.001). Tertile stratification according to global placental DNA methylation levels revealed a significantly higher frequency of LGA births in the third tertile. Furthermore, a multiple logistic regression analysis corrected for known factors influencing birth weight highlighted an independent positive association between global placental DNA methylation and the frequency of LGA births (p = 0.001).

Air pollution during pregnancy and placental adaptation in the levels of global DNA methylation.

BackgroundHealth in early life is crucial for health later in life. Exposure to air pollution during embryonic and early-life development can result in placental epigenetic modification and foetus reprogramming, which can influence disease susceptibility in later life. Objectives: The aim of this paper was to investigate the placental adaptation in the level of global DNA methylation and differential gene expression in the methylation cycle in new-borns exposed to high fine particulate matter in the foetal stage.Study designThis is a nested case-control study. We enrolled pregnant healthy women attending prenatal care clinics in Tehran, Iran, who were residents of selected polluted and unpolluted regions, before the 14th week of pregnancy. We calculated the regional background levels of particle mass- particles with aerodynamics diameter smaller than 2.5 μm (PM2.5) and 10 μm (PM10)—of two regions of interest. At the time of delivery, placental tissue was taken for gene expression and DNA methylation analyses. We also recorded birth outcomes (the new-born’s sex, birth date, birth weight and length, head and chest circumference, gestational age, Apgar score, and level of neonatal care required).ResultsAs regards PM2.5 and PM10 concentrations in different time windows of pregnancy, there were significantly independent positive correlations between PM10 and PM2.5 in the first trimester of all subjects and placental global DNA methylation levels (p-value = 0.01, p-value = 0.03, respectively). The gene expression analysis showed there was significant correlation between S-adenosylmethionine expression and PM2.5 (p = 0.003) and PM10 levels in the first trimester (p = 0.03).ConclusionOur data showed prenatal exposures to air pollutants in the first trimester could influence placental adaptation by DNA methylation.

Time- and Sex-Dependent Associations Between Prenatal Alcohol Exposure and Placental Global DNA Methylation

Epigenetic changes, in particular in the placenta, may mediate the effects of prenatal alcohol exposure (PAE) on children's health. We examined the relationship between PAE patterns, based on dose and timing, and placental global DNA methylation. Using linear regression analysis, we examined the association between different PAE categories and placental global DNA methylation (n=187), using the proxy measure of Alu-interspersed repeats. Following adjustment for important covariates, we found no evidence of an association between PAE and placental global DNA methylation overall. However, when stratifying by newborn sex, PAE throughout pregnancy was associated with higher placental global DNA methylation (1.5%; p=0.01) of male newborns. PAE may have sex-specific effects on placental global DNA methylation if alcohol is consumed throughout pregnancy.

Placental expression of DNA methyltransferase 1 (DNMT1): Gender-specific relation with human placental growth

AimsPlacental physiology and morphology is critically regulated by DNA methylation. As such, placental global DNA methylation and transcript abundance of placental DNA methyltransferases (DNMT1 and DNMT3A) may relate to placental and fetal growth in human pregnancies. We aimed to test correlations of human fetoplacental parameters and birth weight with the placental expression of DNA methyltransferases (DNMT1 and DNMT3A) and placental global methylation. Subjects and methodsPlacentae (n = 109) were collected from small- (SGA) and appropriate- (AGA) for gestational age full-term singleton pregnancies (n = 56 SGA and 53 AGA). Placentae and neonates were weighed at birth. Realtime quantitative PCR was performed to assess placental transcript abundance of DNMT1, DNMT3A and DNTMT3B normalized to a panel of reference genes. LINE-1 methylation was measured using a quantitative MethyLight assay in a subset of samples (n = 68). Associations of placental transcript abundances of DNMT1, DNMT3A and DNMT3B and of LINE-1 methylation levels with maternal, placental and neonatal parameters were tested. ResultsPlacental DNMT1 transcript abundance associated positively with placental weight (β = 10.21, P = 0.013). This association was specific to the AGA births (β = 12.77, P = 0.022) and was absent in the SGA births. Association of DNMT1 expression with placental weight and birth weight within the AGA births was specific to the female gender (Birth weight: β = 83.61, P = 0.043; Placental weight: β = 23.92, P = 0.025). Placental DNMT1 transcript levels were not different according to SGA status or gender. Placental DNMT3A transcript levels and LINE-1 methylation levels did not show any associations with maternal, placental and neonatal parameters. ConclusionsPlacental DNMT1 expression was found to be associated positively with placental weight and birth weight, specifically in the female AGA births. Thus, we hypothesize that placental DNMT1 participates in fetoplacental growth in a fetal gender-specific manner.

Increased global placental DNA methylation levels are associated with gestational diabetes.

BackgroundGestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes. It is known that GDM is associated with an altered placental function and changes in placental gene regulation. More recent studies demonstrated an involvement of epigenetic mechanisms. So far, the focus regarding placental epigenetic changes in GDM was set on gene-specific DNA methylation analyses. Studies that robustly investigated placental global DNA methylation are lacking. However, several studies showed that tissue-specific alterations in global DNA methylation are independently associated with type 2 diabetes. Thus, the aim of this study was to characterize global placental DNA methylation by robustly measuring placental DNA 5-methylcytosine (5mC) content and to examine whether differences in placental global DNA methylation are associated with GDM.MethodsGlobal DNA methylation was quantified by the current gold standard method, LC-MS/MS. In total, 1030 placental samples were analyzed in this single-center birth cohort study.ResultsMothers with GDM displayed a significantly increased global placental DNA methylation (3.22 ± 0.63 vs. 3.00 ± 0.46 %; p = 0.013; ±SD). Bivariate logistic regression showed a highly significant positive correlation between global placental DNA methylation and the presence of GDM (p = 0.0009). Quintile stratification according to placental DNA 5mC levels revealed that the frequency of GDM was evenly distributed in quintiles 1–4 (2.9–5.3 %), whereas the frequency in the fifth quintile was significantly higher (10.7 %; p = 0.003). Bivariate logistic models adjusted for maternal age, BMI, ethnicity, recurrent miscarriages, and familiar diabetes predisposition clearly demonstrated an independent association between global placental DNA hypermethylation and GDM. Furthermore, an ANCOVA model considering known predictors of DNA methylation substantiated an independent association between GDM and placental DNA methylation.ConclusionsThis is the first study that employed a robust quantitative assessment of placental global DNA methylation in over a thousand placental samples. The study provides large scale evidence that placental global DNA hypermethylation is associated with GDM, independent of established risk factors.

Placental oxidative stress and decreased global DNA methylation are corrected by copper in the Cohen diabetic rat

Fetal Growth Restriction (FGR) is a leading cause for long term morbidity. The Cohen diabetic sensitive rats (CDs), originating from Wistar, develop overt diabetes when fed high sucrose low copper diet (HSD) while the original outbred Sabra strain do not. HSD induced FGR and fetal oxidative stress, more prominent in the CDs, that was alleviated more effectively by copper than by the anti-oxidant vitamins C and E. Our aim was to evaluate the impact of copper or the anti-oxidant Tempol on placental size, protein content, oxidative stress, apoptosis and total DNA methylation. Animals were mated following one month of HSD or regular chow diet and supplemented throughout pregnancy with either 0, 1 or 2ppm of copper sulfate or Tempol in their drinking water. Placental weight on the 21st day of pregnancy decreased in dams fed HSD and improved upon copper supplementation. Placental/fetal weight ratio increased among the CDs. Protein content decreased in Sabra but increased in CDs fed HSD. Oxidative stress biochemical markers improved upon copper supplementation; immunohistochemistry for oxidative stress markers was similar between strains and diets. Caspase 3 was positive in more placentae of dams fed HSD than those fed RD. Placental global DNA methylation was decreased only among the CDs dams fed HSD. We conclude that FGR in this model is associated with smaller placentae, reduced DNA placental methylation, and increased oxidative stress that normalized with copper supplementation. DNA hypomethylation makes our model a unique method for investigating genes associated with growth, oxidative stress, hypoxia and copper.

Placental DNA hypomethylation in association with particulate air pollution in early life

BackgroundThere is evidence that altered DNA methylation is an important epigenetic mechanism in prenatal programming and that developmental periods are sensitive to environmental stressors. We hypothesized that exposure to fine particles (PM2.5) during pregnancy could influence DNA methylation patterns of the placenta.MethodsIn the ENVIRONAGE birth cohort, levels of 5’-methyl-deoxycytidine (5-mdC) and deoxycytidine (dC) were quantified in placental DNA from 240 newborns. Multiple regression models were used to study placental global DNA methylation and in utero exposure to PM2.5 over various time windows during pregnancy.ResultsPM2.5 exposure during pregnancy averaged (25th-75th percentile) 17.4 (15.4-19.3) μg/m3. Placental global DNA methylation was inversely associated with PM2.5 exposures during whole pregnancy and relatively decreased by 2.19% (95% confidence interval [CI]: -3.65, -0.73%, p = 0.004) for each 5 μg/m3 increase in exposure to PM2.5. In a multi-lag model in which all three trimester exposures were fitted as independent variables in the same regression model, only exposure to PM2.5 during trimester 1 was significantly associated with lower global DNA methylation (-2.13% per 5 μg/m3 increase, 95% CI: -3.71, -0.54%, p = 0.009). When we analyzed shorter time windows of exposure within trimester 1, we observed a lower placental DNA methylation at birth during all implantation stages but exposure during the implantation range (6-21d) was strongest associated (-1.08% per 5 μg/m3 increase, 95% CI: -1.80, -0.36%, p = 0.004).ConclusionsWe observed a lower degree of placental global DNA methylation in association with exposure to particulate air pollution in early pregnancy, including the critical stages of implantation. Future studies should elucidate genome-wide and gene-specific methylation patterns in placental tissue that could link particulate exposure during in utero life and early epigenetic modulations.

Differential placental methylation and expression of VEGF, FLT- 1 and KDR genes in human term and preterm preeclampsia

BackgroundPreeclampsia, a pregnancy complication of placental origin is associated with altered expression of angiogenic factors and their receptors. Recently, there is considerable interest in understanding the role of adverse intrauterine conditions in placental dysfunction and adverse pregnancy outcomes. Since we have observed changes in placental global DNA methylation levels in preeclampsia, this study was undertaken to examine gene promoter CpG methylation and expression of several angiogenic genes.We recruited 139 women comprising, 46 normotensive women with term delivery (≥37 weeks), 45 women with preeclampsia delivering preterm (<37 weeks) and 48 women with preeclampsia delivering at term. Expression levels and promoter CpG methylation of VEGF, FLT-1 and KDR genes in placentae from respective groups were determined by Taqman-based quantitative real time PCR and by the Sequenom® EpiTYPER™ technology respectively.ResultsWe observed several differentially methylated CpG sites in the promoter regions of VEGF, FLT-1 and KDR between the normotensive and preeclampsia groups. We specifically observed hypomethylated CpGs in the promoter region and an increased expression of VEGF gene between term and preterm preeclampsia. However, mean promoter CpG methylation could not account for the higher expression of FLT-1 and KDR in preterm preeclampsia as compared to normotensive group.ConclusionsOur data indicates altered DNA methylation patterns in the VEGF, FLT-1 and KDR genes in preeclampsia as compared to the normotensive group, which could be involved in the pathophysiology of preeclampsia. Hypomethylation of VEGF promoter and consequent upregulation of VEGF mRNA levels could be a compensatory mechanism to restore normal angiogenesis and blood flow in preterm preeclampsia. This study suggests a role of altered DNA methylation in placental angiogenesis and in determining adverse pregnancy outcomes.

Maternal choline intake alters the epigenetic state of fetal cortisol‐regulating genes in humans

The in utero availability of methyl donors, such as choline, may modify fetal epigenetic marks and lead to sustainable functional alterations throughout the life course. The hypothalamic-pituitary-adrenal (HPA) axis regulates cortisol production and is sensitive to perinatal epigenetic programming. As an extension of a 12-wk dose-response choline feeding study conducted in third-trimester pregnant women, we investigated the effect of maternal choline intake (930 vs. 480 mg/d) on the epigenetic state of cortisol-regulating genes, and their expression, in placenta and cord venous blood. The higher maternal choline intake yielded higher placental promoter methylation of the cortisol-regulating genes, corticotropin releasing hormone (CRH; P=0.05) and glucocorticoid receptor (NR3C1; P=0.002); lower placental CRH transcript abundance (P=0.04); lower cord blood leukocyte promoter methylation of CRH (P=0.05) and NR3C1 (P=0.04); and 33% lower (P=0.07) cord plasma cortisol. In addition, placental global DNA methylation and dimethylated histone H3 at lysine 9 (H3K9me2) were higher (P=0.02) in the 930 mg choline/d group, as was the expression of select placental methyltransferases. These data collectively suggest that maternal choline intake in humans modulates the epigenetic state of genes that regulate fetal HPA axis reactivity as well as the epigenomic status of fetal derived tissues.

Effects of Altered Maternal Folic Acid, Vitamin B12 and Docosahexaenoic Acid on Placental Global DNA Methylation Patterns in Wistar Rats

Potential adverse effects of excess maternal folic acid supplementation on a vegetarian population deficient in vitamin B12 are poorly understood. We have previously shown in a rat model that maternal folic acid supplementation at marginal protein levels reduces brain omega-3 fatty acid levels in the adult offspring. We have also reported that reduced docosahexaenoic acid (DHA) levels may result in diversion of methyl groups towards DNA in the one carbon metabolic pathway ultimately resulting in DNA methylation. This study was designed to examine the effect of normal and excess folic acid in the absence and presence of vitamin B12 deficiency on global methylation patterns in the placenta. Further, the effect of maternal omega 3 fatty acid supplementation on the above vitamin B12 deficient diets was also examined. Our results suggest maternal folic acid supplementation in the absence of vitamin B12 lowers plasma and placental DHA levels (p<0.05) and reduces global DNA methylation levels (p<0.05). When this group was supplemented with omega 3 fatty acids there was an increase in placental DHA levels and subsequently DNA methylation levels revert back to the levels of the control group. Our results suggest for the first time that DHA plays an important role in one carbon metabolism thereby influencing global DNA methylation in the placenta.

225: Placental global DNA methylation in a mouse model of fetal programming

The placenta regulates nutrient transport and plays a central role in fetal growth. Disturbances in placental function during critical periods of development set the stage for fetal programming of adult diseases. DNA methylation of placental genes may be one of the mechanisms by which placental function is altered and fetal adaptations leading to developmental programming occur. Our goal was to evaluate the global DNA methylation status in a previously characterized transgenic mouse model of utero-placental insufficiency and fetal vascular programming.

Sex- and diet-specific changes of imprinted gene expression and DNA methylation in mouse placenta under a high-fat diet.

BackgroundChanges in imprinted gene dosage in the placenta may compromise the prenatal control of nutritional resources. Indeed monoallelic behaviour and sensitivity to changes in regional epigenetic state render imprinted genes both vulnerable and adaptable.Methods and FindingsWe investigated whether a high-fat diet (HFD) during pregnancy modified the expression of imprinted genes and local and global DNA methylation patterns in the placenta. Pregnant mice were fed a HFD or a control diet (CD) during the first 15 days of gestation. We compared gene expression patterns in total placenta homogenates, for male and female offspring, by the RT-qPCR analysis of 20 imprinted genes. Sexual dimorphism and sensitivity to diet were observed for nine genes from four clusters on chromosomes 6, 7, 12 and 17. As assessed by in situ hybridization, these changes were not due to variation in the proportions of the placental layers. Bisulphite-sequencing analysis of 30 CpGs within the differentially methylated region (DMR) of the chromosome 17 cluster revealed sex- and diet-specific differential methylation of individual CpGs in two conspicuous subregions. Bioinformatic analysis suggested that these differentially methylated CpGs might lie within recognition elements or binding sites for transcription factors or factors involved in chromatin remodelling. Placental global DNA methylation, as assessed by the LUMA technique, was also sexually dimorphic on the CD, with lower methylation levels in male than in female placentae. The HFD led to global DNA hypomethylation only in female placenta. Bisulphite pyrosequencing showed that neither B1 nor LINE repetitive elements could account for these differences in DNA methylation.ConclusionsA HFD during gestation triggers sex-specific epigenetic alterations within CpG and throughout the genome, together with the deregulation of clusters of imprinted genes important in the control of many cellular, metabolic and physiological functions potentially involved in adaptation and/or evolution. These findings highlight the importance of studying both sexes in epidemiological protocols and dietary interventions.