Introduction

Fetal growth restriction (FGR) is becoming an increasingly common problem not only in Russia but also worldwide. According to aggregated epidemiological data, around 30 million babies are born with this condition each year worldwide [1, 2]. Fetal growth restriction accounts for around 30% of stillbirths, contributes to high perinatal morbidity and mortality, and is a common cause of preterm delivery, as well as adverse psychoneurological consequences [3–5]. A wide range of fetal, maternal and placental factors have been identified among the causes [6]. Impaired placentation and uteroplacental blood flow, oxidative stress and inflammation associated with various pregnancy complications lead to chronic fetal hypoxia. One of the most common extragenital complications of pregnancy is anemia, which contributes significantly to the development of hypoxic conditions in both the mother and the fetus [7]. Alongside classical etiological factors (iron and folic acid deficiencies), the role of genetic predisposition has been actively investigated in recent years [8]. Of particular interest are the genes involved in adaptation to the physiological hypoxia of pregnancy.

Hypoxia-inducible factor-1α (HIF-1α), encoded by the HIF-1A gene, is a key regulator of the cellular response to hypoxia. It controls the expression of hundreds of genes, including erythropoietin (EPO), the primary stimulator of erythropoiesis [9]. The rs11549465 polymorphism (C>T, Pro582Ser) is functionally significant: the T allele is associated with increased transcriptional activity of the protein and improved adaptation to hypoxia [10]. Previously, its role has primarily been studied in the context of placental dysfunction, pre-eclampsia and fetal growth restriction; however, evidence has emerged suggesting that HIF-1A variants that impair the hypoxic response may disrupt the physiological stimulation of erythropoiesis during pregnancy, predisposing women to anemia [11].

Novokuznetsk is a major industrial centre in the Kemerovo Region (Kuzbass), where enterprises in the metallurgical, coal-processing and chemical industries are concentrated. Just under half of the region’s territory is classified as an environmental crisis zone, and, according to Roshydromet, Novokuznetsk has very high levels of atmospheric pollution. The greatest potential threat to the population of towns in southern Kuzbass is posed by benzo(a)pyrene, formaldehyde, particulate matter, nitrogen dioxide (NO2), hydrogen fluoride, nitrogen monoxide (NO), carbon monoxide (CO), and sulphur dioxide (SO2) [12]. Chronic exposure to air pollutants, typical of industrial centres, disrupts maternal oxygen homeostasis and exerts direct embryotoxic and foetotoxic effects, leading to intrauterine growth restriction. Numerous epidemiological studies and laboratory animal studies confirm that the main mechanisms of this effect are the induction of oxidative stress, direct damage to the DNA of the placenta and fetus, and endocrine disorders [13].

The aim of this study is to identify the association between the HIF-1A gene polymorphism, anemia during pregnancy and fetal growth restriction in women living in a region with high anthropogenic pressure.

Materials and methods

An assessment of total atmospheric emissions and average annual concentrations of pollutants in Novokuznetsk was carried out for the period from 2021 to 2024. Data were obtained from the Kemerovo Centre for Hydrometeorology and Environmental Monitoring, a branch of the Federal State Budgetary Institution ‘West Siberian Directorate for Hydrometeorology and Environmental Monitoring’. Two independent pilot case-control studies were conducted on a cohort of pregnant women living in Kuzbass (Novokuznetsk), a major industrial centre in the Kemerovo Region.

The first group comprised of 37 pregnant women (mean age 28.3 ± 4.1 years) diagnosed with anaemia complicating pregnancy, childbirth and the puerperium (ICD-10 code O99.0) and 53 pregnant women without this condition (mean age 27.8 ± 3.9 years). Venous blood samples for clinical and genetic analysis were collected upon booking at the antenatal clinic. The diagnosis of anemia was made according to WHO criteria (Hb < 110 g/L).

The second group comprised of 23 women, who had given birth to infants with intrauterine growth restriction, and 33 women with uncomplicated pregnancies resulting in the birth of healthy, full-term infants. The neonates born to these women were also examined: 19 neonates with intrauterine growth restriction and 40 healthy neonates. The diagnosis was confirmed in accordance with clinical guidelines, ‘Assessment of the physical development of children and adolescents’ (Ministry of Health Letter No. 15-2/10/2-8090, dated 21 November 2017), whereby the asymmetric form of FGR is diagnosed when body weight is below the 10th percentile for the given gestational age, and the symmetric form (hypoplastic type) is diagnosed when both the infant’s weight and length are below the expected values. Genomic DNA was isolated using the phenol-chloroform extraction method from peripheral blood leucocytes [14]. Gene typing was performed using real-time PCR on a DTprime 4 instrument (NPO DNA-Technology LLC, Russia). Test kits for the molecular genetic analysis of polymorphisms in the HIF-1A (hypoxia-inducible factor-1α) gene were developed by the Institute of Biochemistry and Physiology of Metabolism, Siberian Branch of the Russian Academy of Sciences, and synthesized by SibDNA LLC. A comparison of genotype frequencies to identify an association with the risk of developing anemia and fetal growth restriction was carried out using the χ² test to determine statistically significant differences between observed and expected frequencies. Odds ratios (OR) were calculated to assess the association between the risk factor and the outcome [15]. Deviation from Hardy–Weinberg equilibrium was tested using the exact test [16].

The study was approved by the local ethics committee of the Federal State Budgetary Scientific Institution ‘Research Institute for Complex Problems of Hygiene and Occupational Diseases’ (Minutes No. 4, Section 1, 18 November 2021), and was conducted in accordance with the principles of the World Medical Association’s Declaration of Helsinki ‘Ethical Principles for Medical Research Involving Human Subjects’, as amended in 2013.

Results

The total volume of atmospheric emissions in Novokuznetsk from 2021 to 2024 decreased slightly, from 268,297 to 256,683 thousand tonnes. The reduction amounted to 11,614 thousand tonnes, which is attributable to Novokuznetsk’s participation in the national ‘Clean Air’ project.

Table 1 shows the average annual concentrations of carcinogenic pollutants, expressed in mg/m³ and as a percentage of the maximum permissible concentration (MPC).

The annual average concentrations of benzo(a)pyrene were 6.2 to 11.2 times higher than the established MPC throughout the entire study period; the greatest exceedance (11.2-fold) was observed in 2022. Annual average concentrations of formaldehyde were 1.7 to 3.95 times higher than the corresponding MPC between 2021 and 2024. In 2021, annual average concentrations of carbon (soot) were below the MPC; since 2022, concentrations of this substance have not been measured in the city.

Molecular genetic studies identified polymorphic variants of the HIF-1A gene (rs11549465) that are significantly associated with the development of anemia complicating pregnancy and fetal growth restriction, as well as variants associated with a protective effect against these conditions.

The CC genotype of the HIF-1A gene (rs11549465) was significantly associated with the development of anemia (χ² = 4.73; OR = 3.57), whilst the CT genotype was associated with a reduced risk of anemia (χ² = 4.00; OR = 0.31) (Table 2).

Analysis of the results presented in Table 3 showed that, in the group of patients who gave birth to infants with intrauterine growth restriction, no statistically significant associations with the HIF-1A gene polymorphism were found.

In the group of neonates with FGR a statistically significant association was demonstrated between the homozygous CC genotype of the HIF-1A gene and intrauterine growth restriction (χ² = 4.59; OR = 7.71), and between the heterozygous CT genotype and normal development (χ² = 3.93; OR = 0.15) (Table 4).

Analysis of the combinations of HIF-1A gene variants in the mother and infant revealed a statistically significant association between the combination of homozygous CC/CC genotypes of the HIF-1A gene in both the mother and infant, and intrauterine growth restriction (χ² = 5.76; OR = 6.26), whilst the combination of the homozygous CC genotype in the mother with the heterozygous CT genotype in the infant was associated with normal neonatal development (χ² = 4.90; OR = 0.18) (Table 5).

Thus, a statistically significant association was identified between the CC genotype of the HIF-1A gene and the development of anemia in pregnant women and fetal growth restriction, particularly when these genotypes are combined in the mother and infant, whilst the CT genotype is associated with an increased likelihood of a normal pregnancy and the birth of a healthy child.

Discussion

Pathological endogenous hypoxia develops in virtually all diseases, whether infectious or non-infectious, under conditions of stress, as well as in cases of impaired uteroplacental blood flow, smoking, alcoholism and other adverse factors [17, 18]. A reduction in the efficiency of oxygen delivery to tissues leads to disturbances in the regulation of the microcirculatory bed, extravasation, and the activation of leucocytes and other immune and pro-inflammatory resident cells, which produce reactive oxygen species, leading to oxidative stress and mitochondrial dysfunction [19–22]. The oxygen-sensitive subunit HIF-1α, whose activity is genetically determined, plays a leading role in the body’s adaptation to hypoxia. To date, 16 single-nucleotide polymorphisms (SNPs) in the HIF-1A gene have been identified that influence the subunit’s activity and are associated with various pathologies. One of the most extensively studied is rs11549465 C>T, which results in the substitution of serine for proline (Pro582Ser; C1772T) and affects HIF-1α stability and transcriptional activity, leading to increased expression of the HIF-1α protein [23–28].

Against the backdrop of a slight reduction in overall atmospheric emissions in Novokuznetsk between 2021 and 2024, there has been a persistent exceedance of the maximum permissible concentrations (MPCs) for the annual average concentrations of benzo(a)pyrene and formaldehyde, which are classified as hazard classes 1 and 2. The combined effect of these pollutants is capable not only of causing direct damage to the respiratory system, but also of inducing chronic tissue hypoxia, which is particularly critical during gestation.

One of the key compounds disrupting oxygen homeostasis is formaldehyde. Traditionally, its toxicity has been linked to the formation of DNA–protein cross-links and local inflammation; however, recent studies have revealed its direct destructive effect on the central regulator of adaptation to hypoxia. Formaldehyde is classified as a phototoxic stressor – an agent that causes structural damage to proteins. It has been established that the HIF-1α and HIF-2α subunits contain extensive intrinsically disordered regions, which make them extremely vulnerable to damage by aldehydes. Formaldehyde induces rapid and selective degradation of HIF-1α; in the absence of proteasome degradation, the damaged protein becomes insoluble and denatured. Consequently, rather than activating a protective response to hypoxia, HIF-1α is inactivated, which deprives cells of their ability to respond adequately to oxygen deprivation [29].

Formaldehyde is also capable of switching metabolism to anaerobic glycolysis even in the presence of oxygen, stimulating this process via the induction of HIF-1α. This results in a paradoxical situation of oxygen deprivation amidst an excess of oxygen, which exacerbates mitochondrial stress [30]. Chronic exposure to formaldehyde can lead to the depletion of the normal physiological response to hypoxia.

Benzo(a)pyrene, a typical representative of PAHs and a priority carcinogen in industrial cities, is a potent pro-oxidant. It has been shown that benzo(a)pyrene influences cell proliferation by modulating metabolic genes and the ROS/HIF-1α/HO-1 signaling pathway, which is accompanied by the induction of reactive oxygen species. Furthermore, benzo(a)pyrene simultaneously activates the aryl hydrocarbon receptor (AhR), triggering a signaling cascade that potentiates mitochondria-mediated apoptosis [31].

Thus, living in conditions of chronic exposure to formaldehyde and benzo(a)pyrene creates a synergistic background of mitochondrial dysfunction and oxidative stress. This leads to the depletion of the normal physiological response to hypoxia, mediated by HIF-1A, which is critically important for the realization of the genetic risk associated with the rs11549465 polymorphism, particularly in pregnant women.

In our study, we demonstrated a single, genetically determined basis for impaired adaptation to pregnancy-related hypoxia, which manifests as anemia in the mother and as fetal growth restriction in the fetoplacental unit. In mothers who are carriers of the CC polymorphic variant of the HIF-1A gene, insufficient stimulation of erythropoiesis is observed in response to hemodilution and an increasing demand for oxygen [32]. In the fetus, a weak HIF-1α response in the placenta leads to suboptimal vascularization and impaired trophoblast invasion, which is a known mechanism underlying placental insufficiency and FGR [33–35].

A woman with the CC genotype initially has reduced compensatory reserves for adapting to pregnancy. She is at higher risk of developing anemia, and her body may be less able to support the formation of a fully functional placenta. If the fetus also inherits the low-activity C allele, its own ability to stimulate angiogenesis and growth, even in the context of mild placental insufficiency, is reduced. This leads to a synergistic effect and a high probability of developing FGR (OR = 6.26). In an industrial region, this genetic predisposition may be exacerbated by exogenous hypoxia and other environmental factors.

Limitations. As this was a pilot study, it was limited by a small sample size.

Conclusion

The data obtained suggest that the HIF-1A (rs11549465) polymorphism can be considered a candidate marker for stratifying the risk of developing pregnancy-related adaptation disorders, comprising maternal anemia and placental insufficiency, in environments with high anthropogenic stress. Identifying women with the CC genotype, particularly in cases where the fetus is likely to inherit the C allele, identifies them as being at high risk of developing associated complications (anemia and placental insufficiency). These women require enhanced preventive measures: pre-pregnancy preparation, early monitoring of hemoglobin levels and fetoplacental parameters, and adjustments to working and living conditions.

Prospective studies involving larger cohorts are required to confirm these findings.