Placental DNA Research Articles

Added sugar intake during pregnancy: Fetal behavior, birth outcomes, and placental DNA methylation.

Pregnancy is a critical time for the effects of environmental factors on children's development. The effect of added sugar intake on fetal development and pregnancy outcomes remains understudied despite increasing dietary intake in the United States. This study investigated the effect of added sugar on fetal programming by examining the association between maternal added sugar consumption, fetal movement, birth outcomes, and placental DNA methylation. Further, primary human fibroblasts were cultured under normal or high glucose conditions to assess the effect of high glucose exposure on cells' DNA methylation. We found that higher added sugar intake across pregnancy was associated with reduced 3rd-trimester fetal movement (p<.05) and shorter gestation (p<.01). Our sample size was not powered to detect the alteration of individual placental CpG with genome-wide significance. However, a secondary analysis suggested that added sugar consumption was associated with differential methylation of functionally related gene families across pregnancy. Consistent with this, high glucose exposure in primary cultured human fibroblasts altered the methylation of 17% of all CpGs, providing converging evidence for an effect of sugar on DNA methylation. Our results suggest that diets high in added sugar during pregnancy may have implications for offspring health via prenatal programming effects measurable before birth.

Forever Connected: The Lifelong Biological Consequences of Fetomaternal and Maternofetal Microchimerism.

Originally studied as a mechanism to understand eclampsia-related deaths during pregnancy, fetal cells in maternal blood have more recently garnered attention as a noninvasive source of fetal material for prenatal testing. In the 21st century, however, intact fetal cells have been largely supplanted by circulating cell-free placental DNA for aneuploidy screening. Instead, interest has pivoted to the ways in which fetal cells influence maternal biology. In parallel, an increasing appreciation of the consequences of maternal cells in the developing fetus has occurred. In this review, we highlight the potential clinical applications and functional consequences of the bidirectional trafficking of intact cells between a pregnant woman and her fetus. Fetal cells play a potential role in the pathogenesis of maternal disease and tissue repair. Maternal cells play an essential role in educating the fetal immune system and as a factor in transplant acceptance. Naturally occurring maternal microchimerism is also being explored as a source of hematopoietic stem cells for transplant in fetal hematopoietic disorders. Future investigations in humans need to include complete pregnancy histories to understand maternal health and transplant success or failure. Animal models are useful to understand the mechanisms underlying fetal wound healing and/or repair associated with maternal injury and inflammation. The lifelong consequences of the exchange of cells between a mother and her child are profound and have many applications in development, health, and disease. This intricate exchange of genetically foreign cells creates a permanent connection that contributes to the survival of both individuals.

Prenatal fine particulate matter exposure, placental DNA methylation changes, and fetal growth

This study was designed to examine the impact of prenatal fine particulate matter (PM2.5) exposure on fetal growth and the underlying placental epigenetic mechanism in a cohort of Chinese women. Within the prospective Shanghai Mother-Child Pairs cohort (Shanghai MCPC), 329 women carrying singleton pregnancy with a due date in 2018 were recruited between 2017 and 2018. Maternal PM2.5 exposure levels were estimated using gestational exposure prediction model combining satellite-driven ambient concentrations and personal air sampling. Fetal growth characteristics were evaluated by prenatal ultrasound examinations and anthropometric measurements at birth. In a discovery phase, whole-genome DNA methylation analysis was performed using the Infinium 850 K array. In a validation phase, placental DNA methylation was measured using bisulfite pyrosequencing for five candidate genes that showed the most significant alterations and function relevance in our methylation array screen, including BID (BH3 interacting domain death agonist), FOXN3 (Forkhead box N3), FOXP1 (Forkhead box P1), IGF2 (Insulin-like growth factor 2) and HSD11B2 (Hydroxysteroid 11-beta dehydrogenase 2). Multivariate linear regression models were applied to examine the associations among PM2.5 exposure, fetal growth characteristics and DNA methylation on placental candidate genes. Sobel tests were used to evaluate the mediating role of DNA methylation in multivariable models. After excluding women who withdrew or failed to provide placenta, a total of 287 pregnant women with an average age of 30 entered the final analysis. Increased PM2.5 exposure was significantly associated with reduced biparietal diameter (BPD) (β: −0.136 mm, 95% CI: −0.228 to −0.043), head circumference (HC) (β: −0.462 mm, 95% CI: −0.782 to −0.142), femur length (FL) (β: −0.113 mm, 95% CI: −0.185 to −0.041) and abdominal circumference (AC) (β: −0.371 mm, 95% CI: −0.672 to −0.071) in the second trimester and birth length (β: −0.013 cm, 95% CI: −0.025 to −0.001). Prenatal PM2.5 exposure could lead to aberrant changes in DNA methylation profile of placenta genome, which were mainly enriched in reproductive development, energy metabolism and immune response. DNA methylation of IGF2 and BID showed significant associations with PM2.5 exposures during all exposure windows. In addition, BID methylation was negatively correlated with HC (β: −1.396 mm, 95% CI: −2.582 to −0.209) and BPD (β: −0.330 mm, 95% CI: −0.635 to −0.026) in the second trimester. Further mediation analysis indicated that BID methylation mediated about 30% of the effects of PM2.5 exposure on HC. These findings collectively suggested that prenatal PM2.5 exposure may cause adverse effects on fetal growth by modifying placental DNA methylation.

Abnormal placental DNA methylation variation in spontaneous preterm birth

Objective Preterm birth (PTB) has become a major public health concern as the leading cause of neonatal death, but little is understood about its etiology. Children born preterm are also at increased risk of long-term consequences such as neurodevelopmental disorders, adulthood hypertension and diabetes. Recent studies have indicated that DNA methylation may be involved in the occurrence of PTB as well as related adverse outcomes. The latest Infinium EPIC BeadChip extends the coverage of the genome and provides a better tool to help investigate the involvement of DNA methylation in these conditions. Methods We conducted this case-control study in three Women and Children’s hospitals in South China, and enrolled 32 spontaneous preterm births and 16 term births. We assessed placental DNA methylation profiling of these participants with the Infinium EPIC BeadChip. We identified PTB and gestational age (GA)-associated CpG sites with limma regression model, and applied seqlm to identify PTB-associated regions. We performed gene ontology analysis to further interpret functional enrichment of the identified differentially methylated genes in PTB. Results We identified a total of 8 differentially methylated positions (DMPs) that were significantly associated with PTB (FDR < 0.1) and a total of 15 DMPs that were associated with GA (FDR < 0.1). In the regional analysis, one differentially methylated region in the SLC23A1 gene overlapped with PTB-associated CpG site. The differentially methylated CpG sites in PTB were mapped to the genes involving in biological processes mainly regarding neurodevelopment, regulation of inflammation and metabolism. Conclusion Our findings suggested that preterm placenta have distinct DNA methylation alterations, and these alteration patterns established at birth provide insight into the long-term consequences of preterm birth.

Effects of maternal and fetal choline concentrations on the fetal growth and placental DNA methylation of 12 target genes related to fetal growth, adipogenesis, and energy metabolism.

We performed a birth cohort study involving 124 mother-infant pairs to investigate whether placental DNA methylation is associated with maternal choline status and fetal development. Plasma choline concentration was assayed longitudinally in the 1st and 3rd trimesters and at term-pregnancy in mothers and cord blood. Placental DNA methylation was measured for 12 target candidate genes that are related to fetal growth, adipogenesis, lipid and energy metabolism, or long interspersed nuclear elements. Higher maternal plasma and cord blood choline levels at term tended to associate with lower birthweight (r = -0.246, P < 0.013; r = -0.290, P < 0.002) and body mass index (BMI) at birth (r = 0.344, P < 1E-3; r = -0.360, P < 1E-3). The correlation between maternal plasma choline level and cord blood choline level was relatively modest (r = 0.049, P = 0.639). There was an inverse correlation between placental DNA methylation at the retinoid X receptor alpha (RXRA) gene and maternal plasma choline level (r = -0.188 to r = -0.452, P = 0.043 to P < 1E-3 at three points). RXRA methylation level was positively associated with birthweight and BMI at birth (r = 0.306, P = 0.001; r = 0.390, P < 1E-3). Further, RXRA methylation was inversely correlated with RXRA gene expression level (r = 0.333, P < 1E-3). Our results suggest that the association between maternal choline status and placental RXRA methylation represents a potential fetal programing mechanism contributing to fetal growth.

Guideline No. 409: Intrauterine Fetal Diagnostic Testing in Women with Chronic Viral Infections.

This revised guideline provides updated information for the care of women with chronic viral infections who require intrauterine fetal diagnostic testing. Women with chronic viral infections who are pregnant or planning a pregnancy. Non-invasive screening tests for diagnosis: maternal serum placental analytes with or without nuchal translucency, sonography, maternal serum cell-free placental DNA; and intrauterine fetal diagnostic testing: amniocentesis, chorionic villus sampling, cordocentesis. The recommendations in this guideline have the potential to decrease or eliminate morbidity and mortality in women with chronic viral infections and their infants, which is associated with significant health and economic outcomes. Published literature was retrieved through searches of PubMed, guidelines of national societies (Society of Obstetricians and Gynaecologists of Canada, American College of Obstetricians and Gynecologists, Society for Maternal-Fetal Medicine, other international societies), and the Cochrane Library using appropriate controlled vocabulary (amniocentesis, chorionic villus sampling, cordocentesis, procedure pregnancy loss risk, viral vertical transmission, fetal and neonatal infection) and keywords (maternal infection or exposure, hepatitis B, hepatitis C, human immunodeficiency virus). Results were restricted to systematic reviews, randomized controlled trials or controlled clinical trials (if available), and observational case-control studies or case series from 2012 to 2019 published in English or French. Studies from 1966 to 2002 were previously reviewed in the SOGC guideline No. 123: Amniocentesis and Women with Hepatitis B, Hepatitis C, or Human Immunodeficiency Virus, and those from 2002 to 2012 were previously reviewed in the SOGC guideline No. 309: Prenatal Invasive Procedures in Women With Hepatitis B, Hepatitis C, and/or Human Immunodeficiency Virus Infections. Updated literature searches were completed regularly through August 2019 and were incorporated into this guideline. The authors rated the quality of evidence and strength of recommendations using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. See online Appendix A (Tables A1 for definitions and A2 for interpretations of strong and weak recommendations). The intended users are maternity care providers and women with chronic viral infections. This guideline provides information to educate and counsel these women, and to offer them reproductive options. RECOMMENDATIONS (GRADE RATINGS IN PARENTHESES).

Genetic ancestry modifies the relationship between fine particulate matter and placental mitochondrial mutational load

Introduction: Exposure to ambient fine particulate matter (PM2.5) is linked to changes in placental mitochondrial DNA (mtDNA) copy number. Whether PM2.5 impacts mitochondrial mutational load in placental tissue-another biomarker of oxidative damage and aging has not been studied. Further, genetic ancestry likely impacts this relationship and may inform health disparities. Methods: We examine the association between PM2.5 and placental mtDNA mutational load in an urban multi-ethnic cohort (N=285). Mothers' daily exposure to PM2.5 over gestation was estimated using a satellite-based spatio-temporally resolved prediction model. Whole mtDNA sequencing was performed and mutations and haplogroups were determined. Bayesian Distributed Lag Interaction regression models (BDLIMs) were used to statistically model and visualize the PM2.5 timing-dependent pattern of associations with mtDNA mutations (total load and gene-specific) and explore effect modification by haplogroup. Results: Overall, increased PM2.5 exposure across pregnancy was not associated with total mutational load. However, results varied by mtDNA haplogroup with increases in PM2.5 being associated with higher total mutational load for African (cumulative effect 1.92, 95%CI 0.46, 3.48) and Asian (cumulative effect: 1.33, 95% CI 0.07, 2.73) haplogroups; a critical window was identified between 29 and 35 weeks gestation for African haplogroups only. Gene-wise analyses suggested that increased PM2.5 exposure during mid pregnancy (20-25 weeks) might have a stronger impact on mutations located in genes coding for ATP synthase subunits regardless of haplogroup. Conclusions: Placental mtDNA mutations, associated with increased PM2.5 exposure mid to late pregnancy, may have consequences on placental energy production, aging, and metabolic regulation that may impact offspring development. Understanding how these associations differ based on ancestry may further elucidate the etiology of environmentally-related disease disparities.

Maternal plasma concentrations of persistent organic pollutants and placental DNA methylation

Background/Aim: Prenatal maternal plasma persistent organic pollutants (POPs) concentrations have been associated with neonatal outcomes; however, the underlying mechanisms remain unknown. Placental epigenetics may be involved but no prior epigenome-wide studies have investigated such mechanisms. We studied the association between POPs in early pregnancy and epigenome-wide placental DNA methylation among 262 pregnant women from the NICHD Fetal Growth Studies. Methods: Epigenome-wide analyses using robust adjusted linear regression models were performed for each POP with more than 80% plasma concentration above the limit of quantification; including 3 organochlorine pesticides (hexachlorobenzene, trans-nonachlor, p,p’-dichlorodiphenyldichloroethylene), 1 polybrominated diphenyl ether (PBDE 47), 3 polychlorinated biphenyls (138/158, 153, 180) and 6 poly-and-perfluorinated alkyl substances (PFASs) (perfluorodecanoic acid, perfluorohexanesulfonic acid, perfluorononanoic acid, perfluorooctanesulfonic acid, perfluoroundecanoic acid (PFUnDA)). Genes annotated to the differentially methylated CpGs were tested for enrichment of molecular pathways. Results: At 5% false discovery rate, POPs concentrations were associated with a total of 193 differentially methylated CpGs in the placenta (nominal p-values ranging from 8.37x10-21 to 1.52x10-7). Among them, 21 CpGs were significantly correlated with placental expression of 18 unique genes (p-values&lt;0.05). Notably, PFUnDA was associated with higher methylation of 3 CpGs (cg13996963, cg12089439, cg18145877) annotated to TUSC3. Higher methylation at the three CpGs was correlated with decreased expression of TUSC3 in placenta; and decreased expression of TUSC3 was correlated with smaller birth length. Pathway analysis found that genes annotated to CpGs differentially methylated with PBDE 47 were enriched in birthweight (p-value = 2.06x10-5) and differentiation of embryonic cells (p-value = 2.25x10-5), while genes annotated to CpGs associated with PFASs were enriched in brain size (p-value = 4.09x10-4) and morphology (p-value = 7.29x10-3). Conclusions: The findings give insight into the potential for placental DNA methylation and gene expression mechanism to be involved in the prenatal toxicity of POPs and their associations with fetal growth outcomes.

Immediate and durable effects of maternal tobacco consumption alter placental DNA methylation in enhancer and imprinted gene-containing regions

BackgroundAlthough exposure to cigarette smoking during pregnancy has been associated with alterations of DNA methylation in the cord blood or placental cells, whether such exposure before pregnancy could induce epigenetic alterations in the placenta of former smokers has never been investigated.MethodsOur approach combined the analysis of placenta epigenomic (ENCODE) data with newly generated DNA methylation data obtained from 568 pregnant women, the largest cohort to date, either actively smoking during their pregnancy or formerly exposed to tobacco smoking.ResultsThis strategy resulted in several major findings. First, among the 203 differentially methylated regions (DMRs) identified by the epigenome-wide association study, 152 showed “reversible” alterations of DNA methylation, only present in the placenta of current smokers, whereas 26 were also found altered in former smokers, whose placenta had not been exposed directly to cigarette smoking. Although the absolute methylation changes were smaller than those observed in other contexts, such as in some congenital diseases, the observed alterations were consistent within each DMR. This observation was further supported by a demethylation of LINE-1 sequences in the placentas of both current (beta-coefficient (β) (95% confidence interval (CI)), − 0.004 (− 0.008; 0.001)) and former smokers (β (95% CI), − 0.006 (− 0.011; − 0.001)) compared to nonsmokers. Second, the 203 DMRs were enriched in epigenetic marks corresponding to enhancer regions, including monomethylation of lysine 4 and acetylation of lysine 27 of histone H3 (respectively H3K4me1 and H3K27ac). Third, smoking-associated DMRs were also found near and/or overlapping 10 imprinted genes containing regions (corresponding to 16 genes), notably including the NNAT, SGCE/PEG10, and H19/MIR675 loci.ConclusionsOur results pointing towards genomic regions containing the imprinted genes as well as enhancers as preferential targets suggest mechanisms by which tobacco could directly impact the fetus and future child. The persistence of significant DNA methylation changes in the placenta of former smokers supports the hypothesis of an “epigenetic memory” of exposure to cigarette smoking before pregnancy. This observation not only is conceptually revolutionary, but these results also bring crucial information in terms of public health concerning potential long-term detrimental effects of smoking in women.

Altered DNA methylation in human placenta after (suspected) preterm labor.

Aim: The aim of this study was to determine if alterations in DNA methylation in the human placenta would support suspected preterm labor as a pathologic insult associated with diminished placental health. Methods: We evaluated placental DNA methylation at seven loci differentially methylated in placental pathologies using targeted bisulfite sequencing, in placentas associated with preterm labor (term birth after suspected preterm labor [n=15] and preterm birth [n=15]), and controls (n=15). Results: DNA methylation levels at the NCAM1 and PLAGL1 loci in placentas associated with preterm labor did differ significantly (p<0.05) from controls. Discussion: Specific alterations in methylation patterns indicative of an unfavourable placental environment are associated with preterm labor per se and not restricted to preterm birth.

Placental Epigenome-Wide Association Study Identified Loci Associated with Childhood Adiposity at 3 Years of Age.

The aim of this study was to identify placental DNA methylation (DNAm) variations associated with adiposity at 3 years of age. We quantified placental DNAm using the Infinium MethylationEPIC BeadChips. We assessed associations between DNAm at single-CpGs and skinfold thickness using robust linear regression models adjusted for gestational age, child’s sex, age at follow-up and cellular heterogeneity. We sought replication of DNAm association with child adiposity in an independent cohort. We quantified placental mRNA levels for annotated gene using qRT-PCR and tested for correlation with DNAm. Lower DNAm at cg22593959 and cg22436429 was associated with higher adiposity (β = −1.18, q = 0.002 and β = −0.82, q = 0.04). The cg22593959 is located in an intergenic region (chr7q31.3), whereas cg22436429 is within the TFAP2E gene (1p34.3). DNAm at cg22593959 and cg22436429 was correlated with mRNA levels at FAM3C (rs = −0.279, p = 0.005) and TFAP2E (rs = 0.216, p = 0.03). In an independent cohort, the association between placental DNAm at cg22593959 and childhood adiposity was of similar strength and direction (β = −3.8 ± 4.1, p = 0.36), yet non-significant. Four genomic regions were also associated with skinfold thickness within FMN1, MAGI2, SKAP2 and BMPR1B genes. We identified placental epigenetic variations associated with adiposity at 3 years of age suggesting that childhood fat accretion patterns might be established during fetal life.

Prenatal PM2.5 exposure and vitamin D–associated early persistent atopic dermatitis via placental methylation

BackgroundThe effects of prenatal particulate matter with an aerodynamic diameter ranging from 0.1 μm to 2.5 μm (PM2.5) and vitamin D on atopic dermatitis (AD) phenotypes have not been evaluated. DNA methylation and cord blood (CB) vitamin D could represent a plausible link between prenatal PM2.5 exposure and AD in an offspring. ObjectiveTo determine the critical windows of prenatal PM2.5 exposure on the AD phenotypes, if vitamin D modulated these effects, and if placental DNA methylation mediated these effects on AD in offspring. MethodsMother–child pairs were enrolled from the birth cohort of the Cohort for Childhood Origin of Asthma and allergic diseases (COCOA) study. PM2.5 was estimated by land-use regression models, and CB vitamin D was measured by chemiluminescence immunoassay. AD was identified by the parental report of a physician’s diagnosis. We defined the following 4 AD phenotypes according to onset age (by the age of 2 years) and persistence (by the age of 3 years): early-onset transient and persistent, late onset, and never. Logistic regression analysis and Bayesian distributed lag interaction model were used. DNA methylation microarray was analyzed using an Infinium Human Methylation EPIC BeadChip (Illumina, San Diego, California) in placenta. ResultsPM2.5 exposure during the first trimester of pregnancy, especially during 6 to 7 weeks of gestation, was associated with early-onset persistent AD. This effect increased in children with low CB vitamin D, especially in those with PM2.5 exposure during 3 to 7 weeks of gestation. AHRR (cg16371648), DPP10 (cg19211931), and HLADRB1 (cg10632894) were hypomethylated in children with AD with high PM2.5 and low CB vitamin D. ConclusionHigher PM2.5 during the first trimester of pregnancy and low CB vitamin D affected early-onset persistent AD, and the most sensitive window was 6 to 7 weeks of gestation. Placental DNA methylation mediated this effect.

Associations Between Maternal Lifetime Stress and Placental Mitochondrial DNA Mutations in an Urban Multiethnic Cohort

BackgroundDisrupted placental functioning due to stress can have lifelong implications. Cumulative stress and trauma are likely to have lasting impacts on maternal physiological functioning and offspring development, resulting in increased risk for later-life complex disorders for which racial disparities exist. MethodsThis study examined the association between maternal lifetime stress and placental mitochondrial DNA mutational load in an urban multiethnic cohort. Maternal lifetime exposure to stressful events was assessed using the validated Life Stressor Checklist–Revised. Whole mitochondrial DNA sequencing was performed and mutations were determined for 365 placenta samples with complete exposure and covariate data. Multivariable regression was used to model maternal lifetime stress in relation to placental mitochondrial DNA mutational load. Racial/ethnic differences were examined by cross-product terms and contrast statements. Gene-wise analyses were conducted. ResultsWe identified 13,189 heteroplasmies (Phred score > 10,000, minor allele frequency < 0.5, number of mutant reads > 1). Women experiencing increased psychosocial stress over their lifetime exhibited a higher number of total placental mitochondrial mutations (β = .23, 95% confidence interval = .03 to .42) and heteroplasmic mutations (β = .18, 95% confidence interval = .05 to .31) but not homoplasmic mutations (β = −.008, 95% confidence interval = −.03 to .01); the strongest associations were observed among Black women and genes coding for NADH dehydrogenase and cytochrome c oxidase subunits. ConclusionsCumulative maternal lifetime stress is associated with a greater mitochondrial mutational load, particularly among Black women. The impact of racial/ethnic differences in mutational load on placental function directly affecting offspring development and/or leading to chronic disease disparities warrants further investigation.

Placental mitochondrial DNA mutations and copy numbers in intrauterine growth restricted (IUGR) pregnancy

Intrauterine Growth Restriction (IUGR) is a common and significant complication that arises during pregnancy wherein the fetus fails to attain its full growth potential. Mitochondria being one of the primary sources of energy, plays an important role in placentation and fetal development. In IUGR pregnancy, increased oxidative stress due to inadequate oxygen and nutrient supply could possibly alter mitochondrial functions and homeostasis. In this study, we evaluated the biochemical and molecular changes in mitochondria as biosignature for early and better characterization of IUGR pregnancies. We identified significant increase in mtDNA copy number in both IUGR (p = 0.0001) and Small for Gestational Age (SGA) but healthy (p = 0.0005) placental samples when compared to control. Whole mitochondrial genome sequencing identified novel mutations in both coding and non-coding regions of mtDNA in multiple IUGR placental samples. Sirtuin-3 (Sirt3) protein expression was significantly downregulated (p = 0.027) in IUGR placenta but there was no significant difference in Nrf1 expression in IUGR when compared to control group. Our study provides an evidence for altered mitochondrial homeostasis and paves a way towards interrogating mitochondrial abnormalities in IUGR pregnancies.

Abstract 5780: Developing a screen for O6-alkylguanines in human DNA using the damage-specific binding protein, Atl1

Abstract Humans are widely exposed to a wide variety of DNA alkylating agents (AAs) either directly, for example via cigarette smoke or indirectly via their formation in the gastrointestinal tract. Certain AAs, such as the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) are known human carcinogens but the role that other AAs may play in the aetiology of human cancers is still uncertain due to difficulties in assessing human exposure to these agents. The aim of this work was therefore to develop a screening method to assess exposure to AAs by measuring the formation of O6-alkylguanines (O6-AlkGs) in DNA and applying it to the analysis of human DNA samples. To do this we took advantage of the S. pombe alkyltransferase-like protein (Atl1), which binds to all O6-AlkGs so far studied, to develop a slot blot assay. For this, sonicated heat denatured DNA was bound to Hybond-N+ membranes and probed with horseradish peroxidase-conjugated maltose-binding protein-Atl1 fusion protein. The binding was quantitated via peroxidase-mediated enhanced chemiluminescence using TMZ-methylated calf thymus (CT) DNA standards previously calibrated using a tritium-based competition assay. Using 1 μg DNA, the limit of quantitation and detectability were approximately 1.0 and 0.5 fmoles O6-MeG/μg DNA, respectively. Prior treatment of the TMZ-CT DNA standards with the DNA repair protein, O6-methylguanine DNA methyltransferase, reduced the detectable signal to background levels, confirming that the assay detected O6-methylguanine (O6-MeG) in TMZ-CT DNA. Importantly, CT DNA reacted in vitro with the alkylating agents N-ethyl-N-nitrosourea, N-propyl-N-nitrosourea, N-butyl-N-nitrosourea, N-benzyl-N-nitrosourea, N-allyl-N-nitrosourea and potassium diazoacetate all produced strong signals in the assay demonstrating its ability to detect a range of different O6-AlkGs. In addition, a variety of human DNA samples have been analysed in pilot studies: of 14 human placental DNA samples, 8 had O6-AlkG levels in the range of 1.05-1.71 fmoles/μg DNA, 3 had trace levels and in 3 samples there was no detectable signal. Of 5 human breast tumour DNA samples, 4 had O6-AlkGs levels in the range of 1.07-1.30 fmoles/µg DNA and 1 had trace levels. Two DNA samples from normal human breast tissue had no detectable O6-AlkGs but DNA from the tumours of the same patients had levels &amp;gt;1 fmoles/μg DNA. Of 6 DNA samples from whole blood 2 had detectable levels of O6-AlkGs (1.23 &amp; 1.56 fmoles/μg DNA), 3 had trace amounts and 1 had no detectable level. These data thus show the potential usefulness of this slot blot assay as a screening tool to detect and quantify total O6-alkGs adducts in human DNA samples. Citation Format: Hanum Yaakub, Anthony Howell, Geoffrey Margison, Andrew Povey. Developing a screen for O6-alkylguanines in human DNA using the damage-specific binding protein, Atl1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5780.

Keep Calm and the Placenta Will Carry On

A fully functional placenta is critical for a successful pregnancy. In this issue of Developmental Cell, Singh etal. reveal that excessive placental DNA damage in murine models for Cornelia de Lange syndrome results in an inefficient and senescent placenta that impairs embryonic development.

Concentrations of persistent organic pollutants in maternal plasma and epigenome-wide placental DNA methylation

BackgroundPrenatal maternal plasma persistent organic pollutant (POP) concentrations have been associated with neonatal outcomes. However, the underlying mechanisms remain unknown. Placental epigenetic mechanisms may be involved, but no prior epigenome-wide studies have investigated the impact of maternal POPs on placental DNA methylation. We studied the association between maternal plasma POP concentration in early pregnancy and epigenome-wide placental DNA methylation among 260 pregnant women from the NICHD Fetal Growth Studies.ResultsOur analysis focused on POPs with more than 80% plasma concentrations above the limit of quantification, including 3 organochlorine pesticides (hexachlorobenzene, trans-nonachlor, p,p’-dichlorodiphenyldichloroethylene), 1 polybrominated diphenyl ether (PBDE 47), 3 polychlorinated biphenyls (138/158, 153, 180), and 6 poly- and perfluorinated alkyl substances (PFASs) (perfluorodecanoic acid, perfluorohexanesulfonic acid, perfluorononanoic acid, perfluorooctanesulfonic acid, perfluoroundecanoic acid (PFUnDA)). Using 5% false discovery rate, POPs were associated with a total of 214 differentially methylated CpG sites (nominal p values ranging from 2.61 × 10−21 to 2.11 × 10−7). Out of the 214 CpG sites, 24 (11%) were significantly correlated with placental expression of 21 genes. Notably, higher PFUnDA was associated with increased methylation at 3 CpG sites (cg13996963, cg12089439, cg18145877) annotated to TUSC3, and increased methylation at those 3 CpG sites was correlated with decreased expression of TUSC3 in the placenta. Increased methylation at cg18145877 (TUSC3) and decreased expression of TUSC3 were correlated with shorter birth length. Out of the 214 CpG sites, methylation at 44 CpG sites was correlated (p value < 0.10) with at least one neonatal anthropometry measure (i.e., birth weight, birth length, and head circumference). Seven CpG sites mediated (p value < 0.05) the association between PBDE 47 and neonatal anthropometry measures. Genes annotating the top differentially methylated CpG sites were enriched in pathways related to differentiation of embryonic cells (PBDE 47) and in pathways related to brain size and brain morphology (PFASs).ConclusionsDNA methylation changes in the placenta were significantly associated with maternal plasma POPs concentration. The findings suggest that placental DNA methylation and gene expression mechanism may be involved in the prenatal toxicity of POPs and their association with neonatal anthropometry measures.

The hypomethylation of imprinted genes in IVF/ICSI placenta samples is associated with concomitant changes in histone modifications

ABSTRACT Although more and more children are born by Assisted Reproductive Technologies (ART), ART safety has not fully been demonstrated. Notably, ART could disturb the delicate step of implantation, and trigger placenta-related adverse outcomes with potential long-term effects, through disrupted epigenetic regulation. We have previously demonstrated that placental DNA methylation was significantly lower after IVF/ICSI than following natural conception at two differentially methylated regions (DMRs) associated with imprinted genes (IGs): H19/IGF2 and KCNQ1OT1. As histone modifications are critical for placental physiology, the aim of this study was to profile permissive and repressive histone marks in placenta biopsies to reveal a better understanding of the epigenetic changes in the context of ART. Utilizing chromatin immunoprecipitation (ChIP) coupled with quantitative PCR, permissive (H3K4me3, H3K4me2, and H3K9ac) and repressive (H3K9me3 and H3K9me2) post-translational histone modifications were quantified. The analyses revealed a significantly higher quantity of H3K4me2 precipitation in the IVF/ICSI group than in the natural conception group for H19/IGF2 and KCNQ1OT1 DMRs (P = 0.016 and 0.003, respectively). Conversely, the quantity of both repressive marks at H19/IGF2 and SNURF DMRs was significantly lower in the IVF/ICSI group than in the natural conception group (P = 0.011 and 0.027 for H19/IGF2; and P = 0.010 and 0.035 for SNURF). These novel findings highlight that DNA hypomethylation at imprinted DMRs following ART is linked with increased permissive/decreased repressive histone marks, altogether promoting a more permissive chromatin conformation. This concomitant change in epigenetic state at IGs at birth might be an important developmental event because of ART manipulations.

Expression of iron regulatory proteins in full-term swine placenta.

In swine, even though the pregnant sows were with iron abundance, the inborn iron reserve of piglets was compromised. This indicates the insufficiency of molecular machinery involved in local placental iron flux. Here, we investigated the expression of iron regulatory proteins like hepcidin and ferroportin and also their association with iron reserve, inflammation and oxidative stress in placenta of full-term pregnant sows (n=6). Amplification and sequencing of placental DNA confirmed the presence of hepcidin (MN579557) and ferroportin (MN565887) sequences and their 100% identity with existing GenBank data. Real-time amplification of placental mRNA revealed significant higher expression of hepcidin (p<.05) than ferroportin. Western blot analysis of placental tissues revealed specific bands for both hepcidin (~8kDa) and ferroportin (~62kDa) molecules. Immunohistochemistry revealed the immunoreactivity for both proteins in the cytoplasm and membrane of trophoblastic cells of the placenta. Hepcidin and ferroportin expressions were positively associated with placental non-haem iron reserve (p<.0001; p=.033), lipid peroxidation (p=.0060; p<.0001) and reactive oxygen species level (p=.0092; p=.0292). Hepcidin expression was positively associated with interleukin - 6 (p=.0002) and interferon gamma (p<.0001) expressions but ferroportin expression was negatively associated with interleukin-6 (p=.0005), interleukin-1β (p=.0226) and interferon gamma (p=.0059) expressions. This indicates hepcidin and ferroportin may have a role in controlling the local placental iron flux by acting as a molecular bridge between iron trafficking and inflammation.

DNA methylation loci in placenta associated with birthweight and expression of genes relevant for early development and adult diseases

BackgroundBirthweight marks an important milestone of health across the lifespan, including cardiometabolic disease risk in later life. The placenta, a transient organ at the maternal-fetal interface, regulates fetal growth. Identifying genetic loci where DNA methylation in placenta is associated with birthweight can unravel genomic pathways that are dysregulated in aberrant fetal growth and cardiometabolic diseases in later life.ResultsWe performed placental epigenome-wide association study (EWAS) of birthweight in an ethnic diverse cohort of pregnant women (n = 301). Methylation at 15 cytosine-(phosphate)-guanine sites (CpGs) was associated with birthweight (false discovery rate (FDR) < 0.05). Methylation at four (26.7%) CpG sites was associated with placental transcript levels of 15 genes (FDR < 0.05), including genes known to be associated with adult lipid traits, inflammation and oxidative stress. Increased methylation at cg06155341 was associated with higher birthweight and lower FOSL1 expression, and lower FOSL1 expression was correlated with higher birthweight. Given the role of the FOSL1 transcription factor in regulating developmental processes at the maternal-fetal interface, epigenetic mechanisms at this locus may regulate fetal development. We demonstrated trans-tissue portability of methylation at four genes (MLLT1, PDE9A, ASAP2, and SLC20A2) implicated in birthweight by a previous study in cord blood. We also found that methylation changes known to be related to maternal underweight, preeclampsia and adult type 2 diabetes were associated with lower birthweight in placenta.ConclusionWe identified novel placental DNA methylation changes associated with birthweight. Placental epigenetic mechanisms may underlie dysregulated fetal development and early origins of adult cardiometabolic diseases.Clinical trial registrationClinicalTrials.gov, NCT00912132