Genetic and epigenetic alterations associated with gestational diabetes mellitus and adverse neonatal outcomes

Abstract

Gestational diabetes mellitus (GDM) is a metabolic disorder, recognised during 24-28 weeks of pregnancy. GDM is linked with adverse newborn outcomes such as macrosomia, premature delivery, metabolic disorder, cardiovascular, and neurological disorders. Recent investigations have focused on the correlation of genetic factors such as β-cell function and insulin secretary genes (transcription factor 7 like 2, potassium voltage-gated channel subfamily q member 1, adiponectin etc.) on maternal metabolism during gestation leading to GDM. Epigenetic alterations like DNA methylation, histone modification, and miRNA expression can influence gene expression and play a dominant role in feto-maternal metabolic pathways. Interactions between genes and environment, resulting in differential gene expression patterns may lead to GDM. Researchers suggested that GDM women are more susceptible to insulin resistance, which alters intrauterine surroundings, resulting hyperglycemia and hyperinsulinemia. Epigenetic modifications in genes affecting neuroendocrine activities, and metabolism, increase the risk of obesity and type 2 diabetes in offspring. There is currently no treatment or effective preventive method for GDM, since the molecular processes of insulin resistance are not well understood. The present review was undertaken to understand the pathophysiology of GDM and its effects on adverse neonatal outcomes. In addition, the study of genetic and epigenetic alterations will provide lead to researchers in the search for predictive molecular biomarkers.

Key Words: Gene expression; Gestational diabetes mellitus; Feto-maternal outcome; Epigenetic alteration; Molecular biomarkers

Core Tip: Higher morbidity and mortality rates were reported in neonates born to diabetic mothers. Gestational diabetes mellitus (GDM) is linked to both genetic and epigenetic alterations. Therefore, it would be beneficial to implement a strategy to find molecular biomarkers in GDM, such as genetic and epigenetic variations in genes associated with β-cell function and insulin signaling pathways. Implementing this strategy would result in GDM risk prediction, and improved maternal and newborn pregnancy outcomes while contributing to their future well-being.

INTRODUCTION

Gestational diabetes mellitus (GDM) is a pregnancy-related metabolic complication characterized by the development of persistent hyperglycemia in women who do not have a history of diabetes, arising during their gestational period. The chronic metabolic condition normally resolves after pregnancy. GDM can impact the development of several organ systems, with cardiovascular and neural tube defects being common anomalies. Other problems may include fetal growth abnormalities, pre-eclampsia, premature birth, and perinatal mortality. Neuro-developmental research on offspring born to mothers with diabetes has shown an increased prevalence of attention deficit hyperactivity disorder, learning impairments, motor abnormalities, and maybe autism spectrum disorder[1]. The consequences of maternal hyperglycemia on the developing fetus may be caused by increased hypoxia, oxidative stress, apoptosis, and genetic and epigenetic alterations. On a worldwide scale, GDM affects around 14% to 16% of pregnancies. GDM is most commonly diagnosed between 24-28 weeks of pregnancy[2]. Furthermore, an increase in the incidence of GDM depends upon the study population and the employed diagnostic tests. The incidence of GDM varies based on socio-economic status and ethnicity[3]. Additionally, a study indicated that the incidence of GDM has a seasonal variation, such as a larger number of GDM cases reported during the summer compared to the winter[4]. Based on the findings, Asian women had an 11-fold higher likelihood of developing GDM compared to women from other countries[5].

Annually, 5 million women in India, accounting for 16.55% of the female population, are diagnosed with GDM. According to a Report,1 in 6 pregnant women have GDM, worldwide 21 million newborns are affected by diabetes. It is noteworthy that 83% of these cases are caused by GDM[2,6]. However, there is a significant variation in the frequency of GDM, which can be attributed to disparities in diagnostic criteria, screening methods, and the research environment, such as urban vs rural or hospital vs community settings[7]. A 2-hour 75-gram oral glucose tolerance test is often used for diagnosing GDM between 24-28 weeks of pregnancy[8]. GDM is associated with many risk factors such as high parity, overweight or obese, family history of hyperglycemia, and advanced maternal age (Figure 1). The exact underlying etiology of GDM has not yet been uncovered. During pregnancy, the maternal pancreas fails to adjust the increased insulin requirements during gestation[9]. Hyperglycemia is often a result of impaired glucose tolerance, which is caused by malfunction of the pancreatic β-cells in instances of chronic insulin resistance conditions during gestation. GDM is characterized by a wide range of short- and long-term effects that impact both the mother and the fetal health. However, there is currently no widely recognized treatment or preventative method for GDM, except for lifestyle modifications such as exercise and dietary adjustments[2,10]. Insulin treatment may be used in certain instances; nevertheless, its efficacy is limited by the prevailing insulin resistance. While new oral drugs like Metformin and Glyburide offer the potential for diabetes treatment, concerns persist about the long-term well-being of both the mother and child[11].

Figure 1 Risk factors involved in increased incidence of gestational diabetes mellitus. GDM: Gestational diabetes mellitus; PCOS: Polycystic ovary syndrome.

During pregnancy, the accumulation of genetic and epigenetic changes in genes is linked to insulin secretion[12]. Earlier research indicated that genetic variations in genes involved in essential metabolic pathways such as insulin secretion, lipids, and glucose metabolism are linked to the onset of GDM[13,14]. In addition to genetic factors, epigenetic modifications have been identified as linked to GDM[15,16]. Epigenetics refers to the mechanisms that regulate gene expression without altering the underlying DNA sequence[17]. Epigenetic modifications are crucial in guiding the processes of development and differentiation throughout embryonic stages[18]. They play a crucial role in modifying gene expression that impacts pregnant women and newborns, consequently heightening the likelihood of developing metabolic syndrome and diabetes in the future[19]. Previous studied reported altered genetic variants of several genes that regulate β-cell function and insulin secretion such as transcription factor 7 like 2 (TCF7 L2), potassium voltage-gated channel subfamily q member 1 (KCNQ1), melatonin receptor 1b gene (MTNR1B), insulin-like growth factor 2 messenger RNA-binding protein 2 (IGF2BP2), insulin receptor substrate - 1 (IRS1), adiponectin (ADIPOQ), interleukin-10 (IL-10) genes may increase susceptibility to GDM[2,15]. Epigenetic mechanisms, such as DNA methylation, histone modification, and miRNA expression regulate gene expression and are interconnected[20,21]. Recent studies indicate that DNA methylation patterns in the placenta and cord blood of women experiencing GDM differ from those that were found in normoglycemic pregnancies[22]. Exposure of the fetus to the intrauterine environment in mothers with GDM leads to long-term consequences and increases the likelihood of developing metabolic diseases in the future[23]. Genetic alterations are increasingly linked to GDM, The epigenetic background of GDM focuses on DNA methylation patterns of placental regulatory genes such as leptin, ADIPOQ, proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), ATP-binding cassette transporter A1 (ABCA1), lipoprotein lipase (LPL), inflammatory cytokines, and IGF-binding proteins genes, as well as microRNA levels, including miR-132, miR-29a, miR-222, miR-16, miR-17, miR-19a, miR-19b, and miR-20a[24]. Genetic and epigenetic factors impact maternal metabolism, affecting energy supply and metabolic regulation crucial for fetal development. These alterations may predispose newborns to metabolic disorders like childhood obesity, diabetes, cardiovascular disease, and abnormal neural development making them interesting targets for genomic, genetic, and epigenetic studies[25]. By identifying gaps in the research areas, it is necessary to investigate more using a multidisciplinary strategy[26]. The ultimate goal is to improve the management and care offered to women and neonates.

GDM ASSOCIATED WITH ADVERSE SHORT- AND LONG-TERM NEONATAL COMPLICATIONS

Short term complications

The Pedersen theory explains how GDM affects the fetus due to insulin resistance, inducing fetal hyperglycemia and fetal macrosomia by increased transfer of glucose over the placenta to the fetus. Macrosomia is a birth weight exceeding 4000 or 4500 g, distinguished by a rise in subcutaneous fat, muscular mass, and a larger head circumference. It can result from shoulder dystocia and shoulder injuries, leading to birth complications such as fractures of the humerus and clavicle bones and injury to the brachial plexus (Erb's palsy). Abnormal fetal development is due to the transfer of additional growth substances (amino acids and lipids) through the placenta[27,28]. GDM poses risks to the fetus and newborn, including macrosomia, elevated fetal glucose levels, and increased insulin production. This leads to chronic fetal hypoxia, increasing the risk of intrauterine fetal mortality[29] (Figure 2). Fetal hypoxia leads to polycythemia, an excessive increase in red blood cell volume in venous blood, characterized by a venous hematocrit level of more than 65% and increased blood viscosity, can cause bluish discoloration (cyanosis), hypoglycemia, decreased muscle tone (hypotonia), challenges with eating, difficulty in breathing, skin redness (plethora), and hyperbilirubinemia[30,31]. Hypoglycemia in neonates born to mothers with GDM is caused by high insulin production, hindering the normal responses that counteract the placental insufficiency leads to reduced glucose supply, such as lipolysis, glycogenolysis, β-oxidation of fatty acids, and gluconeogenesis. Additionally, it increases glucose utilization by peripheral tissues. The Hyperglycemia and Adverse Pregnancy Outcome research shows a direct correlation between preeclampsia and glucose tolerance tests, a risk factor for premature delivery, and Neonatal Intensive Care Unit admission of neonates for respiratory support[32,33].

Figure 2 gestational diabetes mellitus linked with adverse maternal and newborn complications (i.e. type 2 diabetes mellitus, pre-eclampsia, hyperglycemia, caesarean delivery, recurrent gestational diabetes mellitus, miscarriage, type 1 diabetes mellitus, macrosmia, metabolic complications, obesity, neonatal hypoglycemia, shoulder dystocia, cardio-vascular diseases). T1DM: Type 1 diabetes mellitus; T2DM: Type 2 diabetes mellitus; GDM: Gestational diabetes mellitus.

Long-term complications

The rising global rates of obesity and GDM have significant public health implications, particularly for future generations. Several risk factors like parity, advanced maternal age, and gestational weight gain during pregnancy are significantly related to GDM and cause long-term consequences on newborn’s health[34]. Neonates born to mothers with GDM are susceptible to developing obesity, insulin resistance, Type 2 Diabetes Mellitus, cardiovascular disorders, and other metabolic illnesses[35,36] (Figure 2). Mothers with high glucose levels during pregnancy have a two-fold higher risk of their children being overweight or obese during childhood. This correlation persisted even after considering the mother's pre-pregnancy body mass index and the neonate's birth weight[37]. Maternal obesity substantially increases the probability of developing GDM and is the most effective predictor of obesity in children throughout the perinatal period[38]. Elevated systolic blood pressure in children increases the risk of hypertension in adulthood, a leading cause of cardiovascular disease[39]. The role of the maternal surrounding environment affects the intrauterine programming to determine the offspring obesity is still unknown. Insulin resistance during pregnancy is linked with GDM which increases the likelihood of metabolic syndromes such as dyslipidemia, obesity, glucose intolerance, and hypertension in children[40,41]. Regardless of the birth weight of a newborn or GDM diagnosis, high insulin levels in the umbilical cord at birth, are related to a 17-fold greater risk of metabolic syndrome at an average age of 15 years[42].

PLACENTAL HORMONES AND CHRONIC INSULIN-RESISTANT CONDITIONS ASSOCIATED WITH ALTERED FETOPLACENTAL GROWTH

Pregnancy is an intricate physiological phenomenon that is controlled by various hormones and signaling molecules, to facilitate embryonic growth and development, which involves significant changes in the circulatory, renal, hematologic, respiratory, and metabolic systems[43-45]. Throughout the pregnancy period, the placenta serves as an endocrine organ by releasing numerous hormones that are crucial for regulating maternal physiology. Moreover, it facilitates the delivery of nutrients and gasses to the growing embryo, thereby encouraging fetal development[46]. The trophoblast of the growing placenta releases hormones [leptin, insulin, glucocorticoid, insulin growth factor (IGF), prostaglandin, gonadotrophin, etc.] in a tightly regulated way to optimize the absorption of these hormones by the endometrium, thus promoting the implantation of the embryo and regulate the fetoplacental growth[47-49]. In normal physiological processes, IGFs and insulin regulate placental development by interacting with their receptors respectively IGF1R and IR in embryonic tissue, affecting placental growth, migration, invasion, transport, angiogenesis, and LPL activity[49-51]. The expression of placental IGF1 and IGF2 is influenced by gestation stages. During the first trimester, IGF2 appears predominantly in the cytotrophoblasts of the villous and extra-villous tissues, indicating its key role in the early stages of embryonic development[52]. Maternal hyperglycemia can cause angiogenesis and hyper-vascularization in the placenta, leading to an increase in blood vessel development and excessive proliferation of blood vessels. The excessive pro-angiogenic effects of insulin, impairment of insulin regulation, and growth hormones in the endothelial cells are the main causes of occurrence. IGFs regulate FABP4 expression, glucose, and amino acid transport across the placenta, and mammalian target of rapamycin (mTOR) signaling. They increase GLUT1 expression and glucose transport across epithelial cells. mTOR detects maternal endocrine signals, regulating fetal development through gene transcription and protein synthesis, and regulating nutrition transporter expression[53,54].

Maternal hormones such as IGF1, insulin, and leptin promote the activation of mTOR signaling, while hypoxia and adiponectin inhibit or reduce its activity. Restricting protein intake during pregnancy has an impact on the metabolism of glucose and insulin in the muscular tissue of the fetus[55]. Obesity and exposure to EDC can cause pregnancy complications, affecting the mTOR signaling pathway and placental nutrient transporter efficiency, and adversely affecting fetal development[49,56]. Placental hormones increase during pregnancy, leading to increased insulin resistance, since these hormones antagonize the effects of insulin. The main aim of the mechanism is to provide an adequate level of glucose to the fetus. Elevated maternal glucose levels cause a rise in insulin production, allowing the mother to maintain normal glucose levels[57]. GDM occurs when insulin production is insufficient to compensate for increased resistance or beta cell dysfunction during pregnancy. β-cell dysfunction is the loss of β-cells' ability to accurately measure glucose levels in blood and secrete enough insulin in response[58]. During gestation, insulin sensitivity changes due to pregnancy demands. In the first gestation, the body's response to insulin enhances glucose absorption into fat reserves, preparing for higher energy demands later in pregnancy. During pregnancy, the body experiences an increase in many hormones including placental lactogen, progesterone, placental growth hormone, cortisol, estrogen, and leptin[59].

These hormones function together to facilitate optimal fetal growth. Nevertheless, the increased level of placental hormones can also induce inflammation inside the body, resulting in oxidative stress generation and damage to DNA and cells. Progesterone levels rise during pregnancy specifically from the first to the third trimester, affecting the phosphoinositide 3-kinase (PI3K) pathway by reducing IRS-1 expression and preventing glucose transport and absorption through GLUT4. During pregnancy, estrogen levels rise and reduce insulin action, while human placental lactogen (hPL) functions similarly to insulin or anti-insulin medications[2,60]. Increased levels of hPL during pregnancy facilitate the absorption of glucose and the synthesis of glycogen in the mother's body. However, pituitary growth hormone and human placental growth hormone have diabetogenic effects, causing hyperinsulinemia, reduced glucose uptake, impaired formation of glycogen, and decreased insulin production[61]. During the third trimester of pregnancy, increased levels of tumor necrosis factor-α, the hormone cortisol, and other cytokines such as interleukins (IL-6 and IL-1β) have been linked with alterations in insulin signaling and inflammation. Glucotoxicity refers to the physiological impact of glucose on the failure of β-cells, resulting in a compromised insulin response and further malfunction of β-cells[62] (Figure 3).

Figure 3 Summarized role of various molecules participate in inflammatory and oxidative stress pathway in the pathophysiology of gestational diabetes mellitus. IL: Interleukin; TNF-α: Tumor necrosis factor-α; IRS: Insulin receptor substrates; mTOR: Mammalian target of rapamycin; TLRs: Toll-like receptor; RONS: Reactive oxygen and nitrogen species; ROS: Reactive oxygen species; AGE: Advanced glycation end products; PBMC: Peripheral blood mononuclear cells; NADPH: Nicotinamide adenine dinucleotide phosphate; NADH: Nicotinamide adenine dinucleotide; NADP+: Nicotinamide adenine dinucleotide phosphate oxidized; NAD+: Nicotinamide adenine dinucleotide oxidized; GPx: Glutathione peroxidase.

GENETIC ALTERATIONS AND GDM

Genetic variations are increasingly being linked with insulin resistance during pregnancy causing GDM. Research findings indicate that the alterations in genes that are crucial for metabolic processes during gestation elevate the probability of developing GDM[20]. The genes most prone to be linked with GDM are mostly those involved in β-cell function, such as regulating the insulin signal transduction, as well as several inflammatory and antioxidant genes[63,64]. The impact of these genes on maternal physiology during gestation has been examined by two different methods including the study of different genetic variants of regulatory genes in pregnant women to investigate the connection between the genes and the risk of GDM development. However, this method has shown little efficacy in identifying the particular genes implicated in the pathogenesis of GDM, mostly due to the limited sample numbers and the scarcity of genetic variations in the genomic region. Another method is Genome-wide association studies that found genetic polymorphism from many loci is linked with the risk of GDM. However, type 2 diabetes mellitus (T2DM) is distinguished by the presence of insulin resistance and shares a common genetic architecture with GDM, various genes that are linked with an increased risk of T2DM have also been examined in individuals with GDM[65]. Research on key genes involved in insulin signaling homeostasis has shown a correlation between single nucleotide polymorphisms (SNPs) and the likelihood of developing GDM[66] (Figure 4).

Figure 4 Genetic and epigenetic alterations are associated with environmental interaction involved in the pathophysiology of gestational diabetes mellitus. miRNAs: MicroRNAs; TCF7 L2: Transcription factor 7 like 2; KCNQ1: Potassium voltage-gated channel subfamily q member 1; MTNR1B: Melatonin receptor 1b gene; GCK: Glucokinase; IGF2BP2: Insulin-like growth factor 2 messenger RNA-binding protein 2; IRS1: Insulin receptor substrate-1; ADIPOQ: Adiponectin; IL-10: interleukin-10.

GENES INVOLVED IN INSULIN HOMEOSTASIS

TCF7 L2

TCF7 L2 gene is situated at the long arm of the 10^th chromosome (10q25.3) and encodes a transcription factor that contains a high mobility group box. This transcription factor is involved in the regulation of blood glucose homeostasis[67,68]. A study on the Chinese women population reported that AT genotypes of SNP rs4506565 and TC genotype rs7901695 are at a considerably higher risk for GDM[69]. Previous research on the populations of Saudi Arabia, Egypt, and Bangladesh has shown that individuals with the CT genotype of the SNP rs7903146 (C>T) had 2-3 times increased risk of developing GDM[70-72].

KCNQ1

KCNQ1 gene is located on the short arm of 11^th chromosome (11p15). The voltage-gated channel is present in pancreatic β-cells and has the ability to control insulin secretion by regulating the electrical potential of the cell membrane and the activity of ion channels. This eventually affects the synthesis of insulin[73]. Two separate study of rs2237892 and rs2237895in Korean population by Shin et al[74], and Kwak et al[75], demonstrated that the C alleles of both SNPs found to be linked with the risk of GDM. These SNPs are also significantly associated with insulin sensitivity[74,75]. Another study conducted on Chinese populations revealed that the presence of the C allele of rs2237892 had an elevated risk of GDM[76]. A study on a group of 960 Caucasian and African American women also confirmed that the SNP rs2237895 was found to be directly linked with an increase in gestational weight gain during pregnancy in women with GDM[77]. Similarly, another research found that the C allele of SNP rs2237895 is associated with a higher risk of GDM in Pakistani women[78].

MTNR1B

The gene encodes a receptor that specifically binds to the melatonin hormone. melatonin has a role in the regulation of the circadian cycle, insulin signaling pathway, and metabolism of glucose, among other physiological processes[79]. The genetic variant rs10830963/G of the MTNR1B gene has a notable influence on the progression of GDM and glycemic traits, such as insulin response and glucose absorption, and has been significantly associated with the risk of GDM in many populations[80,81]. Other studies found that T allele of SNP rs1387153 has a strong correlation with an increased risk of developing GDM[82,83]. A study discovered that the SNP rs4753426 of the MTNR1B gene may enhance the susceptibility to the risk of developing GDM. The study stated that the presence of the C allele is associated with elevated fasting plasma glucose levels and reduced pancreatic β-cell function[84].

Glucokinase

The glucokinase (GCK) gene is situated on the short arm of the 7^th chromosome (7p13). GCK gene encodes a protein, that is a part of the hexokinase protein family and serves as a vital regulating enzyme in the pancreaticβ-cells. Its main function is to catalyze the phosphorylation of glucose and control the secretion of insulin[85]. Research conducted on Brazilian women revealed that those who had the C allele of rs780094 had a 1.41 times higher risk of developing GDM[86]. Another case-control study on the Chinese population found that SNP rs1799884 was linked with the risk for GDM[87]. Additional meta-analyses have shown an association between rs1799884 polymorphisms and GDM in both Caucasian and Asian populations[88]. Another meta-analysis in the Caucasian population reported SNP rs780094 of GCKR was discovered as a contributing factor for GDM[89]. The presence of the C allele of SNP rs780094 in the Asian and Malaysian populations was shown to dramatically enhance the risk of GDM development[90].

IGF2BP2

The gene-coded IGF2BP2 protein, that binds to the messenger RNA (mRNA) of insulin-like growth factor 2. IGF2BP2 is mostly expressed in pancreatic β cells and plays a vital role in embryonic growth and development, cellular differentiation, cellular metabolism, and insulin control[91]. The expression of the IGFBP2 gene is controlled by the interaction of many transcription factors with the regulatory domain located at the promoter site[92]. The SNP rs4402960 has been shown to exhibit a robust association with the susceptibility to developing GDM and weight gain during gestation in Korean and Chinese populations[93,94]. A meta-analysis was performed to identify the major risk alleles that elevate the probability of developing GDM in Asian populations and found that the individuals with the genetic variant rs4402960 had a considerably increased risk of developing GDM[95]. Similarly, another research found that individuals with TT genotypes of SNP rs4402960 and AA genotypes of rs11705701 had a strong association with advanced maternal age and adverse effects on newborns[96].

IRS1

IRS1 gene coded the insulin receptor substrate 1 is a crucial adaptor protein essential for insulin signal transduction by transferring the signals between insulin and insulin-like growth factor-1 receptor to regulate the insulin response[97]. The presence of IRS gene SNP rs1801278 (C>T) variant was found to be associated with GDM women in the Chinese population[98]. Another study in Saudi Arabia found that T allele of rs1801278 was shown to be correlated with a high risk of developing GDM[99]. Similar research was found in the Pakistani population[100]. Another research in the Chinese population found that the T allele of rs1801278 was more prevalent in the GDM groups (8.7%) compared to the control groups (5.1%)[101].

ADIPOQ

The gene is situated in the long arm of the 3^rd chromosome (3q27.3) and is predominantly expressed in adipose tissue. ADIPOQ gene encodes a structural protein that has a resemblance to collagens X and VIII, as well as complement factor C1q. these components are mostly active in the bloodstream and has a role in metabolic and hormonal functions. mutations in this gene cause Adiponectin deficiency[102]. Adiponectin is a hormone that regulates insulin sensitivity by controlling fatty acid oxidation and glucose signaling[103]. During pregnancy, adiponectin is also secreted by the placenta[104]. Adiponectin is found in the bloodstream of healthy persons at significantly elevated levels, making up around 0.05% of all plasma proteins. Its concentration typically ranges from 3 to 30 μg/mL. Obese mothers or GDM women often have reduced levels of adiponectin in their bloodstream. Additionally, they tend to give birth to macrosomic babies. They are prone to experiencing prenatal problems and are at risk of developing metabolic syndrome in the future[105]. A meta-analysis on the ADIPOQ SNPs (rs2241766, rs1501299, and rs266729) discovered that rs2241766 was significantly linked to GDM in Asian women. However, no correlation was found between thers1501299 and rs266729 polymorphisms and GDM in the studied population[106]. Another meta-analysis demonstrated that rs266729 was linked to a higher susceptibility to GDM in both Asian and European populations[107]. A study conducted on the Iranian population demonstrated that The G allele of +45T>G (rs2241766) was found to be more prevalent in GDM women than the T allele[108].

IL-10

IL-10 gene is present on the long arm of 1^st chromosome (1q32.1). It serves as an anti-inflammatory cytokine that is synthesized by activated T cells, monocytes, and B cells[109]. Multiple studies have shown that alteration in the generation of anti-inflammatory and pro-inflammatory cytokines during gestation might result in decreased or increased inflammation levels and be associated with insulin resistance[110]. Research conducted on the Taiwanese population revealed that women with the allele A of SNP rs3021094 of IL-10 gene were found to be significantly related to a higher risk of insulin resistance during pregnancy. Furthermore, the AA genotype was found to elevated level of IL-10 in GDM compared to those with the CC genotype[111]. Another study on the Chinese population reported that CC genotype of SNP rs1800872women with the CC genotype of SNP rs1800872 had a higher risk of developing early-onset preeclampsia compared to women with the AA+AC genotype[112]. Women carrying the IL-10 rs1800896 C allele exhibited a greater vulnerability to GDM compared to those carrying the T allele[113].

EPIGENETIC ALTERATIONS AND GDM

During the crucial period of early development, the control of gene expression is regulated by environmental and lifestyle factors via epigenetic processes. This is facilitated by a collection of genomic imprinters that are specifically programmed to perform their functions. The period of fetoplacental development is flexible and may be modified and influenced by changes in the maternal environment, whether they are induced by internal or external factors. These changes can significantly impact embryonic programming[114]. Epigenetic modifications include a variety of processes, including DNA methylation, microRNAs (miRNAs), and histone changes (Figure 4). The placenta has a crucial function in fetal development. It not only controls the interchange of substances between the mother and fetus, which allows for embryonic development but also actively participates in the mother's metabolism and contributes to fetal programming. Research has shown that the placenta adjusts its DNA methylation on the availability of nutrients[115].

DNA methylation

DNA methylation is a well-researched and well-understood epigenetic process. The process comprises the incorporation of a methyl group (CH3) to the 5^th Carbon inside the cytosine-phosphate-guanine dinucleotides (CpG island)[116]. The process is catalyzed by the DNA methyltransferase enzyme. The S-adenosyl-methionine acts as the methyl donor in the transfer process. Methylation of genes located in CpG-dinucleotide-rich promoter regions is frequently associated with transcriptional repression. The hypermethylation of the promoter region causes alteration in the DNA sequence where the transcription factors bind, which results in altered gene expression[117]. DNA methylation is a process that may be reversed. During pregnancy DNA methylation plays a crucial role in the regulating genome to transcribe genetic information and have the ability for intrauterine epigenetic programming. It is well-established that DNA methylation has a significant effect on gene expression and could change various regulatory signal transduction pathways that are linked to several pathophysiological disorders, including GDM[118,119].

Multiple studies have reported alteration in DNA methylation of certain placental regulatory genes, such as leptin, ADIPOQ, peroxisome PGC-1α, ABCA1, LPL, and important inflammatory cytokines genes. Overall, previous data indicate a robust correlation between altered DNA methylation of several regulatory genes with metabolic diseases such as GDM[120-122]. Pregnancy is a state of various physiological modifications in the DNA methylation pattern of blood plasma. This has led to a growing interest in using maternal blood to test for the identification of significant molecular biomarkers of GDM. Research investigating the DNA methylation patterns throughout the whole genome in fetal cord blood and maternal placenta from GDM pregnancies has shown inconsistent results[123,124]. An analysis of 1030 placenta samples found that women with GDM had a substantial increase in overall placental DNA methylation. Furthermore, there was a significant association reported between overall placental DNA methylation and the prevalence of GDM (P = 0.0009)[125]. A research found that an increase in promoter methylation in the areas of IGFBP-1 and IGFBP-2 is associated with reduced mRNA expression in the placenta of GDM women. These genes are responsible for encoding IGF-binding proteins[126]. Another research found the association between DNA hypermethylation and decreased expression of the insulin receptor and adiponectin gene in GDM mothers and their offspring[127]. A study conducted by Kang et al[128] for genome-wide DNA methylation examination in Chinese women with GDM used the Illumina Infinium Human Methylation EPIC Bead Chip array, the results of this study revealed that the 200 loci with differential methylation were associated with 151 genes[128].

Another study found that offspring of GDM mothers exhibited elevated DNA methylation of the adiponectin gene region at the promoter reduced the expression of the ADIPOQ in subcutaneous fat. Nevertheless, there was no significant modification seen in the methylation level of the adiponectin gene in the blood plasma of the GDM mother[129]. A study reported hypermethylation of the PIK3R5 gene in the blood of pregnant women with GDM during the early stages of pregnancy. This altered hypermethylation causes the alteration of PI3K/AKT/mTOR signal transduction in pregnant women[130]. The constraints posed by sample selection and the presence of methylated cell types have hindered a comprehensive investigation of the causes of GDM. In the future, investigating the differential methylation patterns of genes that regulate insulin will enhance our comprehension of how alterations in DNA methylation in maternal and placental cells contribute to the development of insulin resistance in women with GDM.

miRNAs

miRNAs are small around 19-23 nucleotides highly conserved non coding RNA molecules. miRNAs control the expression of certain genes by binding with 30 untranslated region of corresponding mRNA sequences and lead either to disrupt translation or to facilitate mRNA destruction, at the post-transcriptional level. Because of their inherent stability, selectivity to certain tissues and diseases, and convenient detectability in various body fluids[131]. miRNAs are pivotal regulators that govern several biological processes, including cell proliferation, differentiation, and apoptosis. Furthermore, they control the activity of genes that are responsible for metabolic activities such as maintaining stable glucose levels, transmitting signals for insulin, functioning of pancreatic beta-cells, metabolism of lipids, and inflammation[132,133]. Their dysregulation has been shown in several metabolic disorders, including obesity, T2DM, and cardiovascular disease. Despite their intracellular function, several investigations have discovered external circulating miRNAs, which has generated interest in their potential as disease biomarkers[134,135].

miRNAs and GDM

MiRNAs play a vital role in regulating metabolism and embryonic growth during pregnancy. The differential expression of miRNAs is influenced by the surrounding environment of pregnant women, so the altered epigenetic modification of these miRNAs is associated with the progression of GDM. Genome-wide research conducted in 2013 revealed the expression of over 600 miRNAs in the placenta and their association with maternal physiology[136]. In a recent study, Poirier et al[137] examined placental miRNAs that experience abnormal regulation throughout pregnancy in women with GDM[137]. The placenta plays a crucial role in regulating the mother's metabolism throughout pregnancy, and it is believed that the different levels and expression of certain placental miRNAs contribute to these physiological modifications. Placental miRNAs are circulated into the mother's bloodstream. Consequently, these miRNAs have the potential to serve as indicators of placental malfunction causing GDM[138,139]. In 2011, Zhao et al[140] conducted the first analysis of serum miRNA expression of GDM women, by using Taqman low-density arrays and then confirming the results by applying qRT-PCR technique and identifying three specific miRNAs, namely miR-132, miR-29a, and miR-222, that exhibited substantial downregulation in Chinese GDM women (n = 24) as compared to a control group (n = 24)[140].

The differential expressions of these miRNAs (miR-132, miR-29a, and miR-222) were confirmed by various studies. These miRNAs are believed to have a role in maintaining glucose levels, enhancing insulin responsiveness, and regulating the activity of β-cells[141]. Various research conducted on different populations has reproduced similar trials, with contradictory findings. A recent study by Pheiffer et al[142] found that the levels of miR-132, miR-29a, and miR-222 were lower in the blood plasma of South African women with GDM (n = 28) compared to a control group (n = 53). This difference was statistically significant[142]. These data indicate that the expression of these serum miRNAs is common across both South African and Chinese populations. Unlike the findings of Zhao et al[140], another study by Tagoma et al[143] in the Estonian women population demonstrated that the expression of miR-222 was elevated in the plasma sample of GDM women (n = 13) in comparison to the control group (n = 9)[143]. Wander et al[144] found that there were no discernible variations The study examined the levels of miR-222 and miR-29a in the plasma sample of 36 GDM women of American Caucasian ethnicity and compared with the control group of 80 individuals[144]. Zhu et al[145] used high-throughput DNA sequencing and qRT-PCR techniques to examine miRNAs in plasma samples obtained from Chinese women with GDM (n = 10) and control (n = 10) during the 16^th and 19^th weeks of pregnancy. Another study on individuals with GDM found that the expression of five specific miRNAs (miR-16, miR-17, miR-19a, miR-19b, and miR-20a) was considerably increased in GDM women when compared with the control group[145].

The research emphasizes several miRNAs as biomarkers for early detection of GDM. However, the findings often demonstrate variability, perhaps because of variations in sample kinds and sizes, women’s gestational age, and the analytical techniques used. These disparities might arise from variances in the biological material used (serum or plasma), at the stage of pregnancy, or other unidentified variables that have not been taken into consideration. Currently, there is no agreement on the most effective strategy for quantifying circulating miRNAs during profiling. Various quantification techniques are recognized to differ in terms of sensitivity and specificity. This might potentially affect the precision and understanding of the data. Furthermore, the study of circulating miRNA profiling is significantly challenged by the process of data standardization. In order to improve the precision of molecular biomarkers in predicting the likelihood of GDM, future research should focus on incorporating a combination of these indicators into risk stratification models.

CONCLUSION

Pregnancy is characterized by a period of increased metabolic activity, during which it is crucial to maintain glucose homeostasis. Despite ongoing debate, there is still disagreement on the criteria used for diagnosis. Hence, it is essential to establish a reliable diagnostic criterion for GDM, and all endeavours should be focused on researching effective and secure therapies that can manage maternal insulin homeostasis during pregnancy. Since the genetic and epigenetic factors play a role in the onset of GDM, the current research is to identify novel biomarkers to support insulin balance throughout pregnancy. The regulation of insulin homeostasis genes through genetic variations and epigenetic factors, such as the differential expression of miRNA and promoter methylation, could serve as distinctive methods for forecasting the risk of GDM and its associated outcomes. While these molecular biomarkers hold significant promise, there are several limitations that need to be addressed prior to their application in therapeutic settings. However, rapid improvements in technology could potentially address these challenges and pave the way for a fast, affordable test capable of readily identifying GDM women and neonates at elevated risk in the early stages of pregnancy. Consequently, comprehensive research must be undertaken across various populations, and the establishment of a global organization is essential to oversee the analytical standards for molecular biomarkers in intricate diseases.