Integrative network analysis of differentially methylated regions to study the impact of gestational weight gain on maternal metabolism and fetal-neonatal growth.

Integrative network analysis (INA) is important for identifying gene modules or epigenetically regulated molecular pathways in diseases. This study evaluated the effect of excessive gestational weight gain (EGWG) on INA of differentially methylated regions, maternal metabolism and offspring growth. Brazilian women from "The Araraquara Cohort Study" with adequate pre-pregnancy body mass index were divided into EGWG (n=30) versus adequate gestational weight gain (AGWG, n=45) groups. The methylome analysis was performed on maternal blood using the Illumina MethylationEPIC BeadChip. Fetal-neonatal growth was assessed by ultrasound and anthropometry, respectively. Maternal lipid and glycemic profiles were investigated. Maternal triglycerides-TG (p=0.030) and total cholesterol (p=0.014); fetus occipito-frontal diameter (p=0.005); neonate head circumference-HC (p=0.016) and thoracic perimeter (p=0.020) were greater in the EGWG compared to the AGWG group. Multiple linear regression analysis showed that maternal DNA methylation was associated with maternal TG and fasting insulin, fetal abdominal circumference, and fetal and neonate HC. The DMRs studied were enriched in 142 biological processes, 21 molecular functions,and 17 cellular components with terms directed for the fatty acids metabolism. Three DMGMs were identified:COL3A1, ITGA4 and KLRK1. INA targeted chronic diseases and maternal metabolism contributing to an epigenetic understanding of the involvement of GWG in maternal metabolism and fetal-neonatal growth.

Polyphenol rich fruit attenuates genomic instability, modulates inflammation and cell cycle progression of offspring from fatty acid intake maternal.

The aim of this study was to investigate the effect of juçara (Euterpe edulis Mart.) supplementation on the maternal trans fatty acids intake in the livers of 21-day-old offspring. In order for this to happen, histopathological analysis, cytogenetic status, inflammation (COX-2 and TNF-alpha) and cell cycle progression were investigated in this setting. On the first day of pregnancy, female rats were distributed into four groups, as follows: control diet (C), control diet with 0.5 % juçara supplementation (CJ), diet enriched with hydrogenated vegetable fat, rich in TFAs (T), or T diet supplemented with 0.5 % juçara (TJ) during pregnancy and lactation. Juçara pulp induced liver regeneration in newborns exposed to maternal trans fatty acids. A significant decrease in the number of micronucleated hepatocytes was observed in animals exposed to trans fatty acids and treated with juçara. COX-2 and TNF immunoexpression was reduced in animals treated with juçara pulp. Furthermore, a decrease of Ki-67 immunoexpression was detected after treating trans fatty acids intake with juçara. Taken together, our results demonstrate that juçara pulp is able to prevent tissue degeneration and mutagenicity because it decreases inflammation and cell cycle control induced by maternal trans fatty acids in liver cells of rat offspring.

Excessive Gestational Weight Gain Alters DNA Methylation and Influences Foetal and Neonatal Body Composition.

BACKGROUND: Changes in body weight are associated with the regulation of DNA methylation (DNAm). In this study, we investigated the associations between maternal gestational weight gain-related DNAm and foetal and neonatal body composition. METHODS: Brazilian pregnant women from the Araraquara Cohort Study were followed up during pregnancy, delivery, and after hospital discharge. Women with normal pre-pregnancy BMI were allocated into two groups: adequate gestational weight gain (AGWG, n = 45) and excessive gestational weight gain (EGWG, n = 30). Foetal and neonatal body composition was evaluated via ultrasound and plethysmography, respectively. DNAm was assessed in maternal blood using Illumina Infinium MethylationEPIC BeadChip arrays. Linear regression models were used to explore the associations between DNAm and foetal and neonatal body composition. RESULTS: Maternal weight, GWG, neonatal weight, and fat mass were higher in the EGWG group. Analysis of DNAm identified 46 differentially methylated positions and 11 differentially methylated regions (DMRs) between the EGWG and AGWG groups. Nine human phenotypes were enriched for these 11 DMRs located in 13 genes (EMILIN1, HOXA5, CPT1B, CLDN9, ZFP57, BRCA1, POU5F1, ANKRD33, HLA-B, RANBP17, ZMYND11, DIP2C, TMEM232), highlighting the terms insulin resistance, and hyperglycaemia. Maternal DNAm was associated with foetal total thigh and arm tissues and subcutaneous thigh and arm fat, as well as with neonatal fat mass percentage and fat mass. CONCLUSION: The methylation pattern in the EGWG group indicated a risk for developing chronic diseases and involvement of maternal DNAm in foetal lean and fat mass and in neonatal fat mass.

The relationship between obesity-related H19DMR methylation and H19 and IGF2 gene expression on offspring growth and body composition.

Background and objective: Imprinted genes are important for the offspring development. To assess the relationship between obesity-related H19DMR methylation and H19 and IGF2 gene expression and offspring growth and body composition. Methods: Thirty-nine overweight/obese and 25 normal weight pregnant women were selected from the "Araraquara Cohort Study" according to their pre-pregnancy BMI. Fetal growth and body composition and newborn growth were assessed, respectively, by ultrasound and anthropometry. The methylation of H19DMR in maternal blood, cord blood, maternal decidua and placental villi tissues was evaluated by methylation-sensitive restriction endonuclease qPCR, and H19 and IGF2 expression by relative real-time PCR quantification. Multiple linear regression models explored the associations of DNA methylation and gene expression with maternal, fetal, and newborn parameters. Results: H19DMR was less methylated in maternal blood of the overweight/obese group. There were associations of H19DMR methylation in cord blood with centiles of fetal biparietal diameter (BPD) and abdominal subcutaneous fat thickness and newborn head circumference (HC); H19DMR methylation in maternal decidua with fetal occipitofrontal diameter (OFD), HC, and length; H19DMR methylation in placental villi with fetal OFD, HC and abdominal subcutaneous fat thickness and with newborn HC. H19 expression in maternal decidua was associated with fetal BPD and femur length centiles and in placental villi with fetal OFD and subcutaneous arm fat. IGF2 expression in maternal decidua was associated with fetal BPD and in placental villi with fetal OFD. Conclusion: To our knowledge, this is the first study to demonstrate associations of imprinted genes variations at the maternal-fetal interface of the placenta and in cord blood with fetal body composition, supporting the involvement of epigenetic mechanisms in offspring growth and body composition.

Maternal plasma transforming growth factor-ß1 (TGF-ß1) and newborn size: the Araraquara Cohort Study.

OBJECTIVE: To investigate associations of maternal and cord blood cytokine patterns with newborn size and body composition. METHODS: This cross-sectional study involved 70 pregnant women and their healthy newborns selected from the "Araraquara Cohort Study". Newborn anthropometric measurements were recorded at birth. Body composition was evaluated by air displacement plethysmography. Maternal blood samples were collected from pregnant women between 30 and 36 weeks of gestation, and umbilical cord blood samples were collected immediately after placenta discharge. The concentrations of the cytokines were determined in plasma by ELISA. Multiple linear regression models were used to assess associations between maternal and cord blood cytokine concentrations and newborn anthropometry and body composition measurements. RESULTS: Maternal plasma TGF-ß1 concentration was inversely associated with newborn weight (ß = -43.0; p = 0.012), length (ß = -0.16, p = 0.028), head circumference (ß = -0.13, p = 0.004), ponderal index (ß = -0.32, p = 0.011) and fat-free mass (ß = -0.05, p = 0.005). However, the association persisted just for head circumference (ß = -0.26; p = 0.030) and ponderal index (ß = - 0.28; p = 0.028), after adjusting for pre-gestational BMI, gestational weight gain, gestational age, hours after delivery, newborn sex, smoking and alcohol consumption. CONCLUSIONS: Maternal plasma TGF-ß1 concentration may be involved in the regulation of newborn size, mainly head circumference and ponderal index. Further cohort studies are necessary to investigate the role of TGF-ß1 in different trimesters of pregnancy and its effect during the early stages of fetal development.

Maternal plasma transforming growth factor-β1 (TGF-β1) and newborn size: the Araraquara Cohort Study

Abstract Objective To investigate associations of maternal and cord blood cytokine patterns with newborn size and body composition. Methods This cross-sectional study involved 70 pregnant women and their healthy newborns selected from the "Araraquara Cohort Study". Newborn anthropometric measurements were recorded at birth. Body composition was evaluated by air displacement plethysmography. Maternal blood samples were collected from pregnant women between 30 and 36 weeks of gestation, and umbilical cord blood samples were collected immediately after placenta discharge. The concentrations of the cytokines were determined in plasma by ELISA. Multiple linear regression models were used to assess associations between maternal and cord blood cytokine concentrations and newborn anthropometry and body composition measurements. Results Maternal plasma TGF-β1 concentration was inversely associated with newborn weight (β= -43.0; p= 0.012), length (β= -0.16, p= 0.028), head circumference (β= -0.13, p= 0.004), ponderal index (β= -0.32, p= 0.011) and fat-free mass (β= -0.05, p= 0.005). However, the association persisted just for head circumference (β= -0.26; p= 0.030) and ponderal index (β= - 0.28; p= 0.028), after adjusting for pre-gestational BMI, gestational weight gain, gestational age, hours after delivery, newborn sex, smoking and alcohol consumption. Conclusions Maternal plasma TGF-β1 concentration may be involved in the regulation of newborn size, mainly head circumference and ponderal index. Further cohort studies are necessary to investigate the role of TGF-β1 in different trimesters of pregnancy and its effect during the early stages of fetal development.

Perfil da metilação do DNA do binômio mãe-filho no contexto da obesidade materna, adiposidade fetal e neonatal / DNA methylation profile of the mother-child binomial in maternal obesity, fetal and neonatal adiposity

Introdução: Um mecanismo epigenético pelo qual os efeitos adversos do ambiente intra-uterino são transferidos aos descendentes é a metilação do DNA. Objetivo: Avaliar a relação entre obesidade materna, ganho de peso gestacional (GPG) e alteração da metilação do DNA sobre o desenvolvimento fetal e neonatal. Metodologia: Subpopulação de gestantes do estudo epidemiológico prospectivo "Coorte Araraquara" foram acompanhadas durante os três trimestres da gestação, parto e no pós-parto. Para avaliar o impacto do GPG na metilação do DNA e desenvolvimento fetal e neonatal, as gestantes foram alocadas em 2 grupos: ganho de peso gestacional adequado (n=45) e ganho de peso gestacional excessivo (n=30). Para avaliar o impacto da obesidade materna na metilação do DNA e desenvolvimento fetal e neonatal, as gestantes foram alocadas em 2 grupos: A. IMC pré-gestacional adequado (n=25) e B. IMC pré-gestacional sobrepeso/obesidade (n=39 gestantes). A biometria e composição corporal fetal foram avaliadas por ultrassom Siemens ACUSON X300TM (Siemens®, Mountain View, CA, USA) e a composição corporal do neonato por pletismografia, com o PEA POD (Cosmed®, Concord, CA, USA). A dieta materna ao final da gestação foi investigada por recordatórios de 24 horas e analisada no software Nutrition Data System for Research (NDSR, Minnesota, USA). O DNA dos sangues materno e cordão umbilical, da vilosidade e decídua placentárias foram extraídos utilizando proteinase K pelo método santing-out. As regiões hipo e hipermetiladas foram analisados pela técnica de Digestão Enzimática Sensível à Metilação associada à PCR quantitativa (qPCR) e a expressão gênica por qPCR. Para análise da influência do GPG o DNA do sangue materno de gestantes com GPG adequado (N=8) versus com GPG excessivo (N=8), foi hibridizado na plataforma Illumina com o Human Methylation 850K Bead Chip (Illumina, CA, USA). Para análise estatística aplicou-se o Teste t, Qui-quadrado (X2), ANOVA de medidas repetidas e modelos de regressão linear múltiplo e o nível de significância adotado foi de p≤0,05. Resultados: O excessivo ganho de peso gestacional (EGPG) alterou os triglicerídeos (TGs) e colesterol total (CT) maternos, o diâmetro occipito-frontal (DOF) do feto, a circunferência da cabeça, perímetro torácico, peso e a massa gorda neonatais. A análise de metilação global do DNA materno identificou 46 posições diferencialmente metiladas e 11 regiões diferencialmente metiladas (DMRs). Nove fenótipos humanos foram enriquecidos para essas 11 DMRs localizadas em 13 genes (EMILIN1, HOXA5, CPT1B, CLDN9, ZFP57, BRCA1, POU5F1, ANKRD33, HLA-B, RANBP17, ZMYND11, DIP2C, TMEM232), destacando-se os termos resistência à insulina e hiperglicemia. O DNAm materno foi associado com parâmetros de composição corporal, como: tecido total da coxa e do braço fetais e gordura subcutânea da coxa e do braço fetais, bem como ao percentual de massa gorda e massa gorda neonatais. A metilação do DNA materno também foi associada com os TGs e a insulina de jejum maternos, com circunferência abdominal e circunferência da cabeça fetais (CC) e CC neonatal. As DMRs estudadas foram enriquecidas em 142 processos biológicos, 21 funções moleculares e 17 componentes celulares. Três módulos gênicos diferencialmente metilados foram identificados. Por outro lado, o IMC pré-gestacional sopreso/obesidade alterou os percentis do diâmetro biparietal (DBF), o DOF, a CC, a espessura da gordura subcutânea do abdômen (ETSA), tecido total do braço, massa muscular do braço e gordura subcutânea do braço fetais e a CC do neonato. O gene H19DMR foi significativamente menos metilado no sangue materno do grupo com sobrepeso/obesidade em comparação ao grupo com IMC pré-gestacional adequado, para o sangue do cordão umbilical e tecidos placentários, não houve diferença de metilação entre os grupos, bem como não houve diferença de expressão gênica dos genes H19 e IGF2 nos tecidos placentários entre os grupos. Houve associações entre metilação de H19DMR no sangue do cordão umbilical com os percentis do DBP, com a ETSA e a CC neonatais; na decídua com o DOF, a CC e comprimento fetais; no vilo com o DOF, a CC e a ETSA fetais e com a CC neonatal. A expressão do gene H19 na decídua também foi associada ao DBP e ao percentil do comprimento do fêmur fetais; no vilo com o DOF e gordura subcutânea do braço fetais. A expressão do gene IGF2 na decídua foi associada com o DBP fetal e no vilo com o DOF fetal. Conclusão: A metilação do DNA foi alterada pelo ganho de peso gestacional e obesidade maternos e se associou com a adiposidade e crescimento fetais. O excessivo GPG alterou o metiloma materno e está envolvido com um fenótipo de risco para o desenvolvimento de doenças crônicas e metabólicas, a exemplo da diabetes.

Introduction: An epigenetic mechanism by which the adverse effects of the intrauterine environment are transferred to offspring is DNA methylation. Objective: Evaluate the relationship between maternal obesity, gestational weight gain and DNA methylation alteration on fetal and neonatal development. Methodology: Subpopulation of pregnant women from the prospective epidemiological study "Cohorte Araraquara" were followed during the three trimesters of pregnancy, delivery and postpartum and were allocated into 2 groups: A. adequate weight (N=25) and B. overweight/obesity (N=39 pregnant women). Fetal biometry and adiposity were evaluated by ultrasound and the neonate's body composition by plethysmography, with the PEA POD (Cosmed®, Concord, CA, USA). Maternal diet in the end of pregnancy was investigated using 24-hour recalls and analyzed using the Nutrition Data System for Research software (NDSR, Minnesota, USA). DNA maternal and umbilical cord blood, and placental villus and decidua were extracted using proteinase K by the santing-out method. The hypo and hypermethylated regions were analyzed by the Methylation Sensitive Enzymatic Digestion technique associated with quantitative PCR (qPCR) and gene expression by qPCR. To analyze the influence of gestational weight gain (GWG), maternal blood DNA from pregnant women with adequate AGWG (N=8) versus excessive EGWG (N=8) was hybridized on the Human Methylation 850K Bead Chip (Illumina, CA). For statistical analysis, the t test, chi-square (X2) test, repeated measures ANOVA and multiple linear regression models were applied, and the significance level adopted was p≤0.05. Results: The pre-gestational weight, pre-gestational BMI and BMI during pregnancy were higher in group B. In this same group, higher values of fasting insulin, us-CRP and lipid consumption were found in relation to group A. subcutaneous abdominal fat thickness (SCFT) and subcutaneous fat of the fetal arm was also higher in group B compared to group A. Regarding the body composition of neonates, the amount of fat mass was higher in group B. The H19DMR gene was less methylated in maternal blood from group B. No statistical difference was found in the values of IGF2 and H19 gene expression between the groups. There were associations between methylation of the H19DMR gene in umbilical cord blood with fetal biparietal diameter (BPD) and SCFT and neonatal head circumference (HC); in the decidua with occipito frontal diameter (OFD), fetal length and HC; in the villi with DOF, HC and SCFT and neonatal HC. Excessive gestational weight gain altered 46 CpG sites, 11 differentially methylated regions (DMRs) located in 13 genes, namely: EMILIN1, HOXA5, CPT1B, CLDN9, ZFP57, BRCA1, POU5F1, ANKRD33, HLA-B, RANBP17, ZMYND11, DIP2C, TMEM232. These DMRs were enriched in 142 biological processes, 21 molecular functions, 17 cellular components and 9 human phenotypes. In addition, 3 differentially methylated gene modules were identified to the phenotype of interest. Conclusion: DNA methylation was altered by maternal nutritional status and was associated with fetal adiposity and growth. Excessive GPG altered maternal methylome and was associated with the phenotype of metabolic diseases such as diabetes mellitus.