The role of antioxidant defense enzymes in the placenta and fetal membranes in preterm birth

Preterm birth (PTB) and prematurity remain among the most pressing unresolved global problems and challenges that cannot be adequately addressed using current obstetric methods [1]. Despite advances in medical science, the global incidence of PTB remains consistently high, both worldwide (approximately 9.9% of all births according to a 2023 systematic analysis) [2] and in Russia, affecting the lives of thousands of newborns annually (4.9 %) [1]. This results in substantial socioeconomic costs and imposes long-term burdens on healthcare systems. The need to provide qualified medical care is driven by the consequences of prematurity, including a high risk of neonatal mortality and a broad spectrum of lifelong health impairments, ranging from respiratory distress syndrome, intraventricular hemorrhage, and retinopathy to long-term cognitive, neurological, and metabolic disorders [1, 3–5].

PTB is currently regarded as a multifactorial syndrome in which inflammatory processes, infections, vascular disorders, and associated placental insufficiency play key roles in initiating labor [6]. Despite substantial advances in understanding the pathogenesis of PTB, the precise etiology of spontaneous onset of preterm labor, including PTB associated with preterm prelabor rupture of membranes (PPROM), remains unclear [7].

In recent years, mitochondrial dysfunction in placental tissue and fetal membranes has been recognized in the international scientific literature as one of the key components of the pathological cascade, integrating multiple pathogenic pathways leading to PTB [8, 9]. Mitochondria are not only the primary intracellular structures responsible for energy conversion but also central regulators of cellular metabolism, calcium and reactive oxygen species (ROS) homeostasis, and integrators of cellular signaling that determine whether programmed cell death is induced or inhibited [10]. Impaired mitochondrial function not only reduces the efficiency of energy production and, consequently, may lead to an energy crisis in placental tissue, but also results in excessive ROS generation, oxidative stress, and damage to cell membrane lipids and macromolecules, such as proteins and nucleic acids, in the cytoplasm and nucleus [9, 11]. Mitochondrial damage leads to the accumulation of degradation products in the cell cytoplasm and extracellular space, which possess proinflammatory properties and induce immune cell activation [12]. This creates a vicious cycle in which mitochondria-derived proinflammatory factors (MPFs) induce oxidative stress, which exacerbates mitochondrial damage and further accumulation of MPFs. Ultimately, this may lead to inflammation and premature dysfunction of the placenta and fetal membranes [6, 13], even in the absence of an infectious component, followed by activation of myometrial contractility and initiation of the labor cascade [14–17].

This study aimed to assess the levels of antioxidant defense enzymes involved in regulating oxidative stress in the placenta and fetal membranes during PTB.

Materials and methods

This study included 64 women with spontaneous preterm birth (SPB) at 22.0–37.0 weeks of gestation who delivered their babies vaginally. According to the accepted classification, participants were stratified by gestational age as follows: group 1 – 14 women with extremely preterm birth (PB; 22.0–27.6 weeks of gestation); group 2 – 16 women with early PB (28.0–31.6 weeks); group 3 – 17 women with PB at 32.0–33.6 weeks; and group 4 – 19 women with late PB (34.0–36.6 weeks). Given the different etiologies of SPB and PB associated with preterm premature rupture of membranes (PPROM), the participants were further divided into subgroups: 34 women with PPROM and 32 women with SPB. The comparison group comprised women with uncomplicated pregnancies and spontaneous term deliveries at 37.0–41.0 weeks of gestation (n=20).

The inclusion criteria were singleton pregnancy, threatened PB at 22.0–36.6 weeks of gestation, spontaneous PB in the first stage of labor, and PPROM at 22.0–36.6 weeks of gestation.

The exclusion criteria were multiple pregnancies, severe extragenital disease, severe obstetric complications requiring elective preterm delivery, placental abruption, fetal growth restriction, fetal congenital malformations, sexually transmitted infections, and HIV infection.

The study was reviewed and approved by the Research Ethics Committee of the V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology, and Perinatology. All participants provided written informed consent to participate in this study.

Collection of placental tissue and membrane samples

Samples were obtained within 5 min after delivery. In each case, a placental biopsy was collected 1.5–2.0 cm from the umbilical cord insertion site of the placenta. Membrane fragments were collected concurrently with placental biopsies. Samples intended for subsequent protein expression analysis were placed in 1.5-mL Eppendorf tubes and snap-frozen in liquid nitrogen. The tissues were then pulverized in liquid nitrogen and homogenized in a glass homogenizer using radioimmunoprecipitation assay (RIPA) lysis buffer for western blotting, according to the manufacturer’s recommendations (Santa Cruz Biotechnology).

Electrophoresis and western blot analysis of placental tissue and membrane proteins

Proteins were separated using Laemmli polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes (Millipore, USA). To prevent nonspecific antibody binding, the membranes were incubated for 1 h in 5% nonfat dried milk powder (PanReac-AppliChem, Germany) prepared in Tris-buffered saline with Tween 20 (TBST; Bio-Rad, USA). The membranes were then incubated with primary antibodies against superoxide dismutase 1 (SOD1; catalogue no. ab13498), superoxide dismutase 2 (SOD2; catalogue no. (ab16956), glutathione peroxidase 1 (GPX1; catalogue no. ab108427), catalase (catalogue number: ab76024), and β-actin (catalogue no. A5441), all from Abcam (USA), at 4°C for 12 h with continuous agitation. After washing with TBST to remove unbound antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-mouse ab6789 and goat anti-rabbit ab6721 (Abcam, USA). Chemiluminescent detection was performed using the Novex enhanced chemiluminescence (ECL) kit (Invitrogen, USA) and ChemiDoc imaging system (Bio-Rad, USA). Chemiluminescent signal intensities for the proteins of interest were analyzed using the Image Lab software (Bio-Rad, USA). Relative protein expression was determined by normalizing to the reference protein, actin.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (version 6.0; GraphPad Software Inc., USA). Comparisons between two independent groups were performed using the Mann–Whitney U-test. Statistical significance was set at p<0.05. Data are presented as medians and interquartile ranges [Q1; Q3] as well as minimum and maximum values. The normality of the distribution of continuous variables was assessed using the Shapiro–Wilk test. Fisher’s exact test was used to compare the categorical variables.

Results and discussion

Our analysis of the clinical and medical history data of the patients enrolled in the study identified the characteristic features of patients with PB and key risk factors.

All women included in the study were comparable with respect to clinical characteristics, and their ages ranged from 18–41 years. No deviations from the population norms were identified based on the anthropometric measurements. Analysis of the history of hereditary diseases and childhood infections revealed no significant differences between the groups.

Analysis of the reproductive history of the pregnant women included in the study showed that, compared with women with term deliveries, the group of patients with PB had significantly higher frequencies of spontaneous miscarriage—4/16 (25%) in group 2 (p<0.05) versus 1/20 (5%) among women with term deliveries; missed miscarriage – 4/14 (28.5%) in group 1 versus 1/20 (5%) among women with term deliveries (p<0.05); and a history of PB – 4/14 (28.5%) in group 1 and 5/16 (31.2%) in group 2 versus 1/20 (5%) among women with term deliveries (p<0.05). A history of intrauterine procedures was also significantly more common in groups 1 and 2 (2/14 (14.3 %) and 2/16 (12.5 %), respectively) than in the term-delivery group, in which no such cases were reported (0/20 [0%]) (p<0.05). No differences were identified between the groups in the frequency of induced abortions in their medical history.

A detailed analysis of infectious and inflammatory diseases of the reproductive tract revealed that, among women with PB, bacterial vaginosis and candidiasis were significantly more common in group 2 – 5/16 (31.3%) (p<0.05), and Ureaplasma infection was more common in group 1 – 3/14 (21.4%) (p<0.05)compared with women with term deliveries, among whom none of these infections were reported (0/20).

Analysis of the medical history showed that chronic cystitis was more frequent in groups 2 and 3 – 5/16 (31.3%) and 5/17 (29.4%), respectively – than in the group of women who delivered at term – 1/20 (5%) (p<0.05). Analysis of gynecologic history showed that chronic salpingo-oophoritis was more common among women with PB: 3/14 (21.4%) in group 1 and 4/16 (25.0%) in group 2, compared with 2/20 (10.0%) among women with term deliveries; however, the differences were not statistically significant.

Thus, we found that pregnancies resulting in PB occurred in the setting of adverse obstetric and gynecologic histories. Statistically significant risk factors included a history of spontaneous miscarriage, missed miscarriage, and intrauterine procedures, as well as a high prevalence of infectious and inflammatory diseases of the genital tract, including bacterial vaginosis, vulvovaginal candidiasis, mycoplasma infection, and chronic inflammatory diseases of the urinary tract and pelvic organs.

These findings suggest the possible existence of a spectrum of pathogenetic pathways leading to PB that depend not only on gestational age but also on the type of reproductive tract pathology present before conception or manifesting from the first trimester onward. Therefore, the development of stratified approaches for the prediction, early diagnosis, and timely prevention of PB remains highly relevant. Such approaches should be based on a comprehensive assessment of the individual risk profile, incorporating both historical risk factors and current gestational age.

However, conventional approaches to the prediction and prevention of PB based on clinical and historical risk factors have limited effectiveness, underscoring the need to identify novel pathogenetically relevant targets for early diagnosis and therapeutic intervention using contemporary research approaches and methods. Accordingly, we assessed the role of enzymes regulating oxidative stress in placental mitochondria and fetal membranes in the development of PB of different etiologies: SPB and PPROM.

Analysis of the expression of superoxide dismutase types 1 and 2, catalase, and glutathione peroxidase (I) in the placenta and fetal membranes

One of the key mechanisms maintaining reactive oxygen species (ROS) homeostasis within the uteroplacental unit is the antioxidant system, in which enzymes such as superoxide dismutase type 1 (SOD1), superoxide dismutase type 2 (SOD2), glutathione peroxidase 1 (GPX1), and catalase play central roles.

These enzymes provide a two-stage defense system, are localized in the cytoplasm, mitochondria, and peroxisomes, and function in a strictly ordered sequence within a cascade of reactions that convert different ROS species (Fig. 1). Therefore, the functional balance among these enzymes is critical for maintaining ROS at a safe local level sufficient for signal transduction while preventing the accumulation of oxidative damage to macromolecules and the induction of pathological processes. Disruption of the balance between SOD1 and GPX1/catalase activity underlies the pathogenesis of a broad range of diseases, while an increased SOD1/GPX1 ratio correlates with the accumulation of lipid peroxidation products and damage to cellular structures. In particular, GPX1 deficiency causes oxidative damage and DNA fragmentation, inducing apoptosis through a p53-dependent pathway, whereas excess of this enzyme may disrupt the physiological signaling role of ROS (Fig. 1) [18].

The antioxidant system is particularly important in mitochondrial physiology. Mitochondria are both a major source of endogenous ROS and a primary target of oxidative damage because of their high content of unsaturated molecules [9, 19, 20]. In particular, the mitochondrial compartment contains manganese-dependent SOD2 and GPX1, which protect mitochondrial DNA, lipids, and proteins, thereby maintaining the functional integrity of the respiratory chain and the mitochondria. The placenta and fetal membranes, which are highly energy-dependent tissues, are particularly vulnerable to imbalances between pro-oxidant and antioxidant systems. Dysfunction of these enzymes mediates disturbances in energy metabolism, resulting in both a reduction in membrane potential and intracellular ATP levels, and the activation of the mitochondrial quality-control program. Failure of this program leads to programmed trophoblast cell death, impaired placental transport function, oxidative stress, and secretion of proinflammatory mediators, resulting in PB [18]. Available evidence indicates that the activity of antioxidant enzymes is altered in PB; however, the molecular mechanisms linking the dysfunction of SOD1, SOD2, GPX1, and catalase to mitochondrial damage and the initiation of labor remain insufficiently studied [18, 20, 21].

In view of the above, to identify differences in the pathogenetic mechanisms underlying SPB and PPROM-associated PB, we investigated the expression of proteins regulating oxidative stress in the placenta and fetal membranes.

We found that in PB with PPROM, compared with term delivery, fetal membranes exhibited impaired detoxification of superoxide free radicals (reduced SOD1 levels) and impaired reduction of hydrogen peroxide to water (reduced GPX1 and catalase levels). In cases of extremely preterm PB (22.0–27.6 weeks), the levels of all antioxidant defense enzymes were significantly lower (p<0.05) than those observed in term deliveries (Fig. 2b).

In contrast, in the placenta, PB with PPROM at 28.0–34.0 weeks was associated with reduced catalase levels (p<0.01) compared with term delivery, with no changes in the levels of other antioxidant defense enzymes. This finding may reflect the depletion of the antioxidant system during PPROM at these gestational ages (Figure 2a).

In the SPB group, no changes in SOD1, SOD2, or catalase levels were observed in the fetal membranes, whereas GPX1 levels were reduced in all SPB groups and were significantly lower in the extremely preterm PB group (p<0.05) than in the term delivery group. This finding may indicate the reduced metabolic activity of fetal membrane cells during the SPB (Fig. 3b).

Assessment of antioxidant defense enzyme levels in the placenta in SPB revealed statistically significant reductions in SOD1, GPX1, and catalase compared to term delivery (p<0.05). The most pronounced differences were observed when extremely preterm PB was compared with term deliveries (p<0.01). Thus, in SPB, disruption of the placental antioxidant system may be regarded as one of the key mechanisms initiating PB.

We also assessed the SOD1/GPX1 ratio as an indicator of imbalance in the antioxidant system (Table).

Analysis of the relative content of SOD1 and GPX1 in the placenta and fetal membranes in PB and term deliveries revealed an increased SOD1/GPX1 ratio in the fetal membranes in both SPB (1.95) and PPROM (1.77) compared with that in term delivery (0.9), which may be associated with increased oxidative damage to fetal membrane cells during PB. Assessment of the SOD1/GPX1 ratio in the placenta showed that the ratio was higher in SPB (2.8) than in PPROM (1.93) and term delivery (1.72), which may likewise indicate disruption of the antioxidant system in the placenta in cases of SPB.

PB resulting from PPROM is characterized by a statistically significant decrease in all antioxidant enzymes and an imbalance in the protective system in the fetal membranes. These changes are particularly pronounced in extremely preterm births occurring at 22.0–27.6 weeks of gestation. However, in the placenta, this process is selective, manifesting solely as a reduction in catalase at 28.0–34.0 weeks of gestation. This selective reduction may reflect the depletion of maternal-placental-fetal unit reserves at these gestational ages in cases of PPROM.

Conversely, in SPB with intact membranes, the most pronounced decrease in antioxidant defense enzymes was observed in the placenta. A statistically significant reduction in all key antioxidant defense enzymes in the placenta during extremely preterm birth indicates that placental oxidative stress serves as the initiating trigger for labor onset in SPB, whereas no significant reduction in antioxidant defense enzymes occurs in fetal membranes.

Analysis of the present results, considered alongside established evidence regarding the roles of various factors in PB induction, suggests the following pathogenesis for SPB: impaired mitochondrial quality control mechanisms in the presence of an elevated SOD1/GPX1 ratio lead to a simultaneous increase in hydrogen peroxide levels and arachidonic acid, a degradation product of mitochondrial cardiolipin. Consequently, cyclooxygenase-2, the enzyme responsible for the production of prostaglandins that induce myometrial contractions, is activated, initiating labor. Concurrently, 15-hydroxyprostaglandin dehydrogenase, which inactivates prostaglandins, is inhibited by pro-inflammatory cytokines amid decreased production of its activator, progesterone, due to oxidative stress-induced disruption of placental mitochondria, the primary site of precursor synthesis. A further consequence of mitochondrial damage is the accumulation of DAMPs, which act as secondary inflammatory inducers, even in the absence of an infectious agent.

Conclusion

Thus, PB resulting from PPROM is associated with a statistically significant reduction in antioxidant defense enzyme levels within the fetal membranes. These findings demonstrate that pathological alterations in antioxidant status primarily occur in fetal membranes, whereas placental changes are secondary and arise from the depletion of antioxidant defense reserves at later gestational ages.

In contrast to PB associated with membrane rupture, SPB with intact membranes shows a significant reduction in antioxidant defense enzymes predominantly localized to the placenta. A statistically significant decrease across all key enzymes is observed specifically in placental tissue, with no significant alterations detected in the fetal membranes.

The evaluation of the role of these enzymes in oxidative stress regulation provided in this study offers new perspectives for developing pathogenetically sound approaches to understand the mechanisms driving SPB and PPROM, as well as opportunities for predicting and preventing PB to improve perinatal outcomes.