Preeclampsia (PE) is a complex multisystem disorder of pregnancy characterized by the new onset of hypertension and proteinuria after 20 weeks of gestation. It remains a leading cause of maternal and perinatal morbidity and mortality globally, particularly in low- and middle-income countries, contributing to adverse outcomes such as preterm birth, fetal growth restriction, and placental abruption [1]. Despite advances in obstetric care, the ability to predict and prevent preeclampsia early in gestation remains limited.

The pathogenesis of preeclampsia is multifactorial and not fully understood, but abnormal placentation and impaired trophoblast invasion are widely recognized as key contributors [2]. These placental abnormalities result in hypoxia, oxidative stress, and systemic endothelial dysfunction, all of which manifest clinically as preeclampsia [3]. Consequently, much attention has been directed toward identifying biomarkers of placental dysfunction that could serve as early predictors of the disease. Conventional biochemical markers and Doppler ultrasound assessments have demonstrated moderate predictive value, but are not sufficiently sensitive or specific for routine clinical use [4,5].

Recent studies have highlighted the potential of cell-free fetal DNA (cffDNA) circulating in maternal blood as a non-invasive source of information about placental health [6]. Released into the maternal circulation primarily from trophoblast apoptosis, cffDNA is detectable from as early as the first trimester and reflects the epigenetic and genetic status of the placenta [7]. Among the epigenetic modifications, DNA methylation plays a pivotal role in regulating gene expression during placental development and has emerged as a candidate mechanism underlying many pregnancy-related disorders, including preeclampsia [8,9].

Genome-wide methylation profiling of cffDNA offers a novel avenue for exploring disease-specific methylation signatures. Preliminary investigations have reported differentially methylated regions (DMRs) in the cffDNA of women who later developed preeclampsia, particularly in genes involved in angiogenesis, immune regulation, and trophoblast function [10,11]. However, there is a need for prospective, large-scale studies to validate these findings and assess their predictive performance in early pregnancy.

Therefore, this study aimed to investigate the DNA methylation profiles of circulating cell-free fetal DNA in the first trimester and evaluate their utility as early predictive biomarkers for preeclampsia in a prospective cohort.

Study Design and Participants

A total of 180 pregnant women in their first trimester (gestational age 10–14 weeks) attending the antenatal outpatient clinic were recruited.

Inclusion and Exclusion Criteria

Inclusion criteria comprised singleton pregnancies, maternal age between 18 and 40 years, and absence of pre-existing hypertension, diabetes, renal disease, or autoimmune disorders. Women with multiple gestations, known fetal anomalies, or any chronic illness that could confound study outcomes were excluded.

Sample Collection and Processing

At enrollment, 10 mL of peripheral blood was collected from each participant in EDTA tubes. Samples were processed within 2 hours to isolate plasma by centrifugation at 1600 g for 10 minutes, followed by a second centrifugation at 16,000 g for 10 minutes to remove residual cells. Plasma was aliquoted and stored at −80°C until further analysis.

Cell-Free Fetal DNA Extraction and Bisulfite Conversion

Cell-free DNA was extracted from 2 mL of maternal plasma using a commercially available cffDNA isolation kit following the manufacturer's instructions. To enrich fetal-derived sequences, a size-selection step was applied to preferentially retain DNA fragments of approximately 150 bp. Bisulfite conversion of the isolated DNA was performed using the EZ DNA Methylation Kit, allowing methylation analysis of CpG sites.

Methylation Profiling and Data Analysis

Genome-wide DNA methylation was assessed using the Infinium MethylationEPIC BeadChip (Illumina), covering over 850,000 CpG sites. After scanning, raw intensity data were processed using R software and the minfi package. Quality control steps included background correction, probe filtering, and normalization. Methylation levels were expressed as β-values ranging from 0 (unmethylated) to 1 (fully methylated).

Clinical Follow-Up and Outcome Classification

Participants were monitored until delivery. Based on clinical diagnosis following the ISSHP criteria, participants were categorized into two groups: those who developed preeclampsia (PE group, n=30) and those who had normotensive pregnancies (control group, n=150). Diagnosis of preeclampsia was established based on new-onset hypertension (≥140/90 mmHg) after 20 weeks of gestation accompanied by proteinuria or evidence of organ dysfunction.

Statistical Analysis

Differentially methylated regions (DMRs) between the PE and control groups were identified using linear models for microarray data (limma) with false discovery rate (FDR) correction. Logistic regression models were used to evaluate the predictive value of significant DMRs. Receiver operating characteristic (ROC) curves were plotted to determine diagnostic performance, and the area under the curve (AUC) was calculated. A p-value of <0.05 was considered statistically significant.

A total of 180 pregnant women were enrolled and followed until delivery. Among them, 30 participants (16.7%) developed preeclampsia, while 150 (83.3%) had normotensive pregnancies. Demographic and clinical characteristics between the two groups are summarized in Table 1.

Table 1: Baseline Characteristics of Study Participants

*Statistically significant (p < 0.05)

Analysis of DNA methylation profiles revealed significant differences between the two groups. A total of 127 differentially methylated regions (DMRs) were identified with a false discovery rate (FDR) < 0.05. Among these, 93 were hypomethylated and 34 were hypermethylated in the preeclampsia group. Several DMRs were located in genes related to angiogenesis, trophoblast invasion, and immune regulation.

The top five DMRs showing the highest significance and their associated genes are presented in Table 2.

Table 2: Top Differentially Methylated Regions in cffDNA Between PE and Control Groups

Predictive performance of a panel combining these five top markers was evaluated using ROC curve analysis. The model demonstrated an area under the curve (AUC) of 0.89 (95% CI: 0.82–0.94), indicating excellent discrimination between women who developed preeclampsia and those who did not. At an optimal cut-off value, sensitivity and specificity were 86.7% and 81.3%, respectively (Table 3).

Table 3: Diagnostic Performance of Combined Methylation Markers for Preeclampsia Prediction

These findings suggest that the identified methylation alterations in cell-free fetal DNA during early pregnancy can serve as potential predictive biomarkers for preeclampsia (Tables 2 and 3).

This prospective study demonstrates that distinct DNA methylation signatures in circulating cell-free fetal DNA (cffDNA) during the first trimester are significantly associated with the later development of preeclampsia. Our findings support the growing evidence that epigenetic modifications, particularly DNA methylation patterns, can serve as promising early biomarkers for predicting pregnancy-related complications such as preeclampsia.

Preeclampsia is widely acknowledged to be a placental disorder arising from defective trophoblast invasion and abnormal remodeling of the spiral arteries during early gestation, leading to hypoxia and systemic endothelial dysfunction [1]. This placental pathology is known to precede clinical symptoms by several weeks, making early molecular changes in the placenta—reflected in cffDNA—a critical focus of research [2,3].

In our study, genes such as FLT1, ENG, INHBA, and PPARG exhibited significant hypomethylation in the preeclampsia group. These genes play central roles in angiogenesis, vascular remodeling, and placental development. Previous research has shown that FLT1, which encodes soluble fms-like tyrosine kinase-1 (sFlt-1), is overexpressed in preeclamptic placentas and contributes to endothelial dysfunction through its anti-angiogenic effects [4,5]. Similarly, altered methylation of the ENG gene, which encodes endoglin—a co-receptor for transforming growth factor-beta (TGF-β)—has been implicated in defective placentation and maternal vascular adaptation in PE [6].

Our results are consistent with earlier genome-wide methylation studies that identified hypomethylated regions in placental genes associated with vascular function in PE pregnancies [7,8]. Moreover, the high predictive performance of the combined methylation panel (AUC = 0.89) underscores the potential clinical utility of this approach. Previous studies using cffDNA methylation biomarkers have reported AUC values ranging from 0.75 to 0.87, reinforcing the robustness of our model [9,10].

The biological relevance of altered methylation in immune-regulatory genes, such as HLA-G, is also notable. HLA-G plays a crucial role in maternal-fetal immune tolerance, and its dysregulation has been linked to immune-mediated placental dysfunction in PE [11,12]. The hypermethylation of HLA-G observed in our study supports the hypothesis of epigenetically driven immune dysregulation as a contributing mechanism in preeclampsia.

The use of cffDNA as a non-invasive source of fetal and placental information offers several advantages over traditional serum biomarkers. cffDNA can be detected as early as 7 weeks of gestation and is released predominantly from placental trophoblasts, providing a direct reflection of placental status [13,14]. The epigenetic landscape of cffDNA mirrors that of the fetal genome and is relatively stable, making it suitable for early screening applications [15].

This prospective study highlights the promising role of circulating cell-free fetal DNA (cffDNA) methylation profiles as early, non-invasive biomarkers for predicting preeclampsia. Our findings reveal significant alterations in DNA methylation—particularly hypomethylation in genes related to placental angiogenesis and immune modulation—in women who later developed preeclampsia. The strong diagnostic performance of a panel of top differentially methylated regions (AUC = 0.89) underscores their potential clinical value in early pregnancy screening. Integrating cffDNA methylation analysis into routine antenatal care could enable earlier identification of at-risk pregnancies, guiding closer surveillance and timely interventions to reduce maternal and fetal complications. Further large-scale, multi-center studies are warranted to validate these results and establish standardized protocols for clinical application.

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