Epigenetic Modifications of Umbilical Cord Blood and Preeclampsia: a Systematic Review and Metanalysis Protocol

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Umbilical cord blood offers a valuable substrate to investigate the early molecular imprint of preeclampsia and its potential role in developmental programming. Although numerous studies have reported global and gene-specific DNA methylation changes in cord blood, findings are inconsistent, particularly regarding the direction and magnitude of hypo- or hypermethylation. To address these discrepancies, this protocol outlines a rigorous and reproducible framework for a systematic review and meta-analysis aimed at synthesizing evidence on epigenetic modifications in umbilical cord blood associated with preeclampsia and their relationships with maternal and neonatal characteristics. Methods This study, designed in accordance with the PRISMA 2020 guidelines, will assess epigenetic modifications in umbilical cord blood associated with preeclampsia. Observational studies and clinical trials comparing preeclamptic and normotensive pregnancies and analysing DNA methylation or other epigenetic markers will be included. A comprehensive search will be conducted in PubMed, Web of Science, Scopus, and CENTRAL, supplemented by grey literature sources. Study selection, data extraction, and risk of bias assessment (ROB 2, ROBINS-I) will be performed independently by two reviewers. A meta-analysis will be conducted using RevMan according to heterogeneity, with subgroup analyses, assessment of publication bias, and GRADE evaluation of the certainty of evidence. Ethics and dissemination: The studies included in this review are presumed to have obtained the appropriate ethical approvals in accordance with applicable local and international regulations, umbilical cord blood. Conclusion In conclusion, this study will provide a comprehensive and methodologically robust synthesis of the evidence on epigenetic modifications in umbilical cord blood associated with preeclampsia. By clarifying the direction, agnitude, and consistency of reported alterations, and by exploring sources of heterogeneity across studies, this work will strengthen the understanding of the early-life epigenetic imprint of preeclampsia. The findings are expected to inform future mechanistic research, improve risk stratification strategies, and guide the development of preventive and early-life interventions targeting long-term maternal and offspring health. PROSPERO registration number : CRD420261301497 Epigenetic modification DNA methylation cord blood Pre-eclampsia maternal complication Introduction Preeclampsia remains one of the most perplexing paradoxes in obstetric medicine: a disorder specific to pregnancy, long silent, but capable of progressing to severe hypertension and multiorgan damage, jeopardizing the prognosis for both mother and foetus [ 1 – 4 ]. Affecting 2 to 8% of pregnancies worldwide, it remains one of the leading causes of perinatal morbidity and mortality, with 50,000 to 60,000 deaths reported each year [ 5 – 8 ]. In resource-limited countries, its impact is particularly pronounced, with Africa accounting for up to 9% of preeclampsia-related deaths, with some of the highest rates in Nigeria, South Africa, and Ethiopia [ 9 – 11 ]. Despite decades of research, preeclampsia remains a disease with unclear origins. Its development results from a complex interaction between genetic predispositions, immunological factors, and environmental influences [ 12 – 14 ]. Disruptions in the remodelling of spiral arteries by trophoblasts, placental hypoxia, and oxidative stress are central mechanisms, but they are insufficient to explain the entire clinical picture [ 15 – 17 ]. Traditional risk factors, maternal age, obesity, chronic hypertension, autoimmune or endocrine diseases, are also unable to reliably predict the disease [ 18 – 20 ]. Against this backdrop of uncertainty, the emergence of epigenetics has profoundly changed perspectives. Recent work shows that preeclampsia is associated with epigenetic changes, particularly alterations in DNA methylation, both in the placenta and in the cord blood of newborns exposed in utero [ 21 – 23 ]. Cord blood, rich in hematopoietic cells and nucleated red blood cells, provides a unique model for exploring foetal programming [24]. Several studies have reported differential methylation profiles in children born from preeclamptic pregnancies, identifying genes and pathways involved in the cardiovascular and endothelial systems [ 25 – 27 ]. However, the results remain inconsistent: some teams describe overall hypomethylation [ 28 ], while others report hypermethylation [ 4 , 29 ]. These discrepancies suggest the influence of confounding factors, particularly gestational age [ 30 ]. Furthermore, the role of the maternal environment, particularly nutrition, remains an under-explored area. Nutrients such as folate and choline, which play a key role in the metabolism of methyl donors, are likely to modulate foetal genome methylation [ 31 – 33 ]. However, their involvement in placental programming and in the risk of complications such as preeclampsia remains poorly characterized [ 34 – 36 ]. Thus, despite a wealth of literature, there is currently no rigorous synthesis that provides a coherent overview of the specific epigenetic alterations in cord blood in preeclampsia, nor does it identify their biological or clinical value. Existing studies vary considerably in terms of methodology, the epigenomic platforms used, the covariates included, and the populations studied, making it difficult to arrive at a uniform interpretation. Given these uncertainties, a systematic review is needed to: gather and compare available data on epigenetic modifications in cord blood associated with preeclampsia; assess the consistency of reported signatures; identify recurring methodological limitations; and shed light on the potential of these markers as tools for understanding, prediction, or early diagnosis. By synthesizing the interactions between genetics, maternal environment, and foetal epigenetic programming, this review aims to clarify the mechanisms by which preeclampsia leaves its mark from birth, and potentially far beyond. In the long term, this synthesis could contribute to a better understanding of the biological mechanisms linking preeclampsia to the child’s long-term health trajectories, paving the way for early prevention strategies and the identification of biomarkers useful in translational research. Research Question What is the effect of pre-eclampsia on global and gene specific DNA methylation in cord blood among women with and without pre-eclampsia. Objectives To analyse the global DNA methylation in cord blood of women with and without pre-eclampsia To analyse specific genes which are modified in cord blood of women with and without pre-eclampsia. To investigate relationship between maternal lifestyle and epigenetic modification in cord blood. To investigate relationship between epigenetic modification in cord blood and anthropometric measurements of neonates. Methodology Protocol and Registration This work is in line with the PRISMA-P 2020 guidelines [ 37 ]. The study protocol was prospectively registered in the PROSPERO database (ID: CRD420261301497) on February 05, 2026, to ensure methodological transparency and rigor. Inclusion Criteria Participants consist of mothers and newborns whose umbilical cord blood has been analyzed for epigenetic changes in the context of preeclampsia. Intervention The procedure involves the assessment of epigenetic modifications, including DNA methylation analyses and other epigenetic markers in cord blood. Comparison The comparison must be made between samples from pregnancies complicated by preeclampsia and those from uncomplicated or normotensive pregnancies. Outcomes The primary outcome is the characterization of specific epigenetic alterations associated with preeclampsia in umbilical cord blood. Secondary outcomes focus on exploring the relationship between these epigenetic modifications and maternal and neonatal demographic, clinical, or biological parameters. Study Design Study design includes observational studies such as cohorts, case-control studies, or cross-sectional studies analyzing epigenetic profiles in cord blood in relation to preeclampsia. However, clinical trials that collected umbilical cord blood from children of preeclamptic and normotensive mothers and evaluated epigenetic markers will also be considered relevant. Exclusion Criteria The exclusion criteria for this review will eliminate all studies that do not analyze umbilical cord blood or that do not focus on preeclamptic and normotensive pregnancies, as well as those that do not include an assessment of epigenetic changes. Studies that do not allow for comparison between preeclampsia and uncomplicated pregnancies, those that do not report epigenetic alterations associated with preeclampsia or maternal and neonatal demographic, clinical, or biological parameters, as well as studies whose design does not correspond to the types selected, including animal studies, in vitro research, non-original articles, or protocols without data. Finally, studies that do not provide sufficient information to assess the relationship between preeclampsia and epigenetic modifications in cord blood will be excluded. studies published in non-English languages show be excluded. Literature sources and retrieval strategy Two independent reviewers (names) will conduct a systematic and comprehensive literature search in four major electronic databases: PubMed, Web of Science, Scopus, and the Cochrane Central Register of Controlled Trials (CENTRAL), covering the period from the creation of the databases to the present. The search strategy will be meticulously designed to incorporate both controlled vocabulary (e.g., Medical Subject Headings [MeSH] in PubMed, TITLE-ABS-KEY in Scopus) and relevant free-text terms. This strategy will simultaneously target three key conceptual areas: the target population (“children born to mothers with preeclampsia” OR “umbilical cord blood collected at birth after a pregnancy with preeclampsia”), the specific intervention (“presence of specific epigenetic modifications” OR “DNA methylation levels” OR "microRNA expression profiles (miRNA) expression profiles“ OR ”Post-translational modifications of histones“) and the required study design (”analytical observational studies“ OR ”randomized controlled trial"). In order to ensure methodological rigor and optimize the sensitivity and specificity of the search, the search syntax proposed for each database will undergo formal peer review using the PRESS (Peer Review of Electronic Search Strategies) checklist. The search strategy was conducted using the following keywords: epigenetic or DNA methylation or microRNA or miRNA or histone or acetylation) AND cord blood AND preeclampsia. Aware of the risk that database searches may not identify all eligible evidence, additional search methods will be rigorously used. These will include manual review of reference lists of all included studies and relevant systematic reviews, systematic review of key conference proceedings (e.g., major meetings of obstetric gynecology, Neonatology/Perinatal Medicine associations) Perinatal, and Maternal-Fetal Medicine associations) and searching leading in the International Clinical Trials Registry Platform ( http://apps.who.int/trialsearch/ ), in ClinicalTrials.gov ( www.clinicaltrials.gov ), and in Google Scholar ( https://scholar.google.dk/ ) to identify ongoing or unpublished trials. All documents retrieved using these combined methods will be collated using reference management software (EndNote), and duplicate citations will be systematically removed before the selection process begins. Literature Screening and Data Extraction To streamline the process of review and selection, initially, search findings will be automatically exported to Endnote for duplication removal before being transferred to the Rayyan software [ 38 , 39 ]. Literature screening and data extraction will be performed by two independent reviewers, in accordance with PRISMA recommendations. Titles and abstracts will first be evaluated according to eligibility criteria, followed by a full-text review of potentially eligible studies. Any disagreements will be resolved through discussion or, if necessary, by arbitration by a third reviewer (specify their initials). In the event of missing data, the corresponding authors will be contacted; if no response is received, the analyses will be conducted on the available data, with an assessment of the impact of the missing data using sensitivity analyses. The complete selection process will be illustrated in a PRISMA diagram (Page et al., 2021). Data extraction will be based on a pre-tested standardized form, with both reviewers independently collecting key information before cross-checking. The extracted data will include general study characteristics, diagnostic criteria for preeclampsia, maternal and neonatal characteristics, cord blood collection and analysis methods, and epigenetic approaches used, including methylation measurement techniques or other epigenetic markers. The extracted results will focus on epigenetic alterations associated with preeclampsia (primary outcome) and their relationships with maternal and neonatal demographic, clinical, or biological parameters (secondary outcomes), including relevant analytical and statistical details. The final datasets will be verified for accuracy prior to any quantitative or narrative synthesis. Assessment of Risk of Bias The risk of bias assessment will be performed independently by two reviewers to ensure methodological rigor. For included randomized trials, the Cochrane Collaboration's ROB 2 tool will be applied systematically to examine potential biases related to the randomization process, deviations from planned interventions, missing outcome data, outcome measurement methods, and the risk of selective reporting of results [ 40 ]. For non-randomized studies, including observational studies frequently used in research on epigenetic modifications of cord blood in the context of preeclampsia, ROBINS-I will be used to assess bias [ 41 ] related to confounding factors, participant selection procedures (including identification of preeclampsia cases and normotensive controls), classification of exposures or comparators, deviations from planned methods, missing data, measurement of epigenetic outcomes, and the possibility of selective reporting. Any discrepancies between reviewers will be resolved through discussion, and in the event of persistent disagreement, a third senior reviewer will provide final arbitration. Statistical Analysis Statistical analyses will be performed using Review Manager software (RevMan, version 5.4) for quantitative synthesis and graphical presentation of results in the form of forest plots. When studies provide data that are sufficiently homogeneous for pooling, continuous measures of epigenetic alterations (e.g., methylation percentages or expression levels of epigenetic markers) will be synthesized as mean differences or standardized mean differences with 95% confidence intervals, using the inverse variance method. In cases where studies report categorical results (e.g., presence or absence of significantly differentially methylated regions), these will be pooled using risk ratios and 95% confidence intervals. The choice between fixed-effect and random-effect models will be determined by statistical heterogeneity, assessed using the I² statistic; a random-effect model will be preferred when heterogeneity is substantial [42,43]. I² equal to 25%, 50%, and between 75% to 100% are considered as low, medium, and high heterogeneity respectively. If heterogeneity prevents a robust meta-analysis, a structured narrative synthesis will be performed. Exploration of sources of heterogeneity, including methodological variations between epigenetic platforms, differences in diagnostic criteria for preeclampsia, gestational age, or variations in cord blood collection and processing methods, may be examined using exploratory analyses, including meta-regressions when data allow, using STATA. The risk of publication bias will be assessed by visual inspection of funnel plots and statistical tests such as Egger's test, but only when at least ten studies contribute to the analysis of the same outcome. The identification of publication bias occurs when the p-value is less than 0.05 [ 44 ]. If conducting a meta-analysis is not possible, a critical synthesis will be conducted in accordance with Calderon et al. guidelines [ 45 ]. Lastly, in compliance with PRISMA guidelines, the findings will be painstakingly documented and submitted for publication [ 46 ]. Our research findings will be reliable, transparent, and rigorous thanks to this thorough approach. Subgroup Analysis and Sensitivity Analysis Subgroup analyses will be performed to explore factors that may modify the association between preeclampsia and epigenetic alterations observed in cord blood. These pre-specified analyses will examine, in particular, the influence of the severity of preeclampsia (mild vs. severe preeclampsia), the time of onset (early vs. late), gestational age at delivery, neonatal sex, birth weight, relevant maternal characteristics (age, parity, body mass index), as well as methodological differences such as the type of epigenetic platform used (e.g., methylation chips, bisulfite sequencing, non-coding RNA analyses) or the quality of sample processing (time to freezing, storage conditions). In addition, other parameters such as lifestyle (passive or active smoking, alcoholism), as well as associated pathologies, will be considered. When sufficient studies are available, these subgroups will make it possible to examine whether certain biological, clinical, or technical characteristics influence the magnitude or direction of the epigenetic alterations identified. Certainty of Evidence Assessment The certainty of the evidence will be systematically assessed using the GRADE approach with the GRADEpro GDT online tool [47,48]. This assessment will cover five key areas, including the risk of bias in the included studies, inconsistency of results between studies, indirectness of evidence in relation to the research question, imprecision of effect estimates, and risk of publication bias. Particular attention will be paid to the specific characteristics of epigenetic studies of cord blood, including methodological quality, heterogeneity of analytical platforms, and sample size. For each outcome, whether epigenetic alterations associated with preeclampsia or their links with maternal and neonatal parameters, a level of certainty will be assigned (high, moderate, low, or very low), with justification documented in GRADE evidence profile tables. Patient and Public Involvement This systematic review did not involve patients or the public in its design, conduct, or presentation, as it synthesizes existing published data. All analyses use secondary data from original previous studies, for which patient consent was obtained during the initial conduct of the study. Ethics and Dissemination Since this study is based exclusively on secondary analysis of data already published in the scientific literature, no formal ethical approval or additional informed consent is required. The primary studies included in the review are presumed to have obtained the appropriate ethical approvals in accordance with applicable local and international regulations, particularly with regards to the collection and analysis of biological samples such as umbilical cord blood. The full results of this systematic review will be submitted for publication in peer-reviewed international scientific journals to ensure rigorous and validated dissemination of knowledge. The main findings will also be presented at relevant scientific conferences and meetings in the fields of obstetrics, perinatology, and epigenetics, in order to promote discussion with the scientific community. With a view to ensuring rapid and open dissemination of the results, the manuscript may also be submitted to recognized preprint platforms, such as medRxiv. In accordance with the principles of open science and reproducibility, all extracted data, extraction forms, analysis scripts, and methodological documents will be deposited in the Open Science Framework (OSF) public repository. Supplementary materials for the publication will provide a direct link to this repository to facilitate access, transparency, and reuse of the data by interested researchers. Data Management and Reproducibility Statement Data management and reproducibility of this systematic review will be ensured in accordance with the principles of open science and the PRISMA-P recommendations. All study materials, including anonymized and aggregated data extracted from the included articles, standardized data extraction forms, and analysis scripts used for statistical and narrative synthesis (notably in RevMan version 5.4 and Stata version 19), will be deposited in the Open Science Framework (OSF) public repository at the time of publication. The data will be organized in a structured and documented manner to enable reuse and independent verification of the results, while respecting the confidentiality of the participants in the primary studies. The analytical codes will be fully commented on and accompanied by a clear description of the analytical steps, including the processing of epigenetic data, the management of methodological heterogeneity, and sensitivity analyses. The final manuscript and supplementary materials will provide a direct link to the OSF repository, ensuring free and permanent access to all resources necessary for the complete reproduction of the analyses and transparency of the scientific process. Discussion Preeclampsia remains one of the leading causes of maternal and neonatal morbidity and mortality worldwide, with immediate and long-term consequences for both mother and child [ 49 ]. Beyond perinatal clinical complications, a growing body of evidence suggests that in utero exposure to preeclampsia may induce lasting biological alterations in the newborn, particularly through epigenetic mechanisms [ 50 ]. The epigenetic changes observed in umbilical cord blood thus provide a unique window for studying the early molecular imprint of preeclampsia and better understanding the developmental programming mechanisms that may influence the child's future health. However, the existing literature on epigenetic changes in cord blood associated with preeclampsia remains fragmented and heterogeneous [ 27 , 51 ]. Studies vary considerably in terms of clinical definitions of preeclampsia, sample sizes, epigenetic platforms used (methylation arrays, bisulfite sequencing, non-coding RNA analyses, histone modifications), analytical strategies, and consideration of maternal and neonatal confounding factors. This heterogeneity complicates the overall interpretation of results and limits the translation of knowledge into a clinical or preventive perspective. In this context, conducting a rigorously designed systematic review appears essential to critically synthesize the available data, identify robust epigenetic signatures associated with preeclampsia, and highlight current methodological gaps. This protocol aims to provide a transparent and reproducible methodological framework for the systematic evaluation of existing evidence. By integrating both observational studies and clinical trials that have analysed umbilical cord blood, this review will allow for a comprehensive examination of epigenetic alterations associated with preeclampsia, as well as their relationships with maternal, obstetric, and neonatal characteristics. The planned subgroup and sensitivity analyses will allow exploration of the influence of key factors such as the severity and timing of onset of preeclampsia, gestational age, the newborn's sex, and the methodological approaches used, thus contributing to a better understanding of the sources of heterogeneity between studies. The expected results of this systematic review should help clarify the current state of knowledge regarding epigenetic changes in cord blood associated with preeclampsia, identify potentially relevant epigenetic markers for future research, and provide methodological recommendations for upcoming studies. In the long term, this synthesis could contribute to a better understanding of the biological mechanisms linking preeclampsia to the child’s long-term health trajectories, paving the way for early prevention strategies and the identification of biomarkers useful in translational research. Any changes to the protocol resulting from the final review of the data will be transparently documented when publishing the results in a peer-reviewed scientific journal. Conclusion In conclusion, this systematic review protocol offers a rigorous and transparent methodological framework, in line with PRISMA-P recommendations, aimed at ensuring the reproducibility and reliability of the entire research and data synthesis process. By including observational studies and clinical trials that analysed umbilical cord blood, it allows for a comprehensive evaluation of the available data on epigenetic changes associated with preeclampsia. The involvement of independent reviewers for screening, data extraction, and risk of bias assessment helps to minimize errors and selection bias. Taking into account the methodological specificities inherent to epigenetic studies, as well as integrating subgroup analyses, sensitivity analyses, and a structured assessment of the certainty of evidence according to the GRADE framework, should lead to a critical, robust, and nuanced synthesis. This protocol thus lays the groundwork for a systematic review likely to improve the understanding of the biological mechanisms linking preeclampsia to neonatal epigenetic alterations and to guide future research in this field. Limitations and Challenges This systematic review may face several limitations and challenges inherent to the nature of the available literature on epigenetic changes in cord blood associated with preeclampsia. Firstly, substantial methodological heterogeneity is expected among the included studies, particularly regarding clinical definitions of preeclampsia, characteristics of the populations studied, methods of cord blood collection and storage, as well as the epigenetic platforms and analytical pipelines used. This variability could limit the comparability of the results and restrict the possibility of conducting robust quantitative meta-analyses. Additionally, many epigenetic studies rely on relatively small sample sizes, which can lead to limited statistical power and increased imprecision in effect estimates. Incomplete control of maternal and neonatal confounding factors, such as gestational age, socioeconomic status, maternal comorbidities, or the cell composition of cord blood, represents another challenge that may influence the interpretation of observed associations. Furthermore, the risk of publication bias cannot be excluded, as studies reporting positive results may be overrepresented. Finally, the diversity of epigenetic markers studied and the lack of standardization of outcomes limit the generalizability of the conclusions. These constraints highlight the need to interpret the results with caution and underscore the importance of future large-scale, methodologically harmonized studies to strengthen the evidence in this field. Declarations Competing interests The authors declare that they have no competing interests. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contribution NY searched literature and assisted in writing of the original draft. JJ and CB worked on conceptualizing and writing, GT edited and reviewed the manuscript. Acknowledgements Not applicable References Xiao J, Shen F, Xue Q, Chen G, Zeng K, Stone P, et al. 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DOI: 10.21608/ejcm.2024.298036.1304 Calderon Martinez E, Ghattas Hasbun PE, Salolin Vargas VP, García-González OY, Fermin Madera MD, Rueda Capistrán DE, et al. A comprehensive guide to conduct a systematic review and meta-analysis in medical research. Medicine (Baltimore). 2025;104(33):e41868. doi: 10.1097/MD.0000000000041868 . Shamseer L, Moher D, Clarke M, et al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation. BMJ. 2015; 349:g7647. Werner SS, Binder N, Toews I, Schünemann H J, Meerpohl JJ, & Schwingshackl L. Use of GRADE in evidence syntheses published in high-impact-factor nutrition journals: A methodological survey. Journal of Clinical Epidemiology , 2021, 135 , 54–69. https://doi.org/10.1016/j.jclinepi.2021.02.010 Prasad M, Introduction to the GRADE tool for rating certainty in evidence and recommendations. Clinical Epidemiology and Global Health 2024 Vol. 25 Pages 101484. DOI: https://doi.org/10.1016/j.cegh.2023.101484 World Health Organisation WHO. Pre-eclampsia. World Health Organisation, Geneva Swaziland, 2025. https://www.who.int/news-room/fact-sheets/detail/pre-eclampsia Talpur KR, Bano I, Abdullah MW. Epigenetic mechanisms linking pregnancy complications to cardiovascular disease in offspring. Explor Cardiol. 2024;2:241–52. https://doi.org/10.37349/ec.2024.00037 Ma J, Zhan Z, Li N, Huang Y, Li Y, Liu L, Shen Q, Chu Q, Wang X, Wu B, et al. Preliminary Interpretations of Epigenetic Profiling of Cord Blood in Preeclampsia. Genes. 2022; 13(5):888. https://doi.org/10.3390/genes13050888 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8956740","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Study protocol","associatedPublications":[],"authors":[{"id":601493632,"identity":"6e3f48e9-638d-432f-a6f2-6d5105d07c30","order_by":0,"name":"Nofonta Yamkelani","email":"","orcid":"","institution":"Walter Sisulu University","correspondingAuthor":false,"prefix":"","firstName":"Nofonta","middleName":"","lastName":"Yamkelani","suffix":""},{"id":601493636,"identity":"0ed8d3a7-d1a8-4d45-aff5-4d4cf0e8827a","order_by":1,"name":"Jim Thytharayil Joseph","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYFCCA2BCjngNPFAtxqRogViV2EC0FnvGw4c/fNxxJ33D+TMGDD9qGOz5CWnmYTiWJjnzzLPcDTdyDBh7jjEwSxwgqOWMGTNv2+HcbTd4DBh4GxjYGIjQYvz5b9vhdDOgwxj/NjDwyBOhxUCase1wgtmBHANmoC0SBgS1HAD6pffMYcP9N9IKDssckzAwJKSFfQYwxH7uOCwv2X9448M3NTb2coS0MIACiLEBwgYyJQipBwJQNMC0jIJRMApGwSjACgDcBUUSymt16gAAAABJRU5ErkJggg==","orcid":"","institution":"Walter Sisulu University","correspondingAuthor":true,"prefix":"","firstName":"Jim","middleName":"Thytharayil","lastName":"Joseph","suffix":""},{"id":601493637,"identity":"d334e131-10ae-4d73-96a0-3fc788ca360e","order_by":2,"name":"Charles Businge","email":"","orcid":"","institution":"Walter Sisulu University","correspondingAuthor":false,"prefix":"","firstName":"Charles","middleName":"","lastName":"Businge","suffix":""},{"id":601493641,"identity":"4f6c7bb0-7c86-4cf4-a2e3-5e9e234e0a84","order_by":3,"name":"Gabriel Tchuente","email":"","orcid":"","institution":"Walter Sisulu University","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Tchuente","suffix":""}],"badges":[],"createdAt":"2026-02-24 11:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8956740/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8956740/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104403886,"identity":"25ec1e41-aeae-4e6c-aa93-f073f762fa9f","added_by":"auto","created_at":"2026-03-11 12:19:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":645893,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8956740/v1/1d096256-792b-4384-b651-ce82b702d638.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Epigenetic Modifications of Umbilical Cord Blood and Preeclampsia: a Systematic Review and Metanalysis Protocol","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreeclampsia remains one of the most perplexing paradoxes in obstetric medicine: a disorder specific to pregnancy, long silent, but capable of progressing to severe hypertension and multiorgan damage, jeopardizing the prognosis for both mother and foetus [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. Affecting 2 to 8% of pregnancies worldwide, it remains one of the leading causes of perinatal morbidity and mortality, with 50,000 to 60,000 deaths reported each year [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. In resource-limited countries, its impact is particularly pronounced, with Africa accounting for up to 9% of preeclampsia-related deaths, with some of the highest rates in Nigeria, South Africa, and Ethiopia [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite decades of research, preeclampsia remains a disease with unclear origins. Its development results from a complex interaction between genetic predispositions, immunological factors, and environmental influences [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Disruptions in the remodelling of spiral arteries by trophoblasts, placental hypoxia, and oxidative stress are central mechanisms, but they are insufficient to explain the entire clinical picture [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. Traditional risk factors, maternal age, obesity, chronic hypertension, autoimmune or endocrine diseases, are also unable to reliably predict the disease [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAgainst this backdrop of uncertainty, the emergence of epigenetics has profoundly changed perspectives. Recent work shows that preeclampsia is associated with epigenetic changes, particularly alterations in DNA methylation, both in the placenta and in the cord blood of newborns exposed in utero [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cord blood, rich in hematopoietic cells and nucleated red blood cells, provides a unique model for exploring foetal programming [24]. Several studies have reported differential methylation profiles in children born from preeclamptic pregnancies, identifying genes and pathways involved in the cardiovascular and endothelial systems [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, the results remain inconsistent: some teams describe overall hypomethylation [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e], while others report hypermethylation [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. These discrepancies suggest the influence of confounding factors, particularly gestational age [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, the role of the maternal environment, particularly nutrition, remains an under-explored area. Nutrients such as folate and choline, which play a key role in the metabolism of methyl donors, are likely to modulate foetal genome methylation [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, their involvement in placental programming and in the risk of complications such as preeclampsia remains poorly characterized [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, despite a wealth of literature, there is currently no rigorous synthesis that provides a coherent overview of the specific epigenetic alterations in cord blood in preeclampsia, nor does it identify their biological or clinical value. Existing studies vary considerably in terms of methodology, the epigenomic platforms used, the covariates included, and the populations studied, making it difficult to arrive at a uniform interpretation. Given these uncertainties, a systematic review is needed to: gather and compare available data on epigenetic modifications in cord blood associated with preeclampsia; assess the consistency of reported signatures; identify recurring methodological limitations; and shed light on the potential of these markers as tools for understanding, prediction, or early diagnosis. By synthesizing the interactions between genetics, maternal environment, and foetal epigenetic programming, this review aims to clarify the mechanisms by which preeclampsia leaves its mark from birth, and potentially far beyond.\u003c/p\u003e \u003cp\u003eIn the long term, this synthesis could contribute to a better understanding of the biological mechanisms linking preeclampsia to the child’s long-term health trajectories, paving the way for early prevention strategies and the identification of biomarkers useful in translational research.\u003c/p\u003e\n\u003ch3\u003eResearch Question\u003c/h3\u003e\n\u003cp\u003eWhat is the effect of pre-eclampsia on global and gene specific DNA methylation in cord blood among women with and without pre-eclampsia.\u003c/p\u003e \u003cp\u003e \u003cb\u003eObjectives\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cul\u003e \u003cli\u003e \u003cp\u003eTo analyse the global DNA methylation in cord blood of women with and without pre-eclampsia\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eTo analyse specific genes which are modified in cord blood of women with and without pre-eclampsia.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eTo investigate relationship between maternal lifestyle and epigenetic modification in cord blood.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eTo investigate relationship between epigenetic modification in cord blood and anthropometric measurements of neonates.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e "},{"header":"Methodology","content":"\u003ch2\u003eProtocol and Registration\u003c/h2\u003e\u003cp\u003eThis work is in line with the PRISMA-P 2020 guidelines [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. The study protocol was prospectively registered in the PROSPERO database (ID: CRD420261301497) on February 05, 2026, to ensure methodological transparency and rigor.\u003c/p\u003e\u003ch3\u003eInclusion Criteria\u003c/h3\u003e\u003cp\u003eParticipants consist of mothers and newborns whose umbilical cord blood has been analyzed for epigenetic changes in the context of preeclampsia.\u003c/p\u003e\u003ch3\u003eIntervention\u003c/h3\u003e\u003cp\u003eThe procedure involves the assessment of epigenetic modifications, including DNA methylation analyses and other epigenetic markers in cord blood.\u003c/p\u003e\u003ch3\u003eComparison\u003c/h3\u003e\u003cp\u003eThe comparison must be made between samples from pregnancies complicated by preeclampsia and those from uncomplicated or normotensive pregnancies.\u003c/p\u003e\u003ch2\u003eOutcomes\u003c/h2\u003e\u003cp\u003eThe primary outcome is the characterization of specific epigenetic alterations associated with preeclampsia in umbilical cord blood. Secondary outcomes focus on exploring the relationship between these epigenetic modifications and maternal and neonatal demographic, clinical, or biological parameters.\u003c/p\u003e\u003ch3\u003eStudy Design\u003c/h3\u003e\u003cp\u003eStudy design includes observational studies such as cohorts, case-control studies, or cross-sectional studies analyzing epigenetic profiles in cord blood in relation to preeclampsia. However, clinical trials that collected umbilical cord blood from children of preeclamptic and normotensive mothers and evaluated epigenetic markers will also be considered relevant.\u003c/p\u003e\u003ch3\u003eExclusion Criteria\u003c/h3\u003e\u003cp\u003eThe exclusion criteria for this review will eliminate all studies that do not analyze umbilical cord blood or that do not focus on preeclamptic and normotensive pregnancies, as well as those that do not include an assessment of epigenetic changes. Studies that do not allow for comparison between preeclampsia and uncomplicated pregnancies, those that do not report epigenetic alterations associated with preeclampsia or maternal and neonatal demographic, clinical, or biological parameters, as well as studies whose design does not correspond to the types selected, including animal studies, in vitro research, non-original articles, or protocols without data. Finally, studies that do not provide sufficient information to assess the relationship between preeclampsia and epigenetic modifications in cord blood will be excluded. studies published in non-English languages show be excluded.\u003c/p\u003e\u003ch2\u003eLiterature sources and retrieval strategy\u003c/h2\u003e\u003cp\u003eTwo independent reviewers (names) will conduct a systematic and comprehensive literature search in four major electronic databases: PubMed, Web of Science, Scopus, and the Cochrane Central Register of Controlled Trials (CENTRAL), covering the period from the creation of the databases to the present. The search strategy will be meticulously designed to incorporate both controlled vocabulary (e.g., Medical Subject Headings [MeSH] in PubMed, TITLE-ABS-KEY in Scopus) and relevant free-text terms. This strategy will simultaneously target three key conceptual areas: the target population (“children born to mothers with preeclampsia” OR “umbilical cord blood collected at birth after a pregnancy with preeclampsia”), the specific intervention (“presence of specific epigenetic modifications” OR “DNA methylation levels” OR \"microRNA expression profiles (miRNA) expression profiles“ OR ”Post-translational modifications of histones“) and the required study design (”analytical observational studies“ OR ”randomized controlled trial\").\u003c/p\u003e\u003cp\u003eIn order to ensure methodological rigor and optimize the sensitivity and specificity of the search, the search syntax proposed for each database will undergo formal peer review using the PRESS (Peer Review of Electronic Search Strategies) checklist. The search strategy was conducted using the following keywords: epigenetic or DNA methylation or microRNA or miRNA or histone or acetylation) AND cord blood AND preeclampsia.\u003c/p\u003e\u003cp\u003eAware of the risk that database searches may not identify all eligible evidence, additional search methods will be rigorously used. These will include manual review of reference lists of all included studies and relevant systematic reviews, systematic review of key conference proceedings (e.g., major meetings of obstetric gynecology, Neonatology/Perinatal Medicine associations) Perinatal, and Maternal-Fetal Medicine associations) and searching leading in the International Clinical Trials Registry Platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://apps.who.int/trialsearch/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), in ClinicalTrials.gov (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.clinicaltrials.gov\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and in Google Scholar (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://scholar.google.dk/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify ongoing or unpublished trials. All documents retrieved using these combined methods will be collated using reference management software (EndNote), and duplicate citations will be systematically removed before the selection process begins.\u003c/p\u003e\u003ch2\u003eLiterature Screening and Data Extraction\u003c/h2\u003e\u003cp\u003eTo streamline the process of review and selection, initially, search findings will be automatically exported to Endnote for duplication removal before being transferred to the Rayyan software [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. Literature screening and data extraction will be performed by two independent reviewers, in accordance with PRISMA recommendations. Titles and abstracts will first be evaluated according to eligibility criteria, followed by a full-text review of potentially eligible studies. Any disagreements will be resolved through discussion or, if necessary, by arbitration by a third reviewer (specify their initials). In the event of missing data, the corresponding authors will be contacted; if no response is received, the analyses will be conducted on the available data, with an assessment of the impact of the missing data using sensitivity analyses. The complete selection process will be illustrated in a PRISMA diagram (Page et al., 2021). Data extraction will be based on a pre-tested standardized form, with both reviewers independently collecting key information before cross-checking. The extracted data will include general study characteristics, diagnostic criteria for preeclampsia, maternal and neonatal characteristics, cord blood collection and analysis methods, and epigenetic approaches used, including methylation measurement techniques or other epigenetic markers. The extracted results will focus on epigenetic alterations associated with preeclampsia (primary outcome) and their relationships with maternal and neonatal demographic, clinical, or biological parameters (secondary outcomes), including relevant analytical and statistical details. The final datasets will be verified for accuracy prior to any quantitative or narrative synthesis.\u003c/p\u003e\u003ch2\u003eAssessment of Risk of Bias\u003c/h2\u003e\u003cp\u003eThe risk of bias assessment will be performed independently by two reviewers to ensure methodological rigor. For included randomized trials, the Cochrane Collaboration's ROB 2 tool will be applied systematically to examine potential biases related to the randomization process, deviations from planned interventions, missing outcome data, outcome measurement methods, and the risk of selective reporting of results [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. For non-randomized studies, including observational studies frequently used in research on epigenetic modifications of cord blood in the context of preeclampsia, ROBINS-I will be used to assess bias [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e] related to confounding factors, participant selection procedures (including identification of preeclampsia cases and normotensive controls), classification of exposures or comparators, deviations from planned methods, missing data, measurement of epigenetic outcomes, and the possibility of selective reporting. Any discrepancies between reviewers will be resolved through discussion, and in the event of persistent disagreement, a third senior reviewer will provide final arbitration.\u003c/p\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses will be performed using Review Manager software (RevMan, version 5.4) for quantitative synthesis and graphical presentation of results in the form of forest plots. When studies provide data that are sufficiently homogeneous for pooling, continuous measures of epigenetic alterations (e.g., methylation percentages or expression levels of epigenetic markers) will be synthesized as mean differences or standardized mean differences with 95% confidence intervals, using the inverse variance method. In cases where studies report categorical results (e.g., presence or absence of significantly differentially methylated regions), these will be pooled using risk ratios and 95% confidence intervals. The choice between fixed-effect and random-effect models will be determined by statistical heterogeneity, assessed using the I² statistic; a random-effect model will be preferred when heterogeneity is substantial [42,43]. I² equal to 25%, 50%, and between 75% to 100% are considered as low, medium, and high heterogeneity respectively. If heterogeneity prevents a robust meta-analysis, a structured narrative synthesis will be performed. Exploration of sources of heterogeneity, including methodological variations between epigenetic platforms, differences in diagnostic criteria for preeclampsia, gestational age, or variations in cord blood collection and processing methods, may be examined using exploratory analyses, including meta-regressions when data allow, using STATA. The risk of publication bias will be assessed by visual inspection of funnel plots and statistical tests such as Egger's test, but only when at least ten studies contribute to the analysis of the same outcome. The identification of publication bias occurs when the p-value is less than 0.05 [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. If conducting a meta-analysis is not possible, a critical synthesis will be conducted in accordance with Calderon et al. guidelines [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. Lastly, in compliance with PRISMA guidelines, the findings will be painstakingly documented and submitted for publication [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. Our research findings will be reliable, transparent, and rigorous thanks to this thorough approach.\u003c/p\u003e\u003ch2\u003eSubgroup Analysis and Sensitivity Analysis\u003c/h2\u003e\u003cp\u003eSubgroup analyses will be performed to explore factors that may modify the association between preeclampsia and epigenetic alterations observed in cord blood. These pre-specified analyses will examine, in particular, the influence of the severity of preeclampsia (mild vs. severe preeclampsia), the time of onset (early vs. late), gestational age at delivery, neonatal sex, birth weight, relevant maternal characteristics (age, parity, body mass index), as well as methodological differences such as the type of epigenetic platform used (e.g., methylation chips, bisulfite sequencing, non-coding RNA analyses) or the quality of sample processing (time to freezing, storage conditions). In addition, other parameters such as lifestyle (passive or active smoking, alcoholism), as well as associated pathologies, will be considered. When sufficient studies are available, these subgroups will make it possible to examine whether certain biological, clinical, or technical characteristics influence the magnitude or direction of the epigenetic alterations identified.\u003c/p\u003e\u003ch2\u003eCertainty of Evidence Assessment\u003c/h2\u003e\u003cp\u003eThe certainty of the evidence will be systematically assessed using the GRADE approach with the GRADEpro GDT online tool [47,48]. This assessment will cover five key areas, including the risk of bias in the included studies, inconsistency of results between studies, indirectness of evidence in relation to the research question, imprecision of effect estimates, and risk of publication bias. Particular attention will be paid to the specific characteristics of epigenetic studies of cord blood, including methodological quality, heterogeneity of analytical platforms, and sample size. For each outcome, whether epigenetic alterations associated with preeclampsia or their links with maternal and neonatal parameters, a level of certainty will be assigned (high, moderate, low, or very low), with justification documented in GRADE evidence profile tables.\u003c/p\u003e\u003ch2\u003ePatient and Public Involvement\u003c/h2\u003e\u003cp\u003eThis systematic review did not involve patients or the public in its design, conduct, or presentation, as it synthesizes existing published data. All analyses use secondary data from original previous studies, for which patient consent was obtained during the initial conduct of the study.\u003c/p\u003e\u003ch2\u003eEthics and Dissemination\u003c/h2\u003e\u003cp\u003eSince this study is based exclusively on secondary analysis of data already published in the scientific literature, no formal ethical approval or additional informed consent is required. The primary studies included in the review are presumed to have obtained the appropriate ethical approvals in accordance with applicable local and international regulations, particularly with regards to the collection and analysis of biological samples such as umbilical cord blood. The full results of this systematic review will be submitted for publication in peer-reviewed international scientific journals to ensure rigorous and validated dissemination of knowledge. The main findings will also be presented at relevant scientific conferences and meetings in the fields of obstetrics, perinatology, and epigenetics, in order to promote discussion with the scientific community. With a view to ensuring rapid and open dissemination of the results, the manuscript may also be submitted to recognized preprint platforms, such as medRxiv. In accordance with the principles of open science and reproducibility, all extracted data, extraction forms, analysis scripts, and methodological documents will be deposited in the Open Science Framework (OSF) public repository. Supplementary materials for the publication will provide a direct link to this repository to facilitate access, transparency, and reuse of the data by interested researchers.\u003c/p\u003e\u003ch2\u003eData Management and Reproducibility Statement\u003c/h2\u003e\u003cp\u003eData management and reproducibility of this systematic review will be ensured in accordance with the principles of open science and the PRISMA-P recommendations. All study materials, including anonymized and aggregated data extracted from the included articles, standardized data extraction forms, and analysis scripts used for statistical and narrative synthesis (notably in RevMan version 5.4 and Stata version 19), will be deposited in the Open Science Framework (OSF) public repository at the time of publication. The data will be organized in a structured and documented manner to enable reuse and independent verification of the results, while respecting the confidentiality of the participants in the primary studies. The analytical codes will be fully commented on and accompanied by a clear description of the analytical steps, including the processing of epigenetic data, the management of methodological heterogeneity, and sensitivity analyses. The final manuscript and supplementary materials will provide a direct link to the OSF repository, ensuring free and permanent access to all resources necessary for the complete reproduction of the analyses and transparency of the scientific process.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePreeclampsia remains one of the leading causes of maternal and neonatal morbidity and mortality worldwide, with immediate and long-term consequences for both mother and child [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Beyond perinatal clinical complications, a growing body of evidence suggests that in utero exposure to preeclampsia may induce lasting biological alterations in the newborn, particularly through epigenetic mechanisms [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The epigenetic changes observed in umbilical cord blood thus provide a unique window for studying the early molecular imprint of preeclampsia and better understanding the developmental programming mechanisms that may influence the child's future health.\u003c/p\u003e \u003cp\u003eHowever, the existing literature on epigenetic changes in cord blood associated with preeclampsia remains fragmented and heterogeneous [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Studies vary considerably in terms of clinical definitions of preeclampsia, sample sizes, epigenetic platforms used (methylation arrays, bisulfite sequencing, non-coding RNA analyses, histone modifications), analytical strategies, and consideration of maternal and neonatal confounding factors. This heterogeneity complicates the overall interpretation of results and limits the translation of knowledge into a clinical or preventive perspective. In this context, conducting a rigorously designed systematic review appears essential to critically synthesize the available data, identify robust epigenetic signatures associated with preeclampsia, and highlight current methodological gaps. This protocol aims to provide a transparent and reproducible methodological framework for the systematic evaluation of existing evidence. By integrating both observational studies and clinical trials that have analysed umbilical cord blood, this review will allow for a comprehensive examination of epigenetic alterations associated with preeclampsia, as well as their relationships with maternal, obstetric, and neonatal characteristics. The planned subgroup and sensitivity analyses will allow exploration of the influence of key factors such as the severity and timing of onset of preeclampsia, gestational age, the newborn's sex, and the methodological approaches used, thus contributing to a better understanding of the sources of heterogeneity between studies.\u003c/p\u003e \u003cp\u003eThe expected results of this systematic review should help clarify the current state of knowledge regarding epigenetic changes in cord blood associated with preeclampsia, identify potentially relevant epigenetic markers for future research, and provide methodological recommendations for upcoming studies. In the long term, this synthesis could contribute to a better understanding of the biological mechanisms linking preeclampsia to the child\u0026rsquo;s long-term health trajectories, paving the way for early prevention strategies and the identification of biomarkers useful in translational research. Any changes to the protocol resulting from the final review of the data will be transparently documented when publishing the results in a peer-reviewed scientific journal.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this systematic review protocol offers a rigorous and transparent methodological framework, in line with PRISMA-P recommendations, aimed at ensuring the reproducibility and reliability of the entire research and data synthesis process. By including observational studies and clinical trials that analysed umbilical cord blood, it allows for a comprehensive evaluation of the available data on epigenetic changes associated with preeclampsia. The involvement of independent reviewers for screening, data extraction, and risk of bias assessment helps to minimize errors and selection bias. Taking into account the methodological specificities inherent to epigenetic studies, as well as integrating subgroup analyses, sensitivity analyses, and a structured assessment of the certainty of evidence according to the GRADE framework, should lead to a critical, robust, and nuanced synthesis. This protocol thus lays the groundwork for a systematic review likely to improve the understanding of the biological mechanisms linking preeclampsia to neonatal epigenetic alterations and to guide future research in this field.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLimitations and Challenges\u003c/h2\u003e \u003cp\u003eThis systematic review may face several limitations and challenges inherent to the nature of the available literature on epigenetic changes in cord blood associated with preeclampsia. Firstly, substantial methodological heterogeneity is expected among the included studies, particularly regarding clinical definitions of preeclampsia, characteristics of the populations studied, methods of cord blood collection and storage, as well as the epigenetic platforms and analytical pipelines used. This variability could limit the comparability of the results and restrict the possibility of conducting robust quantitative meta-analyses. Additionally, many epigenetic studies rely on relatively small sample sizes, which can lead to limited statistical power and increased imprecision in effect estimates. Incomplete control of maternal and neonatal confounding factors, such as gestational age, socioeconomic status, maternal comorbidities, or the cell composition of cord blood, represents another challenge that may influence the interpretation of observed associations. Furthermore, the risk of publication bias cannot be excluded, as studies reporting positive results may be overrepresented. Finally, the diversity of epigenetic markers studied and the lack of standardization of outcomes limit the generalizability of the conclusions. These constraints highlight the need to interpret the results with caution and underscore the importance of future large-scale, methodologically harmonized studies to strengthen the evidence in this field.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eNY searched literature and assisted in writing of the original draft. JJ and CB worked on conceptualizing and writing, GT edited and reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXiao J, Shen F, Xue Q, Chen G, Zeng K, Stone P, et al. Is ethnicity a risk factor for developing preeclampsia? an analysis of the prevalence of preeclampsia in China. J Hum Hypertens 2014;28(11):694\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAugust P, and Sibai BM. Preeclampsia: Clinical features and diagnosis. Post TW, UpToDate. Waltham, MA: 2017. UpToDate.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNarkhede AM, and Karnad DR. Preeclampsia and related problems. 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Explor Cardiol. 2024;2:241\u0026ndash;52. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.37349/ec.2024.00037\u003c/span\u003e\u003cspan address=\"10.37349/ec.2024.00037\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa J, Zhan Z, Li N, Huang Y, Li Y, Liu L, Shen Q, Chu Q, Wang X, Wu B, et al. Preliminary Interpretations of Epigenetic Profiling of Cord Blood in Preeclampsia. Genes. 2022; 13(5):888. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/genes13050888\u003c/span\u003e\u003cspan address=\"10.3390/genes13050888\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"systematic-reviews","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sysr","sideBox":"Learn more about [Systematic Reviews](http://systematicreviewsjournal.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/sysr/default.aspx","title":"Systematic Reviews","twitterHandle":"@MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Epigenetic modification, DNA methylation, cord blood, Pre-eclampsia, maternal complication","lastPublishedDoi":"10.21203/rs.3.rs-8956740/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8956740/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePreeclampsia remains a major cause of maternal and neonatal morbidity and mortality worldwide, with growing evidence suggesting that in utero exposure to this condition may induce persistent biological alterations in the offspring through epigenetic mechanisms. Umbilical cord blood offers a valuable substrate to investigate the early molecular imprint of preeclampsia and its potential role in developmental programming. Although numerous studies have reported global and gene-specific DNA methylation changes in cord blood, findings are inconsistent, particularly regarding the direction and magnitude of hypo- or hypermethylation. To address these discrepancies, this protocol outlines a rigorous and reproducible framework for a systematic review and meta-analysis aimed at synthesizing evidence on epigenetic modifications in umbilical cord blood associated with preeclampsia and their relationships with maternal and neonatal characteristics.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study, designed in accordance with the PRISMA 2020 guidelines, will assess epigenetic modifications in umbilical cord blood associated with preeclampsia. Observational studies and clinical trials comparing preeclamptic and normotensive pregnancies and analysing DNA methylation or other epigenetic markers will be included. A comprehensive search will be conducted in PubMed, Web of Science, Scopus, and CENTRAL, supplemented by grey literature sources. Study selection, data extraction, and risk of bias assessment (ROB 2, ROBINS-I) will be performed independently by two reviewers. A meta-analysis will be conducted using RevMan according to heterogeneity, with subgroup analyses, assessment of publication bias, and GRADE evaluation of the certainty of evidence.\u003c/p\u003e\u003ch2\u003eEthics and dissemination:\u003c/h2\u003e \u003cp\u003eThe studies included in this review are presumed to have obtained the appropriate ethical approvals in accordance with applicable local and international regulations, umbilical cord blood.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn conclusion, this study will provide a comprehensive and methodologically robust synthesis of the evidence on epigenetic modifications in umbilical cord blood associated with preeclampsia. By clarifying the direction, agnitude, and consistency of reported alterations, and by exploring sources of heterogeneity across studies, this work will strengthen the understanding of the early-life epigenetic imprint of preeclampsia. The findings are expected to inform future mechanistic research, improve risk stratification strategies, and guide the development of preventive and early-life interventions targeting long-term maternal and offspring health.\u003c/p\u003e\u003ch2\u003ePROSPERO registration number\u003c/h2\u003e \u003cp\u003e: CRD420261301497\u003c/p\u003e","manuscriptTitle":"Epigenetic Modifications of Umbilical Cord Blood and Preeclampsia: a Systematic Review and Metanalysis Protocol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 05:44:47","doi":"10.21203/rs.3.rs-8956740/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-03-03T20:31:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-25T13:13:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-25T13:13:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Systematic Reviews","date":"2026-02-24T11:11:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"systematic-reviews","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sysr","sideBox":"Learn more about [Systematic Reviews](http://systematicreviewsjournal.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/sysr/default.aspx","title":"Systematic Reviews","twitterHandle":"@MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"247e6b4d-8967-4d49-bd02-a5d3bdac03ca","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T05:44:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 05:44:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8956740","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8956740","identity":"rs-8956740","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}