Placental DNA methylation signatures of prenatal air pollution exposure and potential effects on birth outcomes: an analysis of three prospective cohorts.

BACKGROUND: Pregnancy air pollution exposure (PAPE) has been linked to a wide range of adverse birth and childhood outcomes, but there is a paucity of data on its influence on the placental epigenome, which can regulate the programming of physiological functions and affect child development. This study aimed to investigate the association between prenatal air pollutant exposure concentrations and changes in placental DNA methylation patterns, and to explore the potential windows of susceptibility and sex-specific alterations. METHODS: This multi-site study used three prospective population-based mother-child cohorts: EDEN, PELAGIE, and SEPAGES, originating from four French geographical regions (Nancy, Poitiers, Brittany, and Grenoble). Pregnant women were included between 2003 and 2006 for EDEN and PELAGIE, and between 2014 and 2017 for SEPAGES. The main eligibility criteria were: being older than 18 years, having a singleton pregnancy, and living and planning to deliver in one of the maternity clinics in one of the study areas. A total of 1539 mother-child pairs were analysed, measuring placental DNA methylation using Illumina BeadChips. We used validated spatiotemporally resolved models to estimate PM2·5, PM10, and NO2 exposure over each trimester of pregnancy at the maternal residential address. We conducted a pooled adjusted epigenome-wide association study to identify differentially methylated 5'-C-phosphate-G-3' (CpG) sites and regions (assessed using the Infinium HumanMethylationEPIC BeadChip array, n=871), including sex-specific and sex-linked alterations, and independently validated our results (assessed using the Infinium HumanMethylation450 BeadChip array, n=668). FINDINGS: We identified four CpGs and 28 regions associated with PAPE in the total population, 469 CpGs and 87 regions in male infants, and 150 CpGs and 66 regions in female infants. We validated 35% of the CpGs available. More than 30% of the identified CpGs were related to one (or more) birth outcome and most significant alterations were enriched for neural development, immunity, and metabolism related genes. The 28 regions identified for both sexes overlapped with imprinted genes (four genes), and were associated with neurodevelopment (nine genes), immune system (seven genes), and metabolism (five genes). Most associations were observed for the third trimester for female infants (134 of 150 CpGs), and throughout pregnancy (281 of 469 CpGs) and the first trimester (237 of 469 CpGs) for male infants. INTERPRETATION: These findings highlight the molecular pathways through which PAPE might affect child health in a widespread and sex-specific manner, identifying the genes involved in the major physiological functions of a developing child. Further studies are needed to elucidate whether these epigenetic changes persist and affect health later in life. FUNDING: French Agency for National Research, Fondation pour la Recherche Médicale, Fondation de France, and the Plan Cancer.

An integrated omics approach highlights how epigenetic events can explain and predict response to neoadjuvant chemotherapy and bevacizumab in breast cancer.

Treatment with the anti-angiogenic drug bevacizumab in addition to chemotherapy has shown efficacy for breast cancer in some clinical trials, but better biomarkers are needed to optimally select patients for treatment. Here, we present an omics approach where DNA methylation profiles are integrated with gene expression and results from proteomic data in breast cancer patients to predict response to therapy and pinpoint response-related epigenetic events. Fresh-frozen tumor biopsies taken before, during, and after treatment from human epidermal growth factor receptor 2 negative non-metastatic patients receiving neoadjuvant chemotherapy with or without bevacizumab were subjected to molecular profiling. Here, we report that DNA methylation at enhancer CpGs related to cell cycle regulation can predict response to chemotherapy and bevacizumab for the estrogen receptor positive subset of patients (AUC = 0.874), and we validated this observation in an independent patient cohort with a similar treatment regimen (AUC = 0.762). Combining the DNA methylation scores with the scores from a previously published protein signature resulted in a slight increase in the prediction performance (AUC = 0.784). We also show that tumors receiving the combination treatment underwent more extensive epigenetic alterations. Finally, we performed an integrative expression-methylation quantitative trait loci analysis on alterations in DNA methylation and gene expression levels, showing that the epigenetic alterations that occur during treatment are different between responders and non-responders and that these differences may be explained by the proliferation-epithelial-to-mesenchymal transition axis through the activity of grainyhead like transcription factor 2. Using tumor purity computed from copy number data, we developed a method for estimating cancer cell-specific methylation to confirm that the association to response reflects DNA methylation in cancer cells. Taken together, these results support the potential for clinical benefit of the addition of bevacizumab to chemotherapy when administered to the correct patients.

Frequency of natural regulatory T cells specific for factor VIII in the peripheral blood of healthy donors.

Tolerance to self-proteins involves multiple mechanisms, including conventional CD4+ T-cell (Tconv) deletion in the thymus and the recruitment of natural regulatory T cells (nTregs). The significant incidence of autoantibodies specific for the blood coagulation factor VIII (FVIII) in healthy donors illustrates that tolerance to self-proteins is not always complete. In contrast to FVIII-specific Tconvs, FVIII-specific nTregs have never been revealed and characterized. To determine the frequency of FVIII-specific Tregs in human peripheral blood, we assessed the specificity of in vitro expanded Tregs by the membrane expression of the CD137 activation marker. Amplified Tregs maintain high levels of FOXP3 expression and exhibit almost complete demethylation of the FOXP3 Treg-specific demethylated region. The cells retained FOXP3 expression after long-term culture in vitro, strongly suggesting that FVIII-specific Tregs are derived from the thymus. From eleven healthy donors, we estimated the frequencies of FVIII-specific Tregs at 0.17 cells per million, which is about 10-fold lower than the frequency of FVIII-specific CD4+ T cells we previously published. Our results shed light on the mechanisms of FVIII tolerance by a renewed approach that could be extended to other self- or non-self-antigens.

Tunable DNMT1 degradation reveals DNMT1/DNMT3B synergy in DNA methylation and genome organization.

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present cell models that allow inducible and reversible DNAme modulation through DNMT1 depletion. By dynamically assessing whole genome and locus-specific effects of induced passive demethylation through cell divisions, we reveal a cooperative activity between DNMT1 and DNMT3B, but not of DNMT3A, to maintain and control DNAme. We show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin, compartmentalization, and peripheral localization. DNA methylation loss coincides with a gradual reduction of cell fitness due to G1 arrest, with minor levels of mitotic failure. Altogether, this system allows DNMTs and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNAme dysfunction and human disease.

Epigenome-Wide Associations of Placental DNA Methylation and Behavioral and Emotional Difficulties in Children at 3 Years of Age.

The placenta is a key organ for fetal and brain development. Its epigenome can be regarded as a biochemical record of the prenatal environment and a potential mechanism of its association with the future health of the fetus. We investigated associations between placental DNA methylation levels and child behavioral and emotional difficulties, assessed at 3 years of age using the Strengths and Difficulties Questionnaire (SDQ) in 441 mother-child dyads from the EDEN cohort. Hypothesis-driven and exploratory analyses (on differentially methylated probes (EWAS) and regions (DMR)) were adjusted for confounders, technical factors, and cell composition estimates, corrected for multiple comparisons, and stratified by child sex. Hypothesis-driven analyses showed an association of cg26703534 (AHRR) with emotional symptoms, and exploratory analyses identified two probes, cg09126090 (intergenic region) and cg10305789 (PPP1R16B), as negatively associated with peer relationship problems, as well as 33 DMRs, mostly positively associated with at least one of the SDQ subscales. Among girls, most associations were seen with emotional difficulties, whereas in boys, DMRs were as much associated with emotional than behavioral difficulties. This study provides the first evidence of associations between placental DNA methylation and child behavioral and emotional difficulties. Our results suggest sex-specific associations and might provide new insights into the mechanisms of neurodevelopment.

Prenatal exposure to triclosan assessed in multiple urine samples and placental DNA methylation.

A previous study reported positive associations of maternal urinary concentrations of triclosan, a synthetic phenol with widespread exposure in the general population, with placental DNA methylation of male fetuses. Given the high number of comparisons performed in -omic research, further studies were needed to validate and extend on these findings. Using a cohort of male and female fetuses with repeated maternal urine samples to assess exposure, we studied the associations between triclosan and placental DNA methylation. We assessed triclosan concentrations in two pools of 21 urine samples collected among 395 women from the SEPAGES cohort. We used Infinium Methylation EPIC arrays to measure DNA methylation in placental biopsies collected at delivery. We performed a candidate study restricted to a set of candidate CpGs (n = 500) identified in a previous work as well as an exploratory epigenome-wide association study to investigate the associations between triclosan and differentially methylated probes and regions. Analyses were conducted on the whole population and stratified by child's sex. Mediation analysis was performed to test whether heterogeneity of placental tissue may mediate the observed associations. In the candidate approach, we confirmed 18 triclosan-associated genes when both sexes were considered. After stratification for child's sex, triclosan was associated with 72 genes in females and three in males. Most of the associations were positive and several CpGs mapped to imprinted genes: FBRSL1, KCNQ1, RHOBTB3, and SMOC1. A mediation effect by placental tissue heterogeneity was identified for most of the observed associations. In the exploratory analysis, we identified a few isolated associations in the sex-stratified analysis. In line with a previous study on male placentas, our approach revealed several positive associations between triclosan exposure and placental DNA methylation. Several identified loci mapped to imprinted genes.

Identification of extracellular vesicle microRNA signatures specifically linked to inflammatory and metabolic mechanisms in obesity-associated low type-2 asthma.

RATIONALE AND OBJECTIVE: Plasma extracellular vesicles (EVs) represent a vital source of molecular information about health and disease states. Due to their heterogenous cellular sources, EVs and their cargo may predict specific pathomechanisms behind disease phenotypes. Here we aimed to utilize EV microRNA (miRNA) signatures to gain new insights into underlying molecular mechanisms of obesity-associated low type-2 asthma. METHODS: Obese low type-2 asthma (OA) and non-obese low type-2 asthma (NOA) patients were selected from an asthma cohort conjointly with healthy controls. Plasma EVs were isolated and characterised by nanoparticle tracking analysis. EV-associated small RNAs were extracted, sequenced and bioinformatically analysed. RESULTS: Based on EV miRNA expression profiles, a clear distinction between the three study groups could be established using a principal component analysis. Integrative pathway analysis of potential target genes of the differentially expressed miRNAs revealed inflammatory cytokines (e.g., interleukin-6, transforming growth factor-beta, interferons) and metabolic factors (e.g., insulin, leptin) signalling pathways to be specifically associated with OA. The miR-17-92 and miR-106a-363 clusters were significantly enriched only in OA. These miRNA clusters exhibited discrete bivariate correlations with several key laboratory (e.g., C-reactive protein) and lung function parameters. Plasma EV miRNA signatures mirrored blood-derived CD4+ T-cell transcriptome data, but achieved an even higher sensitivity in identifying specifically affected biological pathways. CONCLUSION: The identified plasma EV miRNA signatures and particularly the miR-17-92 and -106a-363 clusters were capable to disentangle specific mechanisms of the obesity-associated low type-2 asthma phenotype, which may serve as basis for stratified treatment development.

Do not be scared of the genome's 5th base-Explaining phenotypic variability and evolutionary dynamics through DNA methylation analysis.

Epigenetic processes have taken center stage for the investigation of many biological processes, and epigenetic modifications have shown to influence phenotype, morphology and behavioural traits such as stress resistance by affecting gene regulation and expression without altering the underlying genomic sequence. The multiple molecular layers of epigenetics synergistically construct the cell type-specific gene regulatory networks, characterized by a high degree of plasticity and redundancy to create cell-type-specific morphology and function. DNA methylation occurring on the 5' carbon of cytosines in different genomic sequence contexts is the most studied epigenetic modification. DNA methylation has been shown to provide a molecular record of the exposure to a large variety of environmental factors, which might be persistent through the entire lifetime of an organism and even be passed onto the offspring. Animals might display altered phenotypes mediated by epigenetic modifications depending on the developmental stage or the environmental conditions as well as during evolution. Therefore, the analysis of DNA methylation patterns might allow deciphering previous exposures, explaining ecologically relevant phenotypic diversity and predicting evolutionary trajectories enabling accelerated adaption to changing environmental conditions. Despite the explanatory potential of DNA methylation integrating genetic and environmental factors to shape phenotypic variation and contribute significantly to evolutionary dynamics, studies of DNA methylation are still scarce in the field of ecology. This might be at least partly due to the complexity of DNA methylation analysis and the interpretation of the acquired data. In the current issue of Molecular Ecology Resources, Laine and colleagues (Molecular Ecology Resources, 2022) provide a detailed summary of guidelines and valuable recommendations for researchers in the field of ecology to avoid common pitfalls and perform interpretable genome-wide DNA methylation analyses.

DNA methylation dynamics during pregnancy.

Pregnancy is a state of multiple physiological adaptations. Since methylation of DNA is an epigenetic mechanism that regulates gene expression and contributes to adaptive phenotypic variations, we investigated methylation changes in maternal blood of a longitudinal cohort of pregnant women from the first trimester of gestation to the third. Interestingly, during pregnancy, we found a gain of methylation in genes involved in morphogenesis, such as ezrin, while we identified a loss of methylation in genes promoting maternal-infant bonding (AVP and PPP1R1B). Together, our results provide insights into the biological mechanisms underlying physiological adaptations during pregnancy.

High-Dimensional Mediation Analysis: A New Method Applied to Maternal Smoking, Placental DNA Methylation, and Birth Outcomes.

BACKGROUND: High-dimensional mediation analysis is an extension of unidimensional mediation analysis that includes multiple mediators, and increasingly it is being used to evaluate the indirect omics-layer effects of environmental exposures on health outcomes. Analyses involving high-dimensional mediators raise several statistical issues. Although many methods have recently been developed, no consensus has been reached about the optimal combination of approaches to high-dimensional mediation analyses. OBJECTIVES: We developed and validated a method for high-dimensional mediation analysis (HDMAX2) and applied it to evaluate the causal role of placental DNA methylation in the pathway between exposure to maternal smoking (MS) during pregnancy and gestational age (GA) and birth weight of the baby at birth. METHODS: HDMAX2 combines latent factor regression models for epigenome-wide association studies with max2 tests for mediation and considers CpGs and aggregated mediator regions (AMRs). HDMAX2 was carefully evaluated using simulated data and compared to state-of-the-art multidimensional epigenetic mediation methods. Then, HDMAX2 was applied to data from 470 women of the Etude des Déterminants pré et postnatals du développement de la santé de l'Enfant (EDEN) cohort. RESULTS: HDMAX2 demonstrated increased power in comparison with state-of-the-art multidimensional mediation methods and identified several AMRs not identified in previous mediation analyses of exposure to MS on birth weight and GA. The results provided evidence for a polygenic architecture of the mediation pathway with a posterior estimate of the overall indirect effect of CpGs and AMRs equal to 44.5g lower birth weight representing 32.1% of the total effect [standard deviation (SD)=60.7g]. HDMAX2 also identified AMRs having simultaneous effects both on GA and on birth weight. Among the top hits of both GA and birth weight analyses, regions located in COASY, BLCAP, and ESRP2 also mediated the relationship between GA and birth weight, suggesting reverse causality in the relationship between GA and the methylome. DISCUSSION: HDMAX2 outperformed existing approaches and revealed an unsuspected complexity of the potential causal relationships between exposure to MS and birth weight at the epigenome-wide level. HDMAX2 is applicable to a wide range of tissues and omic layers. https://doi.org/10.1289/EHP11559.

Application of Genomic Sequencing to Refine Patient Stratification for Adjuvant Therapy in Renal Cell Carcinoma.

PURPOSE: Patients with resected localized clear-cell renal cell carcinoma (ccRCC) remain at variable risk of recurrence. Incorporation of biomarkers may refine risk prediction and inform adjuvant treatment decisions. We explored the role of tumor genomics in this setting, leveraging the largest cohort to date of localized ccRCC tissues subjected to targeted gene sequencing. EXPERIMENTAL DESIGN: The somatic mutation status of 12 genes was determined in 943 ccRCC cases from a multinational cohort of patients, and associations to outcomes were examined in a Discovery (n = 469) and Validation (n = 474) framework. RESULTS: Tumors containing a von-Hippel Lindau (VHL) mutation alone were associated with significantly improved outcomes in comparison with tumors containing a VHL plus additional mutations. Within the Discovery cohort, those with VHL+0, VHL+1, VHL+2, and VHL+≥3 tumors had disease-free survival (DFS) rates of 90.8%, 80.1%, 68.2%, and 50.7% respectively, at 5 years. This trend was replicated in the Validation cohort. Notably, these genomically defined groups were independent of tumor mutational burden. Amongst patients eligible for adjuvant therapy, those with a VHL+0 tumor (29%) had a 5-year DFS rate of 79.3% and could, therefore, potentially be spared further treatment. Conversely, patients with VHL+2 and VHL+≥3 tumors (32%) had equivalent DFS rates of 45.6% and 35.3%, respectively, and should be prioritized for adjuvant therapy. CONCLUSIONS: Genomic characterization of ccRCC identified biologically distinct groups of patients with divergent relapse rates. These groups account for the ∼80% of cases with VHL mutations and could be used to personalize adjuvant treatment discussions with patients as well as inform future adjuvant trial design.

DNA hypomethylation of the host tree impairs interaction with mutualistic ectomycorrhizal fungus.

Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown. By manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) and two demethylases DEMETER-LIKE (DML) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor. We compared the ectomycorrhizas formed between transgenic and wild-type (WT) trees and analyzed their methylomes and transcriptomes. The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi-ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX-dml and RNAi-ddm1) and 120 genes/1441 TEs in the fungal genome suggesting a host-induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes (cis and trans effects) in common with 17 being differentially methylated (cis). Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate-mediated pathways, and immune response but also terpenoid metabolism.

Intranasal administration of Acinetobacter lwoffii in a murine model of asthma induces IL-6-mediated protection associated with cecal microbiota changes.

BACKGROUND: Early-life exposure to certain environmental bacteria including Acinetobacter lwoffii (AL) has been implicated in protection from chronic inflammatory diseases including asthma later in life. However, the underlying mechanisms at the immune-microbe interface remain largely unknown. METHODS: The effects of repeated intranasal AL exposure on local and systemic innate immune responses were investigated in wild-type and Il6-/- , Il10-/- , and Il17-/- mice exposed to ovalbumin-induced allergic airway inflammation. Those investigations were expanded by microbiome analyses. To assess for AL-associated changes in gene expression, the picture arising from animal data was supplemented by in vitro experiments of macrophage and T-cell responses, yielding expression and epigenetic data. RESULTS: The asthma preventive effect of AL was confirmed in the lung. Repeated intranasal AL administration triggered a proinflammatory immune response particularly characterized by elevated levels of IL-6, and consequently, IL-6 induced IL-10 production in CD4+ T-cells. Both IL-6 and IL-10, but not IL-17, were required for asthma protection. AL had a profound impact on the gene regulatory landscape of CD4+ T-cells which could be largely recapitulated by recombinant IL-6. AL administration also induced marked changes in the gastrointestinal microbiome but not in the lung microbiome. By comparing the effects on the microbiota according to mouse genotype and AL-treatment status, we have identified microbial taxa that were associated with either disease protection or activity. CONCLUSION: These experiments provide a novel mechanism of Acinetobacter lwoffii-induced asthma protection operating through IL-6-mediated epigenetic activation of IL-10 production and with associated effects on the intestinal microbiome.

Epigenetically regulated PCDHB15 impairs aggressiveness of metastatic melanoma cells.

The protocadherin proteins are cell adhesion molecules at the crossroad of signaling pathways playing a major role in neuronal development. It is now understood that their role as signaling hubs is not only important for the normal physiology of cells but also for the regulation of hallmarks of cancerogenesis. Importantly, protocadherins form a cluster of genes that are regulated by DNA methylation. We have identified for the first time that PCDHB15 gene is DNA-hypermethylated on its unique exon in the metastatic melanoma-derived cell lines and patients' metastases compared to primary tumors. This DNA hypermethylation silences the gene, and treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine reinduces its expression. We explored the role of PCDHB15 in melanoma aggressiveness and showed that overexpression impairs invasiveness and aggregation of metastatic melanoma cells in vitro and formation of lung metastasis in vivo. These findings highlight important modifications of the methylation of the PCDHß genes in melanoma and support a functional role of PCDHB15 silencing in melanoma aggressiveness.

A meta-analysis of pre-pregnancy maternal body mass index and placental DNA methylation identifies 27 CpG sites with implications for mother-child health.

Higher maternal pre-pregnancy body mass index (ppBMI) is associated with increased neonatal morbidity, as well as with pregnancy complications and metabolic outcomes in offspring later in life. The placenta is a key organ in fetal development and has been proposed to act as a mediator between the mother and different health outcomes in children. The overall aim of the present work is to investigate the association of ppBMI with epigenome-wide placental DNA methylation (DNAm) in 10 studies from the PACE consortium, amounting to 2631 mother-child pairs. We identify 27 CpG sites at which we observe placental DNAm variations of up to 2.0% per 10 ppBMI-unit. The CpGs that are differentially methylated in placenta do not overlap with CpGs identified in previous studies in cord blood DNAm related to ppBMI. Many of the identified CpGs are located in open sea regions, are often close to obesity-related genes such as GPX1 and LGR4 and altogether, are enriched in cancer and oxidative stress pathways. Our findings suggest that placental DNAm could be one of the mechanisms by which maternal obesity is associated with metabolic health outcomes in newborns and children, although further studies will be needed in order to corroborate these findings.

Current and Emerging Technologies for the Analysis of the Genome-Wide and Locus-Specific DNA Methylation Patterns.

DNA methylation is the most studied epigenetic modification, and altered DNA methylation patterns have been identified in cancer and more recently also in many other complex diseases. Furthermore, DNA methylation is influenced by a variety of environmental factors, and the analysis of DNA methylation patterns might allow deciphering previous exposures. A number of techniques to study DNA methylation either genome-wide or at specific loci have been devised using a limited number of principles for differentiating the methylation state: (1) methylation-sensitive/dependent restriction enzymes, (2) antibody or methyl-binding protein-based enrichment, or (3) chemical or enzymatic conversion, (4) direct sequence readout. Second-generation sequencing has largely replaced microarrays as a readout platform and is also becoming more popular for locus-specific DNA methylation analysis. In this chapter, the currently used methods for both genome-wide and locus-specific analysis of 5-methylcytosine as well as its oxidative derivatives such as 5-hydroxymethylcytosine are reviewed in detail and advantages and limitations of each approach are discussed. Furthermore, emerging technologies avoiding PCR amplification and allowing a direct readout of DNA methylation are summarized, together with novel applications, such as the detection of DNA methylation in single cells or in circulating cell-free DNA.

At age 9, the methylome of assisted reproductive technology children that underwent embryo culture in different media is not significantly different on a genome-wide scale.

STUDY QUESTION: Can we detect DNA methylation differences between ART children that underwent embryo culture in different media? SUMMARY ANSWER: We identified no significant differences in site-specific or regional DNA methylation between the different culture medium groups. WHAT IS KNOWN ALREADY: Embryo culture in G3 or K-SICM medium leads to differences in embryonic, neonatal and childhood outcomes, including growth and weight. The methylome may mediate this association as the period of in vitro culture of ART treatments coincides with epigenetic reprogramming. STUDY DESIGN, SIZE, DURATION: This study was conducted as a follow-up to a previous culture medium comparison study in which couples were pseudo-randomized to embryo culture in G3 or K-SICM medium. Of the resultant singletons, 120 (n = 65 G3, n = 55 K-SICM), were recruited at age 9. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ART children provided a saliva sample from which the methylome was analysed using the Infinium MethylationEPIC array. After quality and context filtering, 106 (n = 57 G3, n = 49 K-SICM) samples and 659 708 sites were retained for the analyses. Differential methylation analyses were conducted using mixed effects linear models corrected for age, sex, sample plate and cell composition. These were applied to all cytosine-guanine dinucleotide (CpG) sites, various genomic regions (genes, promoters, CpG Islands (CGIs)) and as a targeted analysis of imprinted genes and birth weight-associated CpG sites. Differential variance was assessed using the improved epigenetic variable outliers for risk prediction analysis (iEVORA) algorithm and methylation outliers were identified using a previously defined threshold (upper or lower quartile plus or minus three times the interquartile range, respectively). MAIN RESULTS AND THE ROLE OF CHANCE: After correcting for multiple testing, we did not identify any significantly differentially methylated CpG sites, genes, promoters or CGIs between G3 and K-SICM children despite a lenient corrected P-value threshold of 0.1. Targeted analyses of (sites within) imprinted genes and birth weight-associated sites also did not identify any significant differences. The number of DNA methylation outliers per sample was comparable between the culture medium groups. iEVORA identified 101 differentially variable CpG sites of which 94 were more variable in the G3 group. LARGE SCALE DATA: Gene Expression Omnibus (GEO) GSE196432. LIMITATIONS, REASONS FOR CAUTION: To detect significant methylation differences with a magnitude of <10% between the groups many more participants would be necessary; however, the clinical relevance of such small differences is unclear. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study are reassuring, suggesting that if there is an effect of the culture medium on DNA methylation (and methylation-mediated diseases risk), it does not differ between the two media investigated here. The findings concur with other methylome studies of ART neonates and children that underwent embryo culture in different media, which also found no significant methylome differences. STUDY FUNDING/COMPETING INTEREST(S): Study funded by March of Dimes (6-FY13-153), EVA (Erfelijkheid Voortplanting & Aanleg) specialty programme (grant no. KP111513) of Maastricht University Medical Centre (MUMC+) and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission. The authors do not report any conflicts of interest relevant to this study. TRIAL REGISTRATION NUMBER: Dutch Trial register-NL4083.

Maternal blood pressure associates with placental DNA methylation both directly and through alterations in cell-type composition.

BACKGROUND: Maternal blood pressure levels reflect cardiovascular adaptation to pregnancy and proper maternal-fetal exchanges through the placenta and are very sensitive to numerous environmental stressors. Maternal hypertension during pregnancy has been associated with impaired placental functions and with an increased risk for children to suffer from cardiovascular and respiratory diseases later on. Investigating changes in placental DNA methylation levels and cell-type composition in association with maternal blood pressure could help elucidate its relationships with placental and fetal development. METHODS: Taking advantage of a large cohort of 666 participants, we investigated the association between epigenome-wide DNA methylation patterns in the placenta, measured using the Infinium HumanMethylation450 BeadChip, placental cell-type composition, estimated in silico, and repeated measurements of maternal steady and pulsatile blood pressure indicators during pregnancy. RESULTS: At the site-specific level, no significant association was found between maternal blood pressure and DNA methylation levels after correction for multiple testing (false discovery rate < 0.05), but 5 out of 24 previously found CpG associations were replicated (p-value < 0.05). At the regional level, our analyses highlighted 64 differentially methylated regions significantly associated with at least one blood pressure component, including 35 regions associated with mean arterial pressure levels during late pregnancy. These regions were found enriched for genes implicated in lung development and diseases. Further mediation analyses show that a significant part of the association between steady blood pressure-but not pulsatile pressure-and placental methylation can be explained by alterations in placental cell-type composition. In particular, elevated blood pressure levels are associated with a decrease in the ratio between mesenchymal stromal cells and syncytiotrophoblasts, even in the absence of preeclampsia. CONCLUSIONS: This study provides the first evidence that the association between maternal steady blood pressure during pregnancy and placental DNA methylation is both direct and partly explained by changes in cell-type composition. These results could hint at molecular mechanisms linking maternal hypertension to lung development and early origins of childhood respiratory problems and at the importance of controlling maternal blood pressure during pregnancy.

DNA methylome combined with chromosome cluster-oriented analysis provides an early signature for cutaneous melanoma aggressiveness.

Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients' outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations.