Maternal caffeine consumption during pregnancy and offspring cord blood DNA methylation: a meta-analysis of epigenome-wide association studies

Background Women are advised to limit caffeine consumption during pregnancy. To uncover the potential epigenetic effects of intrauterine caffeine exposure, we investigated the association of maternal caffeine consumption during pregnancy with genome-wide DNA methylation in cord blood. Methods We meta-analysed results from epigenome-wide methylation studies at individual probes and in differentially methylated region (DMR) analysis across 6 European pregnancy and birth cohorts (ALSPAC, BiB, MoBa, Generation R, INMA, EDEN; total n = 3742). Methylation was assessed with lllumina Infinium 450k or EPIC arrays. Maternal caffeine consumption (mg/day) from coffee, tea and cola was derived from questionnaires between weeks 12 - 22 of pregnancy. We investigated associations of methylation with overall and beverage-specific caffeine intake in models adjusted for maternal education, age, BMI, smoking during pregnancy, parity, cord-blood cell proportions and 20 surrogate variables. Results One CpG site (cg19370043, nearest gene PRRX1 ) was associated with maternal caffeine consumption after FDR adjustment for multiple testing and one CpG sites (cg14591243, nearest gene STAG1 ) was associated with maternal cola consumption. We found evidence for 12-22 DMRs for each of the caffeine models but little overlap between DMRs identified for specific caffeinated beverages. Conclusions In models adjusted for maternal smoking and other potential confounders, we found little evidence to support an intrauterine effect of caffeine on offspring DNA methylation. Comparing associations across different sources of caffeine provided no evidence for caffeine being the causal agent. It is possible that our study did not have adequate statistical power to detect very small associations between maternal caffeine and offspring DNA methylation. Key messages ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the UK Medical Research Council and Wellcome (Grant ref: 217065/Z/19/Z) and the University of Bristol provide core support for ALSPAC. A comprehensive list of grants funding is available on the ALSPAC website (http://www.bristol.ac.uk/alspac/external/documents/grant-acknowledgements.pdf This research was performed in the UK Medical Research Council Integrative Epidemiology Unit (grant number: MC\_UU\_00011/7 and MC\_UU\_00011/5) and also supported by the National Institute for Health Research (NIHR) Bristol Biomedical Research Centre at University Hospitals Bristol NHS Foundation Trust and the University of Bristol. This research was also conducted as part of the CAPICE (Childhood and Adolescence Psychopathology: unravelling the complex etiology by a large Interdisciplinary Collaboration in Europe) project, funded by the European Unions Horizon 2020 research and innovation programme, Marie Sklodowska Curie Actions MSCA-ITN-2016 Innovative Training Networks under grant agreement number 721567. This study was supported by the NIHR Biomedical Research Centre at the University Hospitals Bristol NHS Foundation Trust and the University of Bristol. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health and Social Care. GCS is financially supported by an MRC New Investigator Research (grant code MR/S009310/1), an MRC project grant (MR/W020297/1) and the European Joint Programming Initiative A Healthy Diet for a Healthy Life (JPI HDHL, NutriPROGRAM project, UK MRC MR/S036520/1) BiB receives core funding from the Wellcome Trust (WT101597MA), the British Heart Foundation (CS/16/4/32482), a joint grant from the UK Medical Research Council (MRC) and UK Economic and Social Science Research Council (ESRC) (MR/N024397/1) and the National Institute for Health Research (NIHR) under its Collaboration for Applied Health Research and Care (CLAHRC) for Yorkshire and Humber. The research presented in this paper, including obtaining genome-wide and epigenome-wide DNAm data is supported by the US National Institute of Health (R01 DK10324) and European Research Council under the European Unions Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement no 669545. DAL and GCS work in a unit that receives support from the University of Bristol and UK MRC (MC\_UU\_00011/6) and DAL is an NIHR senior investigator (NF-SI-330 0611-10196). The general design of the Generation R Study is made possible by financial support from the Erasmus MC, Erasmus University Rotterdam, the Netherlands Organization for Health Research and Development and the Ministry of Health, Welfare and Sport. The EWAS data were funded by a grant to VWJ from the Netherlands Genomics Initiative (NGI)/Netherlands Organisation for Scientific Research (NWO) Netherlands Consortium for Healthy Aging (NCHA; project nr. 050-060-810), by funds from the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, and by a grant from the National Institute of Child and Human Development (R01HD068437). V.W.J. received a Consolidator Grant from the European Research Council (ERC-2014-CoG-648916). This project received funding from the European Unions Horizon 2020 research and innovation programme (733206, LifeCycle; 874739, LongITools; 874583, ATHLETE; 824989, EUCAN-Connect) and from the European Joint Programming Initiative A Healthy Diet for a Healthy Life (JPI HDHL, NutriPROGRAM project, ZonMw the Netherlands no.529051022 and PREcisE project ZonMw the Netherlands no.529051023). The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research. For this work, MoBa 1 and 2 were supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Z01-ES-49019) and the Norwegian Research Council/BIOBANK (grant no 221097). This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 262700. MoBa3 epigenomics data analyses were funded by INCA/Plan Cancer-EVA-INSERM, France, and the International Childhood Cancer Cohort Consortium (I4C), and performed by the Epigenetics Group at the International Agency for Research on Cancer (IARC, Lyon, France). Where authors are identified as personnel of the International Agency for Research on Cancer / World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer / World Health Organization. This study was funded by grants from Instituto de Salud Carlos III (Red INMA G03/176; CB06/02/0041; PI041436; PI081151 incl. FEDER funds), Generalitat de Catalunya-CIRIT 1999SGR 00241, Fundacio La marato de TV3 (090430), EU Commission (261357-MeDALL: Mechanisms of the Development of ALLergy), European Research Council (268479-BREATHE: BRain dEvelopment and Air polluTion ultrafine particles in scHool children, and the European Joint Programming Initiative A Healthy Diet for a Healthy Life (JPI HDHL and Instituto de Salud Carlos III) under the grant agreement no AC18/00006 (NutriPROGRAM project). We acknowledge support from the Spanish Ministry of Science and Innovation and the State Research Agency through the Centro de Excelencia Severo Ochoa 2019-2023 Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. Foundation for medical research (FRM), National Agency for Research (ANR), National Institute for Research in Public health (IRESP: TGIR cohorte sante 2008 program), French Ministry of Health (DGS), French Ministry of Research, INSERM Bone and Joint Diseases National Research (PRO-A) and Human Nutrition National Research Programs, Paris-Sud University, Nestle, French National Institute for Population Health Surveillance (InVS), French National Institute for Health Education (INPES), the European Union FP7 programs (FP7/2007-2013, HELIX, ESCAPE, ENRIECO, Medall projects), Diabetes National Research Program (in collaboration with the French Association of Diabetic Patients (AFD), French Agency for Environmental Health Safety (now ANSES), Mutuelle Generale de l Education Nationale complementary health insurance (MGEN), French national agency for food security, French speaking association for the study of diabetes and metabolism (ALFEDIAM), grant # 2012/51290-6 Sao Paulo Research Foundation (FAPESP), EU funded MeDALL project. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: For ALSPAC, ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees. Informed consent for the use of data collected via questionnaires and clinics was obtained from participants following the recommendations of the ALSPAC Ethics and Law Committee at the time. For BiB, ethical approval for the data collection was granted by Bradford Research Ethics Committee (Ref 07/H1302/112). On registration with the study, pregnant mothers gave written informed consent for themselves and on behalf of their child. The establishment of MoBa and initial data collection was based on a license from the Norwegian Data Protection Agency and approval from The Regional Committees for Medical and Health Research Ethics. MoBa is currently regulated by the Norwegian Health Registry Act. The study was approved by the Regional Committee for Ethics in Medical Research, Norway (#2017/1362). In addition, MoBa1 was approved by the Institutional Review Board of the National Institute of Environmental Health Sciences, USA. GenerationR was approved by Medical Ethical Committee of Erasmus MC, University Medical Center Rotterdam and written consent was obtained for all participants. For INMA, informed consent was obtained from all participants and the study was approved by the Hospital Ethics Committees in each participating region. For EDEN, ethics approval for data collection was granted by the ethics committee of Kremlin Bicetre and the Commission Nationale Informatique et Liberte. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Access to all data used in the present study can be requested from the individual cohort studies.