The role of some indicators of the antioxidant system and adipose tissue metabolism in predicting isthmic-cervical insufficiency

Introduction

The problem of premature birth and miscarriage is associated with the group of major obstetric syndromes and still remains unresolved. Every year, about 15 million children are born prematurely [1–8], and approximately 1 million children die due to various complications caused by prematurity worldwide [9][10]. Among the most common causes of miscarriage is isthmic-cervical insufficiency (ICI), which is recognized as the cause of 15–40% of spontaneous abortions and up to 30% of premature birth; that makes this pathology relevant from both medical-biological and social points of view [11][12]. The occurrence of ICI in the population varies from 2 to 15%. Currently, two forms of cervical insufficiency are identified, which are known as functional and traumatic (or anatomical). There is still no clarity in the comprehension of ICI pathogenic mechanisms, as well as in the identification of all the causes provoking this condition [3][13][14]. It is believed that ICI development is associated with two main mechanisms, namely through the initiation of the inflammatory process in the cervix and genetically determined connective tissue inadequacy. Normally, the cervix functions as a physiological barrier and prevents penetration of infection into the uterus due to its special anatomical structure, unique composition, and characteristics of cervical mucus, which provides mechanical and immunological protection.

In recent years, much attention has been paid to the search for early biochemical markers of various obstetric complications. For instance, screening, conducted during the first trimester of pregnancy and related to the determination of the concentration of human chorionic gonadotropin, pregnancy-associated protein type A, and placental growth factor, can predict the development of preeclampsia and fetal growth retardation [15–18]. In addition, the ongoing investigations of “signal” factors offer new methods for early diagnostics and prognosis of ICI and premature birth [1][5][9][14].

Oxidative stress and oxidation processes underlie most pathological processes, including those occurring during pregnancy. The balance between the factors of the oxidative and antioxidant systems during pregnancy is very important, since gestation requires additional resources of antioxidant protection. In cases of pregnancy complications, including placental insufficiency, premature birth, fetal growth retardation syndrome, and preeclampsia, inadequate antioxidant buffering has been found, which manifested itself in cell injuries, loss of their function, and subsequent death [19][9][20][14]. Potential causes of oxidative stress can be explained by a number of physiological changes, mineral deficiency, and increased oxygen consumption during pregnancy. Decreased perfusion and ischemic reperfusion in the placenta lead to placental hypoxia and, as a consequence, to increased production of free radicals in the placenta [21][22]. In physiologically normal pregnancy, the production of free radicals in endothelial cells is relatively low. They are quickly neutralized by active defense systems, including antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Certain changes in the levels of various enzymatic and non-enzymatic antioxidants during pregnancy may affect the pregnancy outcome through changes in maternal and fetal metabolism. It is assumed that free radicals produced during the development of placental insufficiency and major obstetric syndromes, including premature birth and shortening of the cervix, lead to the depletion of the antioxidant system [3][8].

Besides, the adipose tissue metabolism processes are recognized to play an important role in the pathogenesis of premature birth. It is believed that during pregnancy they are responsible for immune and inflammatory reactions in tissues [16][15][21]. Potential modulation of myometrial contractility due to cytokines and inflammatory processes makes a significant contribution to the development of pregnancy complications and a decrease in gestational age at birth [16–18]. It has recently become known that the regulation impairments in the synthesis of leptin, adiponectin, and kisspeptin during pregnancy contribute to the pathological course of pregnancy [17][23].

The aim of the study was to evaluate the role of some indicators of antioxidant protection and adipose tissue metabolism in predicting premature birth in pregnant women with ICI.

Materials and methods

The study included 117 pregnant women divided into two clinical groups: Group I consisted of 57 pregnant women with ICI who underwent its surgical correction, and Group II included 60 women with physiologically progressing pregnancy.

Selection into the prospective study groups was carried out using the random sampling method “Random between”. The criteria for inclusion of patients into the prospective study were related to the functional ICI with a Bishop Scale score of more than 8 points, shortening of the cervix, which was less than 25 mm according to the transvaginal ultrasound examination; and consent to participate in the study. The exclusion criteria were the presence of specific infections, immunodeficiency states, genital and extragenital pathologies in the stage of decompensation, traumatic (anatomical) ICI, and the woman’s refusal to participate in the study.

After obtaining informed written consent to participate in the study, all patients underwent general clinical tests, obstetric and gynecological examinations, and, additionally, special biochemical indicators were assessed in equal volumes according to the order of the Ministry of Health of the Russian Federation dated October 20, 2020 No. 1130n “On approval of the Procedure for the provision of medical care in the profile of ‘Obstetrics and Gynecology’”.

During the first screening (from 11.5 to 13.6 weeks of gestation), blood was collected before suturing to determine indicators of antioxidant protection including superoxide dismutase, catalase, and ceruloplasmin levels using high-performance liquid chromatography-mass spectrometry, as well as adipose tissue metabolism indicators including leptin, homocysteine, resistin, ghrelin, and adiponectin by the ELISA method with DBC (Canada), Biovendor Human Resistin ELISA, and BioVendor (Czech Republic) kits on a TECAN (USA) analyzer.

Results

The age-specific analysis did not reveal any significant differences (p>0.05) between the groups; the average age of patients in Group I was 27.34±4.91 years and 25.75±5.33 years in Group II (p>0.05).

Besides, there were no significant differences in the body weight of pregnant women (p>0.05). The body weight of patients in Group I varied from 50.5 to 110 kg, averaging 63.34±7.85 kg, while in Group II, it was from 52 to 112 kg with average weight of 64.38±10.52 kg, i.e. the groups did not differ significantly (p>0.05). The development of the menstrual cycle in pregnant women of the both comparison groups also did not have a significant difference (p>0.05).

According to medical documentation, namely individual cards of pregnant women, 41 (71.9%) women in Group I and 45 (75%) in Group II planned to become pregnant. So, no significant differences were found by this indicator, too (p>0.05).

The first visit to the antenatal clinic for dispensary registration before 12 weeks of pregnancy was made by women of Group I in 78.9% (45 pregnant women), and in Group II in 70% (42) cases. No significant differences were registered in the intergroup comparison on this indicator (p<0.05).

The parity of pregnancy in Group I varied from 0 to 9; in particular, 9 women (15.8%) were primigravidas; 1 pregnancy in the anamnesis was registered in 13 (22.8%) patients; 10 (17.5%) women had 2 pregnancies; 9 (15.8%) women had 3 pregnancies; 4 or more pregnancies were registered in 16 (28.1%) patients. In Group II, parity ranged from 0 to 6 pregnancies in the anamnesis. There were 35 (58.3%) primigravidas, 9 (15%) patients had 1 pregnancy, 6 (10%) of the examined women had 2 pregnancies according to the anamnesis, 5 (8.3%) patients had 3 pregnancies, and 5 (8.3%) women had 4 or more pregnancies. Thus, we revealed a significant difference in the number of pregnancies between the studied groups (p<0.05): primigravidas were more often registered in Group II (OR=7.467 (3.104-17.959), p<0.05) while patients in Group I had more pregnancies (4 or more).

The number of deliveries in the anamnesis of the individuals in Group I varied from 0 to 4. In particular, 18 (31.6%) patients had no deliveries, 3 (5.3%) women had 1 delivery, 31 (54.4%) patients had 2 deliveries, 4 (7%) patients had 3 deliveries, and 1 (1.8%) woman had 4 deliveries, according to medical records. In Group II, 35 (58.3%) patients had 1 delivery, 19 (31.7%) patients had 2 deliveries, and 2 (3.3%) patients had 3 deliveries. A significant (p<0.05) predominance of the number of deliveries in the anamnesis was found among pregnant women with ICI. The obstetric and gynecological anamnesis revealed that medical abortion occurred in 7 (12.3%) patients in Group I and in 5 (8.3%) patients in Group II; statistical significance between the groups was not revealed (p>0.05). However, a significant (p<0.05) prevalence of cases related to spontaneous premature termination of pregnancy in women with ICI was revealed. In particular, spontaneous abortion was registered in 20 (35.1%) patients in Group I, and in 5 (8.3%) patients in Group II (OR=5.946 (2.05–17.247). Non-developing pregnancy occurred in 4 (7%) patients of Group I and in 3 (5%) women of Group II (p>0.05). Antenatal fetal death was detected only in 1 (1.8%) woman of Group I. Ectopic pregnancy was in the anamnesis of only 2 (3.5%) patients of Group I and in 1 (1.7%) patient in Group II.

The examination of the history of gynecological diseases revealed significant differences (p<0.05) between the groups only in the case of vaginitis. This disorder was found in 37 (65%) women in Group I and in 25 (41.7%) patients in Group II (OR=2.590 (1.226–5.471), and was predominant among the pregnant with ICI. Primary infertility occurred in 2 (3.5%) patients in Group I and in 3 (5%) patients in Group II (p>0.05). Secondary infertility was noted in 5 (8.8%) women in Group I and in 2 (3.3%) patients in Group II. Cervical polyps were diagnosed in 9 patients of Group I (15.8%) and in 6 (10%) women in the comparison group (p>0.05). Polycystic ovary syndrome was detected in 2 (3.5%) women of Group I and in 1 (1.7%) patient of Group II. In addition, 2 (3.5%) cases of benign ovarian neoplasms and 26 (45.6%) cases of chronic adnexitis were registered in Group I, in Group II these indicators amounted to 2 (3.3%) and 29 (48.3%), respectively.

Among cases of extragenital pathology, significant (p<0.05) differences were found for obesity, namely 19 (33.3%) cases in patients of Group I and 11 (18.3%) ones in Group II (OR=2.227 (0.947–5.237). In addition, significant (p<0.05) differences were revealed for pyelonephritis, namely, the diagnosis was confirmed in 29 (50.9%) patients of Group I and 10 (16.7%) patients in Group II (OR=5.179 (2.2–12.174); proportions of these pathologies were higher in women with ICI.

Analysis of the concentrations of free radical-neutralizing enzymes involved in the investigation showed that the levels of superoxide dismutase and ceruloplasmin in the blood plasma did not have significant differences in the studied groups; however, we found a significant increase (p>0.05) in the level of catalase, which was higher in patients with ICI. According to the results of the ROC analysis, the prognostic significance of catalase in the detection of ICI was characterized by a sensitivity of 77.8 and specificity of 57.9 (criterion>90.5405, p<0.028) (Table 1).

This redistribution of levels of antioxidant defense enzymes apparently indicates the activation of membrane-destructive processes in the body of pregnant women with ICI, which explains the increased activity of catalase [24].

Catalase, being the first link in intracellular protection against reactive oxygen species, which does not require energy for activation, metabolizes hydrogen peroxide preventing its accumulation in the cells with the formation of water and oxygen. It is known that the activation of hydrogen peroxide production stimulates natural killers, triggering proinflammatory reactions in tissues. Proinflammatory cytokines promote the initiation of metalloproteinases, which loosen the cervical stroma. The most important cytokines involved in this process are interleukin-1 (IL-1) and interleukin-8 (IL-8) [17]. IL-1 directly stimulates cyclooxygenase 2 activity, which enhances prostaglandin production and increases matrix metalloproteinase activity [25].

One cannot ignore the systemic mechanisms in the initiation of cervical dilation processes, and in this regard, the assessment of adipose tissue metabolism is of interest. As a consequence, the next stage of the study was to compare the levels of some substances, which provide processes of adipose tissue metabolism, namely leptin, ghrelin, resistin, adiponectin, and homocysteine (Table 2). The analysis data revealed that women with ICI had a significantly (p<0.05) elevated leptin level. According to the results of ROC analysis, the prognostic significance of the leptin level in predicting ICI corresponded to a sensitivity equal to 98.2 and specificity equal to 89.5 (criterion>19.84, p<0.001) (Table 2).

Concurrently, a significant (p<0.05) increase in the contents of ghrelin and resistin was noted. According to the results of ROC analysis, the prognostic significance of the ghrelin level in predicting ICI corresponded to a sensitivity equal to 35.2 and a specificity equal to 88.3 (criterion<0.15, p<0.014), while for resistin, the sensitivity was 75.6, and the specificity was 61.9 (criterion<2.85, p<0.001).

Other indicators did not have statistically significant (p>0.05) differences between groups.

The data obtained from the investigation of fat metabolism component elements are consistent with the already described results on the content of antioxidant defense enzymes, the shift in the levels of which indicates a local manifestation of the proinflammatory status in the body of pregnant women with ICI.

Leptin is synthesized in adipose tissue and is considered an energy expenditure hormone; due to its direct action on leptin receptors and plasma membranes of various cell types in the human body, leptin indirectly modulates the effects of many biologically active substances, especially cytokines. Resistin belongs to the group of secretory proteins enriched in cysteine [23] and is secreted by immune and epithelial cells. It primarily contributes to the development of insulin resistance. Previously, it has been shown that leptin and resistin have powerful proinflammatory properties due to the activation of proinflammatory cytokines [16][15][17]. However, the results obtained indicate a decrease in the resistin level in women with ICI. This fact confirms the pathological course of pregnancy in women with ICI, since it has been established that during the period of embryonic development, the resistin gene is expressed by placental trophoblasts, and its content in the blood plasma of pregnant women with a physiological course of pregnancy is significantly higher than in the cases of obstetric complications. Meanwhile, ghrelin concentrations are interconnected with the content of leptin in the blood serum; consequently, a decrease in ghrelin levels against the background of high concentrations of leptin in patients of the main group is natural.

Homocysteine and adiponectin levels did not differ statistically significantly in the studied groups (p>0.05).

Discussion

Oxidative stress underlies the pathogenesis of many pregnancy complications and represents to an essential extent a typical general pathological reaction of the human body [16][15][26]. Disruption of the balance between components of the prooxidant and antioxidant systems with the release of free radicals and the development of oxidative stress leads to cell injury and apoptosis, which can clinically manifest itself in the development of any form of major obstetric syndromes, including premature birth and pregnancy loss [9]. The mechanism of pregnancy termination is based on infectious and inflammatory processes; specifically, the critical duration and severity of these processes lead to the accumulation of lipid peroxidation products and the long-term effects of reactive oxygen species on cell membranes.

Catalase is one of the enzymes of the antioxidant defense system, which catalyzes the decomposition of hydrogen peroxide, formed during biological oxidation, into water and molecular oxygen. According to the literature data, in groups of women with the threat of premature birth and with the realized threat of premature birth, the level of catalase increased at the advance in the gestational age. Moreover, upon the threat of premature birth at 28–32 weeks of pregnancy, catalase activity below 150 U/ml of plasma was a negative prognostic factor [27]. According to the obtained results, at the time of the first screening, the level of catalase was higher in the group of women who subsequently manifested ICI and the threat of premature birth compared to patients with physiological pregnancy.

The examined lipid metabolism indicators can also be used as admissible early markers of cervical shortening. According to earlier studies, the leptin level in overweight women was significantly higher in cases of premature birth compared to patients with similar overweight and full-term pregnancies [16][23]. In addition, the leptin level was higher at premature births and in patients with preeclampsia compared to healthy pregnant women. The received data on the increased leptin level in the group of patients with ICI confirm previously obtained results on the role of lipid metabolism factors in the development of miscarriage birth [1][28].

Leptin and ghrelin belong to a group of peptide hormones with counter-directional action, which regulate lipid metabolism and have an immunomodulatory function, and as a consequence, they are necessary for the realization of fertility [16][23]. During pregnancy, leptin decreases the proportion of activated Th cells and lymphocyte apoptosis, as well as provides a multidirectional dose-dependent effect on the content of CD16⁺CD56⁺NK and CD16⁺CD56⁺NKT cells, without affecting the number of CD4⁺CD25^bright T lymphocytes. Ghrelin, on the contrary, dose-dependently increases the rate of activated Th cells and lymphocyte apoptosis without affecting the levels of CD16⁺CD56⁺NK, CD16⁺CD56⁺NKT cells, and CD4⁺CD25^bright T lymphocytes [29]. In accordance with previously conducted studies, during pregnancy, the main function of ghrelin is the regulation of maternal appetite and fetal nutrition, as well as the regulation of myometrial contractility [16][8]. The obtained results indicated a significant decrease in the ghrelin level in women with shortening of the cervix and subsequent threat of pregnancy termination, which was probably associated with its effect on the contractile activity of uterine smooth muscle cells and a decrease in the cervix length.

The contribution of resistin to the inflammatory response makes this hormone important to the study, especially in the context of the pathogenesis of preterm labor. Some cytokines, such as IL-6 and TNF-, stimulate its expression. Resistin activates endothelial production of endothelin-1 in vitro. In scientific databases, there is only limited information on the resistin level at preterm labor. For instance, in 2008, Kusanovic et al. analyzed the composition of amniotic fluid, namely the level of resistin in 648 pregnant women in the second and third trimesters of pregnancy, as well as in the postpartum period [29], and found that resistin was present in amniotic fluid in both the second and third trimesters, and its concentration increased with advancing pregnancy term. In women with the threat of premature birth and signs of intrauterine infection, the level of resistin was higher regardless of the integrity of the fetal membranes. Meanwhile, the onset of labor did not affect amniotic fluid resistin levels in full-term pregnancies. The authors concluded that resistin may play an important role in initiating the inflammatory response in the mother and fetus and triggering preterm labor. Our data, on the contrary, attest to a lower level of resistin in the blood serum of pregnant women with ICI and the threat of pregnancy termination. Perhaps such a discrepancy between the abovementioned data and our results is explained by the different biological media, in which the resistin level was determined (amniotic fluid and blood serum). Undoubtedly, further studies are necessary to identify the role of resistin in the pathogenesis of pregnancy termination.

Conclusion

The active development and implementation of personalized preventive medicine poses a complex task for practicing physicians in terms of the early diagnostics and prevention of obstetric complications. This area is of particular importance in connection with complications associated with demography, including ICI and premature birth, which stipulates high rates of prematurity, childhood morbidity, disability, and mortality. Trends in obstetric care worldwide are shifting medical focuses to the first pregnancy trimester, when the first prenatal screening can make a prognosis for the remaining term of pregnancy and its outcome. In this regard, the search for early markers of cervical shortening and, as a consequence, premature birth is of great practical importance.

The patterns of changes in the levels of oxidative stress markers and lipid metabolism indicators, revealed in the study in the first trimester of pregnancy, can be used to identify risk groups and early diagnosis of ICI in women who demand more careful monitoring of the cervix length and timely preventive and therapeutic measures.