{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,21]],"date-time":"2026-02-21T04:34:03Z","timestamp":1771648443086,"version":"3.50.1"},"reference-count":48,"publisher":"Public Library of Science (PLoS)","issue":"1","license":[{"start":{"date-parts":[[2025,1,9]],"date-time":"2025-01-09T00:00:00Z","timestamp":1736380800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Department of Obstetrics and Gynaecology and the Department of Developmental Biology, Erasmus University Medical Centre, Rotterdam, The Netherlands"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Introduction<\/jats:title>\n<jats:p>Placental DNA methylation differences have been associated with timing in gestation and pregnancy complications. Maternal cell-free DNA (cfDNA) partly originates from the placenta and could enable the minimally invasive study of placental DNA methylation dynamics. We will for the first time longitudinally investigate cfDNA methylation during pregnancy by using Methylated DNA Sequencing (MeD-seq), which is compatible with low cfDNA levels and has an extensive genome-wide coverage. We aim to investigate DNA methylation in placental tissues and cfDNA during different trimesters in uncomplicated pregnancies, and in pregnancies with placental-related complications, including preeclampsia and fetal growth restriction. Identified gestational-age and disease-specific differentially methylated regions (DMRs) could lead to numerous applications including biomarker development.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methods and analysis<\/jats:title>\n<jats:p>Our study design involves three sub-studies. Sub-study 1 is a single-centre prospective, observational subcohort embedded within the Rotterdam Periconception cohort (Predict study). We will longitudinally collect maternal plasma in each trimester and during delivery, and sample postpartum placentas (n = 300). In sub-study 2, we will prospectively collect first and second trimester placental tissues (n = 10 per trimester). In sub-study 3 we will retrospectively collect plasma after non-invasive prenatal testing (NIPT) in an independent validation case-control cohort (n = 30\u201360). A methylation-dependent restriction enzyme (LpnPI) will be used to generate DNA fragments followed by sequencing on the Illumina NextSeq2000 platform. DMRs will be identified in placental tissues and cell types, and in cfDNA related to gestational-age or placental-related complications. (Paired) placental methylation profiles will be correlated to DMRs in cfDNA to aid tissue-of-origin analysis. We will establish a methylation score to predict associated diseases.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Discussion<\/jats:title>\n<jats:p>This study will provide insights in placental DNA methylation dynamics in health and disease, and could lead to clinical relevant biomarkers.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pone.0310019","type":"journal-article","created":{"date-parts":[[2025,1,9]],"date-time":"2025-01-09T18:48:08Z","timestamp":1736448488000},"page":"e0310019","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["Methylome profiling of cell-free DNA during the early life course in (un)complicated pregnancies using MeD-seq: Protocol for a cohort study embedded in the prospective Rotterdam periconception cohort"],"prefix":"10.1371","volume":"20","author":[{"given":"Marjolein M.","family":"van Vliet","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sam","family":"Schoenmakers","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3377-2897","authenticated-orcid":true,"given":"Ruben G.","family":"Boers","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4537-5645","authenticated-orcid":true,"given":"Lotte E.","family":"van der Meeren","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joost","family":"Gribnau","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4353-5756","authenticated-orcid":true,"given":"R\u00e9gine P. M.","family":"Steegers-Theunissen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,1,9]]},"reference":[{"issue":"1","key":"pone.0310019.ref001","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1038\/npp.2012.112","article-title":"DNA methylation and its basic function","volume":"38","author":"L Moore","year":"2012","journal-title":"Neuropsychopharmacology"},{"issue":"4 Suppl","key":"pone.0310019.ref002","doi-asserted-by":"crossref","first-page":"S182","DOI":"10.1016\/j.ajog.2015.07.011","article-title":"Epigenetic regulation of human placental function and pregnancy outcome: considerations for causal inference","volume":"213","author":"V Januar","year":"2015","journal-title":"Am J Obstet Gynecol"},{"issue":"3","key":"pone.0310019.ref003","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1093\/humupd\/dmq052","article-title":"Epigenetics and the placenta","volume":"17","author":"E Nelissen","year":"2011","journal-title":"Hum Reprod Update"},{"key":"pone.0310019.ref004","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1186\/1471-2164-12-529","article-title":"Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental\/stochastic factors","volume":"12","author":"B Novakovic","year":"2011","journal-title":"BMC Genomics"},{"issue":"1","key":"pone.0310019.ref005","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/s12864-020-07186-6","article-title":"Cell-specific characterization of the placental methylome","volume":"22","author":"V Yuan","year":"2021","journal-title":"BMC Genomics"},{"issue":"9","key":"pone.0310019.ref006","doi-asserted-by":"crossref","first-page":"1238","DOI":"10.1016\/j.devcel.2021.04.001","article-title":"Human placental cytotrophoblast epigenome dynamics over gestation and alterations in placental disease","volume":"56","author":"B Zhang","year":"2021","journal-title":"Dev Cell"},{"key":"pone.0310019.ref007","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.placenta.2020.09.004","article-title":"Global DNA methylation in placental tissues from pregnant with preeclampsia: A systematic review and pathway analysis","volume":"101","author":"OJ Cruz de","year":"2020","journal-title":"Placenta"},{"issue":"1","key":"pone.0310019.ref008","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/s13293-020-00313-8","article-title":"Systematic review supports the role of DNA methylation in the pathophysiology of preeclampsia: a call for analytical and methodological standardization","volume":"11","author":"A Cirkovic","year":"2020","journal-title":"Biol Sex Differ"},{"issue":"Suppl 1","key":"pone.0310019.ref009","doi-asserted-by":"crossref","first-page":"S23","DOI":"10.1016\/j.placenta.2017.12.013","article-title":"Utility of DNA methylation to assess placental health","volume":"64","author":"S Wilson","year":"2018","journal-title":"Placenta"},{"issue":"4","key":"pone.0310019.ref010","doi-asserted-by":"crossref","first-page":"530","DOI":"10.1080\/14767058.2017.1291620","article-title":"Epigenetic modifications of human placenta associated with preterm birth: a systematic review","volume":"31","author":"D Toure","year":"2018","journal-title":"J Matern Fetal Neonatal Med"},{"issue":"1","key":"pone.0310019.ref011","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12920-018-0466-3","article-title":"Comparison of DNA methylation profiles associated with spontaneous preterm birth in placenta and cord blood","volume":"12","author":"X Wang","year":"2019","journal-title":"BMC Med Genomics"},{"issue":"8","key":"pone.0310019.ref012","doi-asserted-by":"crossref","first-page":"1143","DOI":"10.1177\/1933719118766265","article-title":"Placental Defects: An Epigenetic Perspective","volume":"25","author":"S Deshpande","year":"2018","journal-title":"Reprod Sci"},{"issue":"1","key":"pone.0310019.ref013","doi-asserted-by":"crossref","first-page":"R23","DOI":"10.1530\/REP-16-0014","article-title":"Recent progress towards understanding the role of DNA methylation in human placental development","volume":"152","author":"T Bianco-Miotto","year":"2016","journal-title":"Reproduction"},{"issue":"7","key":"pone.0310019.ref014","doi-asserted-by":"crossref","first-page":"920","DOI":"10.4161\/epi.6.7.16079","article-title":"Infant growth restriction is associated with distinct patterns of DNA methylation in human placentas","volume":"6","author":"C Banister","year":"2011","journal-title":"Epigenetics"},{"key":"pone.0310019.ref015","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/j.placenta.2017.08.070","article-title":"Early- and late-onset preeclampsia and the tissue-specific epigenome of the placenta and newborn","volume":"58","author":"E Herzog","year":"2017","journal-title":"Placenta"},{"issue":"11","key":"pone.0310019.ref016","doi-asserted-by":"crossref","first-page":"2837","DOI":"10.3390\/ijms20112837","article-title":"The Role of Epigenetics in Placental Development and the Etiology of Preeclampsia","volume":"20","author":"C Apicella","year":"2019","journal-title":"Int J Mol Sci"},{"issue":"19","key":"pone.0310019.ref017","doi-asserted-by":"crossref","first-page":"2307","DOI":"10.1042\/CS20190070","article-title":"Epigenetic processes during preeclampsia and effects on fetal development and chronic health","volume":"135","author":"U Ashraf","year":"2021","journal-title":"Clin Sci (Lond)"},{"key":"pone.0310019.ref018","doi-asserted-by":"crossref","first-page":"759","DOI":"10.2174\/1566524016666161013142335","article-title":"Clinical experience of non-invasive prenatal chromosomal aneuploidy testing in 190,277 patient samples","volume":"16","author":"H Hu","year":"2016","journal-title":"Curr Mol Med"},{"issue":"2","key":"pone.0310019.ref019","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.molmed.2014.12.006","article-title":"Noninvasive fetal genomic, methylomic, and transcriptomic analyses using maternal plasma and clinical implications","volume":"21","author":"A Wong","year":"2015","journal-title":"Trends Mol Med"},{"issue":"2","key":"pone.0310019.ref020","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1097\/GCO.0000000000000252","article-title":"Noninvasive prenatal testing beyond genomic analysis: what the future holds","volume":"28","author":"W Hui","year":"2016","journal-title":"Curr Opin Obstet Gynecol"},{"issue":"1","key":"pone.0310019.ref021","doi-asserted-by":"crossref","first-page":"19837","DOI":"10.1038\/s41598-022-24348-6","article-title":"Hypertensive disorders of pregnancy share common cfDNA methylation profiles","volume":"12","author":"M Spinelli","year":"2022","journal-title":"Sci Rep"},{"issue":"9","key":"pone.0310019.ref022","doi-asserted-by":"crossref","first-page":"2206","DOI":"10.1038\/s41591-023-02510-5","article-title":"Cell-free DNA methylome analysis for early preeclampsia prediction","volume":"29","author":"M De Borre","year":"2023","journal-title":"Nat Med"},{"issue":"2","key":"pone.0310019.ref023","doi-asserted-by":"crossref","first-page":"2282100","DOI":"10.1080\/01443615.2023.2282100","article-title":"Epigenetic phenotype of plasma cell-free DNA in the prediction of early-onset preeclampsia","volume":"43","author":"Dec","year":"2023","journal-title":"J Obstet Gynaecol"},{"key":"pone.0310019.ref024","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.diabres.2018.05.002","article-title":"The first-trimester of pregnancy\u2014A window of opportunity for prediction and prevention of pregnancy complications and future life","volume":"145","author":"L Poon","year":"2018","journal-title":"Diabetes Res Clin Pract"},{"issue":"1","key":"pone.0310019.ref025","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1038\/s41572-023-00417-6","article-title":"Pre-eclampsia","volume":"9","author":"E Dimitriadis","year":"2023","journal-title":"Nat Rev Dis Primers"},{"issue":"3","key":"pone.0310019.ref026","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1016\/j.ajog.2017.11.561","article-title":"Aspirin for the prevention of preterm and term preeclampsia: systematic review and metaanalysis","volume":"218","author":"S Roberge","year":"2018","journal-title":"Am J Obstet Gynecol"},{"issue":"12","key":"pone.0310019.ref027","doi-asserted-by":"crossref","first-page":"4238","DOI":"10.18632\/aging.102049","article-title":"Placental epigenetic clocks: estimating gestational age using placental DNA methylation levels","volume":"11","author":"Y Lee","year":"2019","journal-title":"Aging (Albany NY)"},{"issue":"2","key":"pone.0310019.ref028","doi-asserted-by":"crossref","first-page":"606","DOI":"10.1373\/clinchem.2016.265702","article-title":"Gestational Age Assessment by Methylation and Size Profiling of Maternal Plasma DNA: A Feasibility Study","volume":"63","author":"P Jiang","year":"2017","journal-title":"Clin Chem"},{"issue":"2","key":"pone.0310019.ref029","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/S1701-2163(15)30664-2","article-title":"Determination of gestational age by ultrasound.","volume":"36","author":"K Butt","year":"2014","journal-title":"J Obstet Gynaecol Can"},{"issue":"1","key":"pone.0310019.ref030","doi-asserted-by":"crossref","first-page":"5068","DOI":"10.1038\/s41467-018-07466-6","article-title":"Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease","volume":"9","author":"J Moss","year":"2018","journal-title":"Nat Commun"},{"issue":"2","key":"pone.0310019.ref031","doi-asserted-by":"crossref","first-page":"198","DOI":"10.1038\/s41431-018-0271-7","article-title":"Fetal fraction evaluation in non-invasive prenatal screening (NIPS)","volume":"27","author":"M Hestand","year":"2019","journal-title":"Eur J Hum Genet"},{"key":"pone.0310019.ref032","doi-asserted-by":"crossref","first-page":"812781","DOI":"10.3389\/fped.2022.812781","article-title":"Factors Affecting the Fetal Fraction in Noninvasive Prenatal Screening: A Review","volume":"10","author":"C Deng","year":"2022","journal-title":"Front Pediatr"},{"issue":"13","key":"pone.0310019.ref033","doi-asserted-by":"crossref","first-page":"E65","DOI":"10.1093\/nar\/29.13.e65","article-title":"Bisulfite genomic sequencing: systematic investigation of critical experimental parameters","volume":"29","author":"C Grunau","year":"2001","journal-title":"Nucleic Acids Res"},{"issue":"105\u2013115","key":"pone.0310019.ref034","article-title":"Strategies for analyzing bisulfite sequencing data","volume":"261","author":"K Wreczycka","year":"2017","journal-title":"J Biotechnol"},{"key":"pone.0310019.ref035","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1016\/j.ymeth.2020.06.022","article-title":"Modern epigenetics methods in biological research","volume":"187","author":"Y. Li","year":"2021","journal-title":"Methods"},{"issue":"3","key":"pone.0310019.ref036","doi-asserted-by":"crossref","first-page":"744","DOI":"10.1002\/acn3.51997","article-title":"Distinctive cell-free DNA methylation characterizes presymptomatic genetic frontotemporal dementia","volume":"11","author":"L Giannini","year":"2024","journal-title":"Ann Clin Transl Neurol"},{"issue":"18","key":"pone.0310019.ref037","doi-asserted-by":"crossref","first-page":"4607","DOI":"10.3390\/cancers15184607","article-title":"Liquid Biopsies for Colorectal Cancer and Advanced Adenoma Screening and Surveillance: What to Measure?","volume":"15","author":"E Eikenboom","year":"2023","journal-title":"Cancers (Basel)"},{"issue":"5","key":"pone.0310019.ref038","doi-asserted-by":"crossref","first-page":"1374","DOI":"10.3390\/cancers15051374","article-title":"Menke-van der Houven van Oordt W, Boers R, Boers J, et al. Methylated Cell-Free DNA Sequencing (MeD-seq) of LpnPI Digested Fragments to Identify Early Progression in Metastatic Renal Cell Carcinoma Patients on Watchful Waiting","volume":"15","author":"M Bos","year":"2023","journal-title":"Cancers (Basel)"},{"issue":"11","key":"pone.0310019.ref039","doi-asserted-by":"crossref","first-page":"1582","DOI":"10.1038\/s41587-023-01683-1","article-title":"Retrospective analysis of enhancer activity and transcriptome history","volume":"41","author":"R Boers","year":"2023","journal-title":"Nat Biotechnol"},{"issue":"1","key":"pone.0310019.ref040","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1101\/gr.222885.117","article-title":"Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI","volume":"28","author":"R Boers","year":"2018","journal-title":"Genome Res"},{"issue":"1","key":"pone.0310019.ref041","doi-asserted-by":"crossref","DOI":"10.1186\/s13148-021-01177-4","article-title":"High-throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by methylated DNA sequencing (MeD-seq) of LpnPI digested fragments","volume":"13","author":"T Deger","year":"2021","journal-title":"Clinical Epigenetics"},{"key":"pone.0310019.ref042","article-title":"Cohort Profile Update: the Rotterdam Periconceptional Cohort and embryonic and fetal measurements using 3D ultrasound and virtual reality techniques","author":"M Rousian","year":"2021","journal-title":"Int J Epidemiol"},{"issue":"2","key":"pone.0310019.ref043","doi-asserted-by":"crossref","first-page":"374","DOI":"10.1093\/ije\/dyv147","article-title":"Cohort Profile: The Rotterdam Periconceptional Cohort (Predict Study)","volume":"45","author":"R Steegers-Theunissen","year":"2016","journal-title":"Int J Epidemiol"},{"key":"pone.0310019.ref044","doi-asserted-by":"crossref","first-page":"1091","DOI":"10.1016\/j.ajhg.2019.10.005","article-title":"TRIDENT-2: national implementation of genome-wide non-invasive prenatal testing as a first-tier screening test in the Netherlands","volume":"105","author":"K van der Meij","year":"2019","journal-title":"Am J Hum Genet"},{"issue":"7","key":"pone.0310019.ref045","doi-asserted-by":"crossref","first-page":"1265","DOI":"10.1111\/aogs.14091","article-title":"Uptake of fetal aneuploidy screening after the introduction of the non-invasive prenatal test: A national population-based register study","volume":"100","author":"K Van der Meij","year":"2021","journal-title":"Acta Obstet Gynecol Scand"},{"key":"pone.0310019.ref046","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1016\/j.preghy.2021.09.008","article-title":"The 2021 International Society for the Study of Hypertension in Pregnancy classification, diagnosis & management recommendations for international practice","volume":"27","author":"L Magee","journal-title":"Pregnancy Hypertens"},{"issue":"20","key":"pone.0310019.ref047","doi-asserted-by":"crossref","DOI":"10.1242\/dev.199626","article-title":"RNA profiling of laser microdissected human trophoblast subtypes at mid-gestation reveals a role for cannabinoid signaling in invasion","volume":"148","author":"M Gormley","year":"2021","journal-title":"Development"},{"key":"pone.0310019.ref048","article-title":"DNA methylation database for gynecological cancer detection, classification and assay development","author":"J Boers","year":"2024","journal-title":"bioRxiv"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0310019","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,1,9]],"date-time":"2025-01-09T18:48:22Z","timestamp":1736448502000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0310019"}},"subtitle":[],"editor":[{"given":"Monica","family":"Miozzo","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2025,1,9]]},"references-count":48,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,1,9]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0310019","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,9]]}}}