Reduced Methylation of IRF5 Gene as a Biomarker for Gestational Diabetes Mellitus

Introduction

Diabetes mellitus is a global metabolic disease, and gestational diabetes mellitus (GDM) remains one of the most common complications of pregnancy. According to the American Diabetes Association, GDM is defined as diabetes recognized in the second or third trimester, provided that explicit diabetes has been excluded prior to or, at the latest, in early pregnancy. Gestational diabetes predisposes to a wide range of complications hazardous to both the mother and the fetus. GDM nearly triples the risk of preeclampsia, increases the rate of respiratory distress in the newborn by 4-fold, and raises the risk of postpartum hypoglycemia by 50% [1–3]. Additionally, it substantially aggravates the risk of fetal macrosomia [4], which doubles the risk of shoulder dystocia [5], thereby significantly increasing the incidence of complications such as clavicle fractures, brachial plexus paresis, birth canal injuries, and postpartum hemorrhage [6]. Pregnancies complicated by GDM are also more likely to result in preterm delivery [7], to be delivered by cesarean section [8], and to have a perinatal death risk of approximately 0.5% [9]. Beyond its immediate obstetric risks, GDM has major implications for long-term maternal and offspring health, making it a critical public health concern and underscoring the need for novel diagnostic and preventive strategies.

The pathophysiology of GDM is not fully understood, with the prevailing hypothesis linking abnormal placental hormone expression to maternal metabolic dysfunction and the synthesis and function of insulin [10].

Increased concentrations of prodiabetogenic hormones and pro-inflammatory changes at the cellular level contribute to an overall increase in insulin resistance, which is aimed at ensuring an adequate supply of glucose to the fetus [11,12]. In the course of GDM, however, an increased concentration of pro-inflammatory markers, such as C-reactive protein (CRP), tumor necrosis factor alpha (TNF-alpha), and interleukin (IL)-6, along with disturbed adaptation of pancreatic beta cells to altered metabolic conditions and impairments in the insulin signaling pathway, are observed [13,14]. An insufficient compensatory increase in insulin secretion is observed, which leads to hyperglycemia under conditions of a greater than normal decrease in insulin sensitivity [15]. Although inflammation is a recognized hallmark of GDM, the molecular mechanisms linking immune dysregulation with impaired glucose metabolism remain poorly elucidated, particularly at the epigenetic level.

The interferon regulatory factors (IRFs) family is composed of transcription factors that play a role in innate and adaptive immune responses. Mammalian IRFs consist of 9 members (IRF1–9) and their main function is to activate the expression of interferon (IFN) genes and promote the expression of IFN-related genes. Thus, they are also responsible for increasing the expression of inflammatory molecules such as ILs and chemokines [16,17].

Among these IRFs, IRF5 plays the most important role in inflammation. Recent studies have shown that this molecule is related to the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-23, and TNF-alpha and is also associated with various conditions, including insulin sensitivity issues, vascular diseases, obesity and rheumatoid arthritis. The IRF5 gene is located on chromosome 7. IRF5 is expressed in a variety of immune cells, including dendritic cells, macrophages, monocytes, B cells, and T cells. IRF5 plays a crucial role in shaping the immune response by promoting the polarization of macrophages toward the pro-inflammatory M1 phenotype and modulating T cell migration, proliferation, and differentiation. Its overexpression has been documented in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE) and in murine models of the disease. Similarly, increased IRF5 expression has been detected in inflamed mucosal tissues and PBMCs of patients with inflammatory bowel disease (IBD), where it enhances Th1/Th17-mediated immune responses. Importantly, the expression of IRF5 is regulated, at least in part, through DNA methylation mechanisms, which are known to be tissue-specific and responsive to inflammatory cues. Aberrant methylation of the IRF5 promoter may therefore contribute to its dysregulated expression in chronic inflammatory conditions [18–20, 33–34]. In addition to IRF5, IRF7 is another key regulator of innate immunity, yet its potential role in GDM remains largely unexplored. To date, only a few studies have investigated the methylation status of these IRFs in pregnancy-related disorders, and data on GDM are scarce and inconclusive.

Epigenetic modifications are defined as changes in gene expression that occur without alterations to nucleotide sequence. It is believed that the essential function of epigenetics in the genomic environment is to provide a response to internal and external environmental factors through dynamic, mostly reversible, changes in chromatin structure and gene expression. The basic epigenetic processes are considered to be DNA methylation, histone modifications, noncoding RNA regulation, and chromatin remodeling.

The most frequently studied mechanism is the methylation of cytosine nitrogenous bases in the DNA chain. Methylation is directly related to the inhibition of gene expression. Functionally active genes are hypomethylated, which enables the synthesis of proteins necessary for the metabolic tasks of a specified cell. Methylation in the promoter region of a gene reduces its expression, whereas methylation within the repressor region enhances its expression. Given that DNA methylation has been increasingly implicated in metabolic and inflammatory disorders, investigating its role in GDM could provide novel insights into disease mechanisms and biomarker development.

Although GDM generally resolves after delivery, it can also cause long-term complications such as type II diabetes mellitus (T2DM) and cardiovascular disease in the mother, or obesity, hyperlipidemia, non-alcoholic fatty liver disease, impaired glucose metabolism, or hypertension in the offspring. Moreover, there is a constantly increasing body of evidence suggesting that the long-term metabolic consequences of diabetes can be epigenetically mediated [32]. Therefore, the search for specific epigenetic marks, particularly in pregnant women with GDM, seems valuable. These epigenetic marks could become crucial for preventive measures, early diagnosis, and adequate management.

This study aimed to assess the DNA methylation levels of selected IRF family genes, including IRF5 and IRF7, to elucidate their potential involvement in the pathogenesis of gestational diabetes mellitus (GDM).

Material and Methods

SAMPLE SIZE CALCULATION:

The sample size analysis was estimated based on our experience from previous studies on epigenetic markers, including methylation [20], and assumptions regarding the study design: probability of type I error: 0.05, probability of type II error: 0.2; enrolment ratio HC: GDM - 1: 2, and increased/decreased mean target methylation value in RA group: 50%. Based on the pre-defined statistical assumptions, the minimum required sample size was estimated to be 19 participants in the control group and 38 participants in the GDM group.

SUBJECTS:

We collected data at a single point in time. A total of 62 pregnant women, including 39 with GDM, aged 32.3±5.36 years, and 23 controls, aged, 30.1±3.61 years, were enrolled in the study. Participants were eligible for inclusion if they were pregnant women aged 18 to 45 years, in their second or third trimester, and diagnosed with GDM (the primary exposure variable). GDM was diagnosed according to the oral glucose tolerance test (OGTT, 75 g, at 24 to 28 weeks). The guidelines for glucose testing during pregnancy are stated by the Polish Society of Gynecology and Obstetrics, which involves fasting overnight, followed by drinking a solution containing 75 grams of glucose. Blood samples are taken before ingestion and at 1 and 2 hours afterward. Gestational diabetes is diagnosed if at least 1 of the following thresholds is met: fasting: ≥5.1 mmol/L (≥92 mg/dL), 1 hour: ≥10.0 mmol/L (≥180 mg/dL), 2 hours: ≥8.5 mmol/L (≥153 mg/dL).

Exclusion criteria included pre-existing type 1 or type 2 diabetes, serious autoimmune disorders, inflammatory conditions, and severe comorbidities (including active infections, malignancies, advanced heart failure, or end-stage renal disease). The study was carried out according to the Declaration of Helsinki and was approved by the Bioethics Committee of the University of Rzeszów, protocol number 04/02/2020. Prior to conducting any procedures related to this study, individuals were informed about the potential benefits and risks associated with participation, and it was clearly communicated that involvement in the study was entirely voluntary. The individuals provided their written informed consent. The characteristics of the subjects are presented in Table 1. The clinical variables included in the patient characteristics were obtained from medical records and encompassed, among others, pharmacological treatment and dietary. The ethnic background of the patients was homogeneous (all were White), and all participants were recruited from a single clinical center. The study cohort consisted of consecutive hospital admissions, without stratification or preselection based on age, BMI, or other clinical parameters. To reduce the risk of selection bias, participant recruitment was carried out using a standardized and validated protocol, ensuring consistency and representativeness of the study population. This approach ensured the inclusion of a representative sample of patients with GDM, enhancing the generalizability of the findings.

One 9-ml tube of maternal whole-blood samples with EDTA was collected from the antecubital vein after OGTT (24–28 weeks). Peripheral blood mononuclear cells (PBMCs) were isolated in a density gradient using the Gradisol L reagent (Aqua Med, Poland, d=1.077 g/cm³). Briefly, whole blood was diluted with phosphate-buffered saline solution (PBS) in a 1: 3 ratio. The samples were centrifuged for 20 minutes at 20°C and 2000 rpm. PBMCs were collected and were washed twice with 10 ml of PBS. The cells were then suspended in 1 ml of PBS and stored at −80°C until further analysis. Genomic DNA was extracted from 200 μl of samples according to the manufacturer’s protocol using the GeneMATRIX Quick Blood DNA Purification Kit (Eurx, Poland). DNA quantity and purity were assessed by spectrophotometry (Nanodrop, Thermo Fisher Scientific). A total of 500 ng of DNA was converted by sodium bisulfite using the EZ DNA Methylation Gold Kit (Zymo Research, USA) according to the manufacturer’s recommendations with the elution volume of 50 μl. Converted DNA was stored at −80°C until downstream analysis.

METHYLATION ANALYSIS:

The primary outcome variables included DNA methylation levels of selected genes from the IRF family, specifically IRF2, IRF3, IRF5, IRF7, and IRF9. The IRF promoters’ regions were found in the Eukaryotic Promoter Database [21]. Primers for the IRF5 gene were previously described [20]. The flanking region of −1000 bp to +100 bp relative to the transcription start site was used for primer design. Oligonucleotides complementary to other targets were designed using the MethPrimer Software, version 1.0 [22]. Their sequences and CpG sites are presented in Table 2. The primers were designed to ensure that at least 1 contained 2 CpG sites, with 1 positioned within the final 3 nucleotides at the 3’ end. The specificity of primers was initially tested in-silico using the BiSearch e-polymerase chain reaction (e-PCR) online tool [23]. Then, 4 samples were randomly selected to test the ability to amplify the selected targets. The specificity of PCR products was confirmed using fully methylated and unmethylated DNA controls (EpiTect PCR Control DNA Set, Qiagen, Germany). The PCR reactions were performed according to the conditions described below, and amplification products were visualized on a 2% agarose gel. After this phase of the experiment, the following targets were selected for further analysis: IRF5 and IRF7–2. The promoter regions of both genes are shown in Figures 1 and 2. The detailed sequence of the promoter regions is provided in the supplementary materials. Quantitative real-time methylation-specific PCR (Q-MSP) was used to analyze methylation levels of selected regions. The methylation status of the selected promoters was evaluated using primers complementary to the methylated target sequence. The Q-MSP reaction contained 300 nM of each primer for all targets. To normalize the input of DNA after bisulfide conversion, the promoter region free of CpG sites in the beta-actin gene (ACT-B) was used as previously described [24]. Q-MSP reactions were performed using either SG onTaq qPCR Master Mix (Eurx, Poland) or ExiLENT SYBR Green master mix (EXICON, Denmark) in the QuantStudio 5 Real-Time PCR System (Applied Biosystems) under the thermal cycling conditions specified in the mix manual, in 40 amplification cycles. Only amplification curves with Ct values below 35 were evaluated. The reaction was performed using the ExiLENT SYBR Green master mix (EXICON, Denmark) for IRF7 gene and SG onTaq qPCR Master Mix for IRF5 gene under the thermal cycling conditions specified in the mix manual, except that the annealing/elongation step for IRF5 was performed in 2 steps at 61°C for 30 seconds and at 72°C for 30 seconds. For IRF7, 1 step at 61° for 60 seconds. was used. Both PCRs were conducted in 45 amplifications cycles. Following each PCR reaction, melting curve analysis was performed to verify the specificity of the amplification products. Each reaction plate included molecular-grade water as a negative control to detect potential reagent contamination. A calibrator previously prepared from 5 randomly selected samples was used to normalize the variability between plates. Q-MSP efficiency was evaluated as previously described [5]. The data were collected and analyzed using the comparative Ct method (ΔΔCt method; QuantStudio Design and Analysis Software v1.5.2, Applied Biosystems; Thermo Fisher Scientific, CA, USA). The expression results are presented as relative quantitation (RQ).

Depending on the data distribution, assessed using the Shapiro-Wilk W test, quantitative variables with normal distribution were presented as mean ± SD otherwise, the median (25^th–75^th percentile) was used. Differences between 2 independent groups were compared using either Student’s t test or the Mann-Whitney U test. The relationship between 2 continuous variables was analyzed using Spearman’s rank correlation coefficient (r_s). Qualitative variables were presented as numbers with percentages and were analyzed using contingency tables with a χ² test, including Yates’s correction where appropriate. Since only 2 pre-defined comparisons were performed (for IRF5 and IRF7-2), correction for multiple testing was not applied.

A P value <0.05 was considered statistically significant. The analysis was performed with STATISTICA, Version 13.1 (Dell, Inc., 2016, Tulsa, OK, USA).

Results

This study employed a cross-sectional design, with data collected at a single point in time. Methylation levels of the IRF5 and IRF7 genes were analyzed in GDM patients and healthy controls. A significantly lower methylation level of the IRF5 gene was observed in the GDM group compared to controls (median [25th–75th percentile]: 0.26 [0.18–0.43] vs 0.42 [0.30–0.66], P=0.027; Figure 3). No significant differences were found in IRF7 methylation between the groups (mean±SD: 1.01±0.27 vs 1.07±0.22, P=0.35; Figure 4).

Correlation analyses revealed no significant associations between the methylation levels of IRF5 and IRF7 and FPG levels, maternal BMI, or birth weight (Spearman’s rs ranged from −0.17 to 0.14). Similarly, no significant correlations were found within the GDM group between C-reactive protein (CRP) levels and methylation of either gene (rs=0.17 for IRF5, rs=−0.16 for IRF7; Figure 5). Figure 6 illustrates the heterogeneity within the GDM group, showing that while some patients exhibit high methylation – particularly for IRF5 – and CRP levels (red area), the majority present with low to intermediate levels across all parameters.

No significant differences in methylation levels of IRF5 and IRF7 were observed between primiparous and multiparous women (P=0.27 and P=0.58, respectively). Likewise, among GDM patients, methylation levels did not differ significantly between those treated with insulin (n=9) and those managed with dietary interventions (n=30; P=0.77 for IRF5, P=0.59 for IRF7).

In a subgroup analysis of primiparous women (10 women in GDM group and 10 individuals in control group), those with GDM exhibited significantly lower IRF5 methylation compared to healthy controls (mean±SD: 0.26±0.19 vs 0.46±0.21, P=0.043; Figure 7). No significant differences were observed for IRF7 methylation in this subgroup (P=0.62; Figure 8).

Discussion

This study demonstrated for the first time that women with GDM have a significant reduction in IRF5 gene methylation compared to healthy controls, with this effect particularly evident among primiparous women. In contrast, IRF7 methylation did not differ between groups, and no associations were observed between IRF5 or IRF7 methylation and common clinical variables such as fasting plasma glucose, BMI, or neonatal birth weight. These findings suggest that altered IRF5 methylation may be an independent epigenetic feature of GDM.

Our findings contribute to a growing body of research on epigenetic modifications associated with metabolic and inflammatory conditions [25–27]. IRF5 and IRF7 are transcription factors involved in immune responses, particularly in regulating inflammation. Although no direct studies on IRF5 and IRF7 methylation in GDM are available, research in other metabolic conditions has shown associations between altered methylation patterns and inflammatory gene regulation [20,28,29].

The observed lower methylation level of the IRF5 gene in GDM patients could suggest increased expression of this gene in the context of GDM. Studies in autoimmune diseases and other inflammatory disorders have reported that IRF5 hypomethylation is associated with increased transcriptional activity, leading to enhanced expression of pro-inflammatory cytokines. Since GDM is characterized by chronic low-grade inflammation, this hypomethylation could reflect a similar pro-inflammatory shift. The lack of significant differences in IRF7 methylation suggests that its role in GDM may be limited or that this gene’s regulation in the context of GDM is not heavily influenced by methylation. Alternatively, IRF7 may be regulated by other epigenetic mechanisms or post-transcriptional modifications not captured in this study. Further investigation is warranted to determine if IRF7 plays a unique role in other metabolic or immune conditions associated with pregnancy. The hypomethylation of the IRF5 gene observed in GDM patients may indicate an epigenetic adaptation or maladaptation to the inflammatory environment characteristic of GDM. The absence of significant correlations between IRF5 or IRF7 methylation and FPG, BMI, or birth weight suggests that these methylation differences may be independent of traditional metabolic markers. This could imply that IRF5 methylation reflects a more fundamental immunological change in GDM rather than being merely a consequence of hyperglycemia or increased adiposity. While our study did not find an association, future studies with larger sample sizes may help clarify whether there is a meaningful relationship between systemic inflammation and IRF5 methylation in GDM.

If validated in larger, prospective cohorts, IRF5 methylation could be developed as a minimally invasive diagnostic tool or used to stratify patients according to complication risk. This may ultimately support more personalized management of GDM. Moreover, interventions known to influence epigenetic profiles, such as dietary modification or pharmacological agents, could be explored as strategies to reverse or mitigate IRF5-related inflammatory responses in GDM [19,30,31].

A limitation of our methylation study was the biological material. We evaluated the frozen PBMCs, including all mononuclear cells, such as T and B cells and monocytes, while the analysis of the individual subpopulations or systemically circulating cells or circulating nucleic acids such as cell-free DNA may be helpful for a more in-depth understanding of the role of IRFs in GDM. On the other hand, collection of peripheral blood is a minimally invasive procedure and is easy to perform. The use of the Q-MSP method as a technique for searching for potential biomarkers also has its justification. Techniques based on the PCR reaction are characterized by high sensitivity and specificity and are not costly. Gene expression analysis based on RNA quantification may be more complicated than analysis at the DNA level. RNA is more labile, sensitive to nucleases, and more easily hydrolyzed. Moreover, classic protein-based biomarkers also have some limitations. The challenge is to identify candidate proteins which will be highly specific and sensitive. Another problem is the complexity of protein structures, the multitude of post-translational modifications, and their often low concentration in the available biological material, which makes their detection difficult [35,36,37].

This was an initial study, and further studies should aim to replicate these findings in larger, multi-center cohorts and explore other genes involved in immune regulation to provide a broader understanding of epigenetic changes in GDM. Furthermore, functional analysis is needed to determine if lower IRF5 methylation directly contributes to the pathophysiology of GDM or if it is merely a marker of the disease. Investigating how lifestyle factors such as diet, physical activity, and medication may impact IRF5 methylation could also offer insights into preventive or therapeutic approaches.

Conclusions

This study demonstrated a significant reduction in IRF5 methylation levels in women with gestational diabetes mellitus (GDM), particularly among primiparous women, while IRF7 methylation levels remained unaffected. Importantly, these alterations appeared to be independent of fasting plasma glucose, BMI, and neonatal birth weight, suggesting that IRF5 methylation is an epigenetic feature of GDM beyond traditional clinical parameters.

Although IRF5 hypomethylation may contribute to enhanced inflammatory signaling in GDM, this interpretation remains speculative and requires further confirmation. Thus, the observed association should be viewed primarily as a preliminary finding rather than evidence of causality.

Our results provide new insight into the molecular mechanisms potentially linking immune dysregulation and GDM. However, the absence of methylation changes in IRF7 highlights the complexity of interferon regulatory factors in pregnancy and underscores the need for broader epigenetic profiling.

Future studies should include longitudinal cohorts to determine the temporal relationship between IRF5 hypomethylation and GDM onset, as well as mechanistic investigations to clarify whether altered methylation directly drives inflammatory pathways. Research is needed to evaluate whether IRF5 methylation can serve as a clinically useful biomarker or a target for preventive interventions.

Taken together, our findings suggest that IRF5 methylation is a promising epigenetic signal in GDM, warranting further investigation to improve early detection and refine our understanding of disease pathophysiology.