Low birth weight, DNA methylation patterns in cord blood, and inflammation at birth

Introduction

Low birth weight (LBW) (≤2,500 g) is a robust predictor of long-term risk for various chronic diseases, including metabolic, cardiovascular, neurodevelopmental and immune disorders (1-7). Although epidemiologically established, the molecular mechanisms underlying this association remain poorly understood and represent a major challenge in biomedical research. Both genetic and epigenetic mechanisms underpin the link between LBW and adult disease susceptibility. Genetic factors establish a baseline risk, either through direct effects or via gene-environment interactions. Meanwhile, early-life intrauterine exposures dynamically shape phenotypic outcomes through epigenetic reprogramming. This process acts as a molecular memory connecting the intrauterine environment to long-term gene regulation and developmental plasticity. This process drives lasting adaptations in physiological functions and metabolic processes, thereby shaping an individual’s lifelong health trajectory (8,9). DNA methylation, an important epigenetic modification, has emerged as a key mediator that regulates genes in response to early-life environments without altering the DNA sequence (10-12). Investigating differential DNA methylation patterns associated with LBW at birth is crucial for understanding the epigenetic regulation of development by the intrauterine environment. This investigation can facilitate the discovery of early biomarkers, help identify neonates at elevated risk for chronic diseases, and guide targeted prenatal interventions.

Previous studies have found that DNA methylation patterns at birth partially explain variations in birth weight (BW) (13,14). Genome-wide epigenetic alterations observed in LBW infants have been implicated in processes such as immune regulation, sphingolipid metabolism, and cellular maturation (15,16). However, these correlative findings lack validation of physiological relevance, and future work should investigate the specific physiological pathways through which these epigenetic changes contribute to disease pathogenesis. Moreover, the genetic backgrounds of study subjects often confound causal inference. Monozygotic (MZ) twins, who share nearly identical genetic backgrounds, exhibit BW discordance primarily due to distinct intrauterine conditions. Therefore, MZ twins with discordant BW provide a unique natural model to distinguish the effects of the prenatal environment on DNA methylation. Study of MZ twins minimizes genetic confounding factors and allows for high statistical power even with a small sample size (17,18). Umbilical cord blood, with its non-invasive collection and capacity to mirror perinatal fetal physiology, is a valuable sample for neonatal epigenetic research. Thus, examining specific differential DNA methylation patterns in the cord blood of MZ twins discordant for BW provides an ideal approach to elucidate the influence of LBW-related intrauterine epigenetic programming on long-term health outcomes.

In this study, we utilized a selected cohort of neonatal MZ twin pairs discordant for BW to identify differentially methylated genes (DMGs) in cord blood mononuclear cells (CBMCs) through genome-wide DNA methylation profiling using methylated DNA immunoprecipitation sequencing (MeDIP-seq). Using gene set linkage analysis (GSLA) among the top DMGs, we identified the predominant functional networks enriched in immune-related biological processes, with interferon (IFN)-γ emerging as a central mediator. We then validated the messenger RNA (mRNA) levels of key DMGs by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in CBMCs from an independent neonatal cohort. To functionally validate the findings, we further characterized the immune phenotype by measuring the expression of cytokines in monocytes and lymphocytes isolated from CBMCs of LBW and normal birth weight (NBW) neonates following in vitro stimulation with either lipopolysaccharide (LPS) or phytohemagglutinin (PHA). Our study leverages the natural model of BW-discordant MZ twin neonates and employs multi-level validation to elucidate how LBW influences immune function through epigenetic mechanisms at birth. We present this article in accordance with the STROBE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-954/rc).

Methods

Samples

We employed a cohort of BW-discordant MZ twins for genome-wide DNA methylation profiling. Zygosity was confirmed in 112 twin pairs through analysis of 16 polymorphic short tandem repeat (STR) loci, which identified 19 MZ twin pairs. Fetuses with malformations, conceptions achieved through assisted reproductive technology, as well as pregnancies complicated by severe maternal conditions were excluded. Ultimately, four MZ twin pairs (two male, two female) at 35–37 gestational weeks were selected. Of these, two pairs exhibited significant BW discordance (>20% difference), while the other two pairs showed concordant BW (<5% difference) (Table S1). Although the sample size was limited due to inherent recruitment difficulties, this carefully selected cohort offered a unique opportunity for in-depth mechanistic exploration.

For validation, 197 independent neonates were recruited and stratified into LBW (≤2,500 g) and normal birth weight (NBW, >2,500 and <4,000 g) groups (characteristics are shown in Table 1). First, in a subset of 60 neonates (30 LBW, 30 NBW), we measured the mRNA levels of key DMGs involved in relevant biological processes in CBMCs. Second, in another subset of 137 neonates (54 LBW, 83 NBW), we analyzed the transcript and protein levels of eight cytokines in monocytes and lymphocytes, both at baseline and following in vitro immune stimulation.

This study was conducted at the Women’s Hospital of Zhejiang University (Hangzhou, China) with approval from the hospital’s Ethics Committee (No. 20130027). Written informed consent was obtained from all parents prior to participation. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. An overview of the study design and analytical workflow is provided in Figure 1.

Figure 1 Schematic overview of the study design. DMG, differentially methylated gene; GSLA, gene set linkage analysis; IFN, interferon; IL, interleukin; LBW, low birth weight; LPS, lipopolysaccharide; NBW, normal birth weight; PHA, phytohemagglutinin; TNF, tumor necrosis factor.

Methylated DNA immunoprecipitation and sequencing (MeDIP-seq) profiling of genome-wide DNA methylation

Cord blood samples were collected at birth, and CBMCs were immediately isolated and frozen at –80 ℃. Genomic DNA was extracted from CBMCs using the QIAamp DNA Mini Kit (Qiagen, Duesseldorf, Germany) with standard protocol. 5 μg of gDNA was fragmented by sonication (Covaris, Woburn, USA). Adaptor-ligated DNA was immunoprecipitated by an anti-5mC antibody in a MeDIP kit (Diagenode, Catalog: mc-magme-048, Liège, Belgium). MeDIP products were sequenced on an Illumina HiSeq 2000 platform. The MeDIP-seq results of the four pairs of MZ twins are accessible at the NCBI Sequence Read Archive (SRA) database with accession SRP048587.

Paired-end reads were aligned to the human reference genome (NCBI CRGh37.p5) using Bowtie2 (19), retaining only concordant and uniquely mapped reads. Precipitated DNA coverage (PDC), defined as uniquely mapped reads per base pair, was calculated for individual and combined 500 bp promoter regions. We applied sample-specific normalization to obtain a normalized PDC (NPDC) value of 1.0 across all promoters. DMGs were identified by comparing NPDC values within these promoter regions. The corresponding normalization factors are provided in Table S1.

Measurement of mRNA levels and protein levels

Total RNA was extracted using the RNAiso Plus kit (TAKARA Bio, Shiga, Japan), and its concentration was quantified with a NanoDrop spectrophotometer. Reverse transcription was performed using the PrimeScript RT Reagent/gDNA Eraser kit (TAKARA Bio). qRT-PCR was conducted with SYBR Premix Ex Taq (TAKARA Bio) on an Applied Biosystems 7900HT Fast Real-time PCR system to measure relative mRNA levels, using beta-actin as an endogenous control.

Supernatant protein levels of cytokine were measured using Quantikine Enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems; catalogs DIF50, DIP100, Minneapolis, USA). Optical densities were determined with a Varioskan Flash Multimode Reader (Thermo Fisher Scientific, Waltham, USA).

Measurement of cytokine expression in CBMCs following immune stimulation

CBMCs were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Little Chalfont, UK), washed three times, and resuspended in RPMI 1640 (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (Gibco). Cells were plated in 24-well plates (Corning Costar, New York, USA) at 1×10⁶ cells/well and incubated at 37 ℃ with 5% CO₂ for 1 h to allow monocyte adhesion. Non-adherent cells, primarily lymphocytes, were transferred to separate wells. Monocyte purity was assessed by flow cytometry using PE-conjugated anti-CD14 (eBioscience; catalog 12-0149, San Diego, USA), with over 90% of adherent cells CD14⁺. Three technical replicates were used per subject.

Monocytes were treated with medium alone or 0.1 μg/mL LPS (Sigma, St. Louis, USA) for 3 hours. Cells were lysed in TRIzol (Invitrogen, Carlsbad, USA) to extract RNA, and mRNA levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were quantified by qRT-PCR. For cytokine secretion analysis, monocytes were treated similarly for 12 hours, and IL-1β, IL-6, and TNF-α protein levels in the supernatant were measured by Enzyme-Linked Immunosorbent Assay (ELISA).

Lymphocytes were treated with medium alone or 1 µg/mL PHA (Sigma) for 24 hours, after which cells were harvested, lysed in TRIzol, and RNA was extracted. mRNA expression of IL-4, IL-5, IL-10, IFN-γ, and IP-10 was determined by qRT-PCR. For cytokine secretion analysis, lymphocytes were treated with medium or PHA for 48 hours, and cytokine supernatant protein levels were assessed by ELISA.

Statistical analysis

Two complementary statistical tests, Q1 and Q2, were designed to identify DMGs associated with BW. Q1 used a classical group comparison ignoring twin pairs, while Q2 used the twin design to control for genetic confounding. Both tests used Student’s t-tests. DMGs with P values <0.03 in Test Q2 were considered significant, and we reported the top 50 DMGs ranked by the smallest P values from Test Q1. The synergistic functional impacts of the top 50 DMGs were analyzed using GSLA, a tool used to assess whether a group of genes collectively influence biological processes (20). Analysis of covariance (ANCOVA) was used to compare mRNA and protein levels between the LBW and NBW groups in the validation cohorts, adjusting for gestational age, sex, and mode of delivery.

Results

Identification of top 50 DMGs associated with LBW

To investigate DMGs associated with LBW, we examined genome-wide DNA methylation patterns in gene promoter regions of CMBCs derived from four pairs of MZ twins (Table S1). These regions are defined as the 1,500 bp upstream of transcription start sites, including 5' UTRs (21). Methylation levels were assessed in 500 bp windows and were normalized relative to the average promoter methylation level in each sample. This approach enabled evaluation of functional coordination between genes at the DNA methylation level. The top 50 DMGs with the most significant differences are distributed across distinct functional categories, as listed in Table S2. Seven DMGs demonstrate associations with inflammation and cytokine signaling pathways, including TRAF3IP2, JAK1, and FBXW7. Eight genes are involved in metabolic regulation and energy homeostasis, notably LEPR, MC3R, and GLIS3. Eleven DMGs are associated with neurodevelopment and cognition-related pathways, such as TTN, NRXN3, and MYT1L. Additionally, twelve genes were linked to cell cycle control and genomic stability, including BACH2, FHIT, and CENPL. The remaining twelve genes, including LRRC3B, ZCCHC16, and TMEM178, currently lack well-defined functional annotations and require further investigation (Table S2).

Identification of 11 DMGs that functionally interact with ten biological processes

GSLA of the top 50 DMGs found 11 DMGs functionally interact with ten biological processes (Table 2). GSLA leverages a molecular interaction network covering about 20% of the human interactome to efficiently uncover functional connections between biological processes and to interpret molecular phenotypes (20). Eleven DMGs are JAK1, FHIT, PACSIN2, FRS3, RNF19A, LEPR, NRXN3, NLGN4X, PPP2R2B, FBXW7, and RFX3, respectively. These DMGs were documented in the literature as participating in inflammatory and immune processes, neurodevelopment, energy metabolism, and cell cycle progression. 10 biological processes were classified into four functional themes: “IFN-γ-mediated immune response”, “synaptogenesis”, “translational regulation”, and “pancreas development”. The IFN-γ-mediated immune response was the central theme of the molecular interaction network, which included five processes: regulation of IFN-γ signaling, regulation of the JAK-STAT cascade, regulation of type I IFN signaling, the viral replication, and the viral infectious cycle. Together, these processes accounted for half of the ten significantly enriched biological processes identified, highlighting a close association between LBW-associated DMGs and inflammatory immune mechanisms at birth (Table 2).

Transcript levels of 11 DMGs in CBMCs of independent LBW neonates

We analyzed the transcriptional levels of 11 DMGs in CBMCs collected from independent 30 LBW and 30 NBW neonates. Among these, eight DMGs (73%) exhibited significant differential expression (P<0.05) (Table S3 and Figure 2). Specifically, three DMGs (NRXN3, RNF19A, and RFX3) were upregulated, while five (JAK1, FRS3, FBXW7, NLGN4X, and PACSIN2) were downregulated (22). Currently, there is no direct evidence linking the methylation patterns, gene activity levels, and disease associations of JAK1, PACSIN2, FRS3, RNF19A, NRXN3, and RFX3 to LBW. Existing literature highlights FBXW7 as critical for neurological function, and its hypermethylation-mediated suppression could lead to loss of neurological function (23). NLGN4X, a core synaptic molecule, is implicated in neurodevelopmental disorders such as autism spectrum disorders (24).

Figure 2 Validation of the transcript levels of 11 DMGs in independent neonates with LBW and NBW. ANCOVA was used to adjust for gender, gestational age, and mode of delivery in the comparison between the LBW and NBW groups. ANCOVA, analysis of covariance; DMG, differentially methylated gene; LBW, low birth weight; NBW, normal birth weight.

Cytokine expressions in monocytes and lymphocytes of independent LBW neonates in response to immune stimulation

CBMCs were collected from 54 LBW and 83 NBW independent neonates at the time of delivery, after which we analyzed the transcript and protein expression levels of eight cytokines before and after stimulating monocytes with LPS and lymphocytes with PHA, respectively.

At the transcript level, IFN-γ and IP-10 exhibited significantly enhanced PHA-induced responses in lymphocytes from LBW neonates compared with NBW controls (P<0.001), although their baseline expression levels were comparable. In contrast, while the baseline levels of IL-4 and IL-10 in lymphocytes prior to PHA stimulation showed no significant difference between the two groups, their expression was significantly reduced following PHA stimulation in the LBW group (P<0.01). Additionally, IL-1β and TNF-α mRNA levels were consistently and significantly elevated in monocytes from LBW neonates compared with NBW controls (P<0.05), and this difference was independent of LPS stimulation. Overall, stimulation increased the mRNA levels of pro-inflammatory cytokines (IFN-γ, IL-1β, TNF-α) while decreasing those of anti-inflammatory cytokines (IL-4, IL-10) in LBW neonates (Table S4 and Figure 3).

Figure 3 The relative mRNA levels of eight key cytokines in monocytes and lymphocytes before and after LPS and PHA stimulation. The responses of IFN-γ, IP-10, IL-4, IL-5, and IL-10 were measured by stimulating lymphocytes with 1 µg/mL PHA for 24 hours. The responses of IL-1β, IL-6 and TNF-α were measured by stimulating monocytes with 0.1 µg/mL LPS for 3 hours. 54 LBW and 83 NBW neonates were examined. ANCOVA was used to adjust for gender, gestational age, and mode of delivery in the comparison between the LBW and NBW groups. ANCOVA, analysis of covariance; IFN, interferon; IL, interleukin; LBW, low birth weight; LPS, lipopolysaccharide; NBW, normal birth weight; PHA, phytohemagglutinin; TNF, tumor necrosis factor.

ELISA analysis of cytokine protein levels in culture supernatant showed that immune stimulations induced a significantly higher production of IFN-γ, IL-5, and IL-6 in LBW neonates than NBW controls (P<0.05), whereas their baseline cytokine levels were comparable between groups. Consistent with transcriptional findings, LBW status was independently associated with increased IFN-γ protein levels in neonatal lymphocytes stimulated in vitro (Table S5 and Figure 4).

Figure 4 Supernatant protein levels (pg/mL) of eight key cytokines in monocytes and lymphocytes before and after LPS and PHA stimulation. The response of IFN-γ, IP-10, IL-4, IL-5, and IL-10 were measured by stimulating lymphocytes with 1 µg/mL PHA for 48 hours. The responses of IL-1β, IL-6 and TNF-α were measured by stimulating monocytes with 0.1 µg/mL LPS for 12 hours. 54 LBW and 83 NBW neonates were examined. ANCOVA was used to adjust for gender, gestational age, and mode of delivery in the comparison between the LBW and NBW groups. ANCOVA, analysis of covariance; IFN, interferon; IL, interleukin; LBW, low birth weight; LPS, lipopolysaccharide; NBW, normal birth weight; PHA, phytohemagglutinin; TNF, tumor necrosis factor.

Discussion

This study employed a MZ twin model discordant for BW to identify genome-wide DNA methylation patterns associated with LBW. This approach revealed 50 DMGs significantly linked to LBW. Functional analysis showed that 11 of these DMGs were enriched in 10 biological processes, with the IFN-γ-mediated immune response representing the predominant theme. These methylation findings were further validated in independent neonatal cohorts. Among the 11 DMGs, eight showed significant changes in mRNA levels in CBMCs from LBW neonates compared to those from NBW controls. In vitro immune stimulation assays provided further evidence that LBW neonates exhibit an inflammatory state at birth, characterized by a hyperresponsive IFN-γ signaling after stimulation in lymphocytes, elevated mRNA levels of pro-inflammatory cytokines (IL-1β, TNF-α) in monocytes both at baseline and after stimulation, and reduced mRNA levels of anti-inflammatory cytokines (IL-4, IL-10) in lymphocytes following stimulation. These findings demonstrate that LBW neonates exhibit altered DNA methylation patterns associated with immune dysregulation characterized by a pro-inflammatory state and sensitization of the IFN-γ response, which may predispose them to an exaggerated inflammatory phenotype and a higher risk of chronic diseases.

The pro-inflammatory state observed in LBW neonates highlights the significance of early life epigenetic programming in the development of chronic inflammatory diseases. Genetic predisposition underlies initial susceptibility, whereas adverse intrauterine exposures (e.g., malnutrition or hypoxia) can induce persistent alterations in gene expression through epigenetic programming. This programmed change serves as a key mechanistic link connecting early developmental insults to an increased risk of long-term diseases (25,26). Due to epigenetic drift, which refers to the gradual changes in epigenetic marks caused by environmental factors and aging, the differentially methylated CpG sites in LBW adults likely differ from those in LBW neonates, making DNA methylation in LBW neonates a more direct reflection of intrauterine influences on fetal development (27,28). It has been found that genome-wide methylation alterations in term LBW infants are associated with immune function and cellular maturation, though these lack functional validation (15). A meta-analysis revealed neonatal BW-associated methylation sites overlapping with those linked to maternal smoking and body mass index (BMI), leaving unclear whether these reflect confounding or true fetal programming (14). A twin epigenome-wide study confirms that the intrauterine environment has a major influence on neonatal methylation patterns, linking LBW-associated methylation to metabolic and biosynthetic genes as well as elevated cardiometabolic risk, but the relevant biological processes and physiological implications remain unexplored (29). Comparing MZ twins discordant for BW helps control genetic confounding, offering clearer insight into intrauterine growth restriction (IUGR)-specific epigenetic changes. This study was designed not only to identify the biological processes underlying LBW at birth, but also to validate the expression of LBW-associated DMGs and the corresponding immune responses in CBMCs.

 We found that ten biological processes were enriched among 11 of the top 50 LBW-associated DMGs. Half of the biological processes are IFN-γ-related, such as IFN-γ/JAK-STAT/type I interferon signaling and viral transcription and infection pathways. The JAK-STAT pathway not only mediates IFN-γ and type I interferon antiviral responses but also integrates signals from metabolism and the immune system, and its dysregulation could increase the risk of cardiometabolic disease (30,31). Furthermore, the understanding of IFN-γ has evolved from an antimicrobial cytokine to a key regulator of chronic inflammation in non-communicable diseases. In LBW neonates, impaired IFN-γ responsiveness leads to diminished viral clearance, but this defect can be reversed by IFN-γ stimulation (32,33). Although the compensatory IFN-γ-mediated immune response in LBW neonates helps fight viral pathogens, this hyperinflammatory state can also harm the cardiovascular system and other organs. A study showed that IFN-γ promotes atherosclerosis, while blocking its pathway significantly reduced plaque volume (34). Other research revealed that IFN-γ activates inflammatory pathways in heart tissue, worsens autoimmunity in type 1 diabetes, accelerates insulin resistance in type 2 diabetes, and sustains neuroinflammation via glial activation in Parkinson’s disease models (35-38). We hypothesize that LBW triggers compensatory IFN-γ hyper-responsiveness through JAK-STAT pathway dysregulation, which in turn propels chronic inflammation and adverse outcomes. This aligns with evidence that LBW increases the risk of early-life infections, neurodevelopmental deficits, and later cardiometabolic syndrome (39-41). Our finding provides an epigenetic mechanistic basis for understanding the origins of chronic inflammatory diseases related to LBW.

IFN-γ acts as the central orchestrator of the Th1 immune response. It induces CXCL10 (IP-10) and suppresses Th2 cytokines, cooperating with pro-inflammatory factors to maintain immune homeostasis (42). Excessive IFN-γ signaling promotes a cytokine imbalance that sustains a chronic tissue-damaging inflammation. Our study showed that LBW individuals from birth may be predisposed to increased production of pro-inflammatory cytokines (IFN-γ, IL-1β, TNF-α), alongside a reduced anti-inflammatory response (IL-4, IL-10). This immunological phenotype detectable at birth, may represent a crucial connection between LBW and a heightened susceptibility to chronic inflammatory diseases. Notably, IFN-γ alterations were specifically observed after stimulation with PHA, suggesting that LBW neonates may have a reduced threshold for immune activation, potentially causing exaggerated inflammatory responses to environmental stimuli throughout life. Evidence showed that maternal systemic inflammation can be transmitted and amplified through the placenta, leading to intrauterine immune priming and fetal epigenetic reprogramming. This process not only contributes to the development of IUGR and LBW, but also to the promotion of a persistent pro-inflammatory phenotype in LBW neonates (43,44). It enables targeted interventions during critical developmental windows, including anti-inflammatory prenatal maternal nutrition and inflammation monitoring, as well as postnatal neonatal immunomodulatory strategies informed by immune screening. These interventions aim to modulate epigenetic-immune pathways and mitigate chronic inflammation in LBW individuals at birth.

Our findings highlight LBW-associated DNA methylation alterations as a key mechanism in early immune developmental programming. The epigenetic and functional profile of CBMCs from LBW neonates reveals a systemic programming toward a low-grade inflammatory state at birth. Although observed in immune cells, these alterations are implicated in the pathogenesis of cardiometabolic diseases, primarily through the establishment of a chronic pro-inflammatory milieu, characterized by epigenetically driven hyperresponsive IFN-γ signaling and a dysregulated cytokine profile. This sustained systemic inflammation could promote insulin resistance, endothelial dysfunction, and atherosclerosis, thereby linking the neonatal “pro-inflammatory immune phenotype” to later-life metabolic syndrome and cardiovascular disease.

In addition to immune alterations, we identified three additional biological process themes: synaptogenesis, translational regulation, and pancreas development. Epigenetic changes in CBMCs associated with LBW appear to drive dysregulation within these processes, potentially contributing to neurodevelopmental deficits and metabolic dysfunction, such as impaired cognitive performance and disrupted glucose metabolism (6,45,46). These findings suggest that prenatal epigenetic programming in LBW perturbs fundamental developmental pathways across multiple organ systems, offering an integrative molecular framework for the diverse clinical outcomes observed in LBW individuals.

A major strength of this study lies in its distinctive MZ twin cohort design and multi-level validation, which collectively minimize genetic confounding and enhance the generalizability of the findings. We identified an epigenetic association with the LBW in neonatal MZ twin cohort, then validated transcripts of key DMGs, and confirmed functional relevance by analyzing ex vivo cytokine responses in cord blood immune cells in independent neonatal cohorts. This integrated approach reveals dysregulation of the IFN-γ-JAK-STAT pathway alongside a pro-inflammatory cytokine imbalance as epigenetically programmed mechanisms present at birth in LBW neonates. These findings provide a mechanistic basis for developing targeted perinatal interventions (e.g., immunomodulation) and suggest that these inflammatory markers could aid in early risk stratification for future chronic inflammatory diseases.

There are limitations in this study. First, the small size of the discordant neonatal MZ twin cohort (only four pairs) may reduce statistical power and increase the risk of false positives. To mitigate this issue, we used MeDIP-seq as a hypothesis-generating approach to identify potential methylation differences. In addition, we applied two complementary statistical tests (Q1 and Q2) to detect LBW-associated DMGs with robust biological relevance. Encouragingly, the candidate DMGs and pathways identified were successfully validated at high rates in independent neonatal cohorts. Although the sample size was constrained by recruitment challenges, this well-characterized cohort, analyzed with function-oriented methods, still proved informative and generated high-quality hypotheses. Second, the absence of direct within-subject methylation-expression correlation analysis restricts causal inferences at the gene level. Nevertheless, our complementary two-phase strategy, which involves controlling the genetic background in MZ twins, followed by functional validation of the implicated IFN-γ pathway via immunostimulation, provides the key pathway-level evidence. Third, analysis confined to CBMCs limits insights into tissue-specific epigenetic mechanisms. Future studies should use larger neonatal MZ twin cohorts, incorporate multi-tissue analyses, and employ targeted mechanistic approaches to construct a comprehensive map of the neonatal epigenome and elucidate the functional pathways linked to LBW at birth.

Conclusions

This study demonstrates that distinct DNA methylation patterns in CBMCs from LBW neonates are associated with exaggerated IFN-γ-mediated immune responses and a pro-inflammatory state at birth. These epigenetic alterations may contribute to the increased long-term risk of chronic inflammatory diseases in LBW individuals. Our findings provide mechanistic insights into the ways in which adverse intrauterine conditions can epigenetically program long-term health outcomes, thereby highlighting potential targets for early risk assessment and prevention.

Acknowledgments

We gratefully acknowledge all the people who have given us help with our experiments, especially Wanxia Gai for her valuable assistance and insightful comments on data processing during her time at Zhejiang University.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-954/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-954/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-954/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-954/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study protocol was approved by the Ethics Committee of the Women’s Hospital of Zhejiang University (No. 20130027). Informed consent was obtained from the parents of all participating neonates involved in the study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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