https://orcid.org/0000-0002-2783-3032
Abstract
Objective
To evaluate a level of expression of endoglin (Eng), leptin (Lep), placental growth factor (PlGF), and hypoxia-inducible factor-1alpha (HIF-1α) in placenta among women with pre-eclampsia and diabetes mellitus (DM), considering the method of glycemia correction and preconception care.
Materials and methods
A retrospective cohort study was conducted. A total of 124 women were divided into following groups: type 1 DM (n = 40), type 2DM (n = 31), gestational DM (n = 33), pre-eclampsia without DM (PE) (n = 10) and the control group (n = 10). The histochemical study was performed by using primary monoclonal antibodies to Eng, PlGF, Lep, and HIF-1α (Abcam, UK).
Results
The highest level of placental expression of Eng was observed in the PE group (20.34%). The same trend was also typical for T1DM (not planned) and insulin-treated groups: T2DM and GDM. An amount of cell with an PlGF expression was significantly higher in the control group (12.2%), while the lowest was observed in the pre-eclampsia group (1.18%) and T1DM (not planned) (1.26%). The placental leptin expression within each DM group was increased among the patients with unplanned pregnancy and those who received insulin therapy. We observed the lowest Lep expression in the PE group (6.3%). High level of HIF-1α expression was detected in T1DM (not planned) (30.44%) and PE (29.64%) as compared to the control group (11.62%). In T2DM and GDM insulin groups, the HIF-1α expression was significantly higher as compared to diet groups.
Conclusion
The obtained data show that DM and pre-eclampsia are associated with changes in angiogenic and metabolic placental factor expressions. The degree of changes depends on preconception care and the control of glycemia level during pregnancy.
Introduction
Today diabetes mellitus (DM) remains a global healthcare problem, and its prevalence in the population increases steadily [Citation1]. According to the latest data, hyperglycemia among pregnant women is revealed in 16.9% cases worldwide, and the greatest number of cases (86.4%) falls on gestational diabetes mellitus (GDM) [Citation1]. Pre-gestational types of DM are less common (up to 2%) but they are frequently associated with adverse maternal and perinatal outcomes [Citation1].
The placenta reflects various metabolic disorders in the pregnant woman's body. Hyperglycemia, insulin resistance, and oxidative stress underlie the pathogenesis of DM and negatively affect the mother-placenta-fetus system. This results in changes of morphology and disturbance of production of biologically active substances, the changed expression of which may signal of different pathologic processes [Citation2]. Previously, we have shown that increased expression levels of kisspeptin (KISS1) in pregnant women with DM may play a role in impaired placentation processes and lead to the subsequent development of PE [Citation3]. Placental growth factor (PlGF) and endoglin (Eng) are one of the primary placental function mediators. They exhibit antagonistic effects and are involved in angiogenesis and vascular tone control [Citation2]. Hypoxia-induced factors (HIF and, in particular HIF-1α) during pregnancy act as key mediators of the development and functioning of the placenta by the expression of diverse genes. These genes play a critical role in erythropoiesis, angiogenesis, glycolysis, and glucose transport [Citation4]. Another essential biomarker is leptin which is synthesized, mainly, in the fatty tissue and placenta. It regulates energy metabolism [Citation5]. Excessive leptin enhances the synthesis of proinflammatory cytokines (TNF-α, IL-6), affects insulin resistance and endothelial dysfunction, underlying the formation of placental ischemia [Citation5].
Despite the critical role of these substances in regulation of placental function, there are only a few papers in the literature dedicated to their contents in DM. Therefore, the purpose of our study was to examine the level of expression of endoglin (Eng), leptin (Lep), placental growth factor (PlGF), and hypoxia-induced factor-1 alpha (HIF-1α) in pregnant women with various types of diabetes mellitus (DM) taking into account the method of glycemia correction and preconception care.
Materials and methods
A retrospective, single center cohort study was conducted. The recruitment of patients was carried out at D.O. Ott Research Institute of Obstetrics, Gynecology, and Reproductive Medicine, Saint Petersburg, Russia. Preconception planning among T1DM patients was carried out no later than six months before conception (HbA1c during the pregnancy was less than 6.5%).
A total of 124 pregnant women were divided into eight comparison groups, as follows: T1DM (not planned) (n = 20); T1DM (planned) (n = 20); T2DM (diet) (n = 11); T2DM (insulin) (n = 20); GDM (diet) (n = 14); GDM (insulin), (n = 19); PE (n = 10); control (n = 10).
For immunohistochemical analysis, we used primary monoclonal antibodies to Eng, PlGF, Lep, and HIF-1α (Abcam, UK). The quantitative assessment of the results of immunohistochemical studies was carried out on micrographs obtained using a microscopic image fixation system consisting of an Olympus BX46 microscope and the Cell Sens 47 Entry software. The photography was performed at a magnification of × 200, × 400. The fields of view containing tissue defects, staining defects, and artifacts were excluded from the photography. An amount of the cells with an expression of the studied marker was calculated using the VideoTest-Morphology 5.2 program (Videotest, Russia).
Statistical analysis was performed using SPSSV.23.0 software and Prism 8-Graph Pad (USA). For the evaluation of the distribution, the Kolmogorov–Smirnov test was performed. In case of the normal distribution, the mean value was calculated by the ANOVA test with 95% confidence interval (95% CI). Nonparametric data distribution was done by Kruskal–Wallis N-test, median test, and Dann method. Statistic processing of qualitative signs was accomplished using of χ2 criterion. Hypothesis of average values equality in the groups was rejected when the significance level was p < .05.
Results
Clinical characteristics of groups
The characteristics of the study groups are presented in . The age of patients and body mass index (BMI) were higher in T2DM and GDM groups (p = .01). Women in the T1DM, PE groups and the control group were nulliparous more often, while patients with T2DM and GDM were multiparous. Among patients with T1DM, vascular complications were more common. Those subjects had the highest levels of HbA1c throughout gestation, which indicates severe metabolic disorders in these women. Patients with various types of DM tended to develop complications of pregnancy such as gestational hypertension and PE more frequently. Fetal macrosomia occurred in one-third of patients with T1DM without preconception planning (35%) and in pregnant women with T2DM and GDM on insulin therapy (p =.043). Fetal growth restriction was found in only two women in the GDM groups on insulin therapy and the PE group (p =.04). Small gestational age (SGA) was more frequently observed in patients with various types of DM and PE, while in the control group, SGA was not recorded (p = .049).
Table 1. Clinical characteristics for study groups.
Immunohistochemical study
Placental endoglin expression
The significantly high amount of expression of Eng was revealed in the PE group (20.34% (95% CI 7.09–22.09)). It exceeded this parameter in the control group more than two times (8.37%) (p <.0001). Eng expression was significantly higher in the T1DM (not planned) group (16.98% (95% CI 15.28–18.68)), T2DM (insulin)−14.78% (95% CI 13.12–16.43) and GDM (insulin) − 15.8% (95% CI 14.1–17.5), respectively, comparing to the control group (8.37% (95% CI 7.09–9.66)) (p < .001).
Expression of placental growth factor (PlGF)
The area of PlGF expression was significantly higher in the control group (12.12% (95% CI 5.47–15.76)), and the minimum amount of cells with an expression was observed in the PE group (1.18% (95% CI 0.15–2.2)), and T1DM (no planned) group (1.26 (95% CI 0.21–3.31)). For T2DM and GDM on a diet, the expression levels were significantly higher comparing to those who received insulin: 7.2% (95% CI 2.8–12.19) and 9.36% (95% CI 6.17–13.55) versus 4.45% (95% CI 2.33–8.57) and 6.39% (95% CI 3.29–9.49), respectively.
Placental leptin expression
We discovered the significantly high Lep expression level in the placenta in T1DM (not planned) group (18.63% (95% CI 14.72–22.54)) as compared to the PE group (6.3 (95% CI 4.65–8.42)). The Lep expression area in the T2DM (insulin) group was significantly higher than in the T2DM (diet) group: 13.98% (95% CI 10.83–17.13) versus 9.86% (95% CI 8.64–11.09). We found a similar trend in the GDM group: 12.23% (95% CI 10.59–13.88) on insulin and 8.34% (95% CI 3.74–12.95) as compared to the diet (p < .001). Lep expression in the control group in average was 8.02% (95% CI 7.65–8.38).
Placental HIF-1αExpression
Significantly high HIF-1α expression was observed in the T1DM (not planned) group (30. 44% (95% CI 27.72–33.15)) and PE (29.64% (95% CI 23.62–35.66)), in comparison to the control group (11.62 (95% CI 7.45–15.8)). In T2DM and GDM groups receiving insulin therapy, the expression level of this marker was significantly higher comparing to the diet group: 25.26% (95% CI 22.63–27.88) and 24.17% (95% CI 21.67–26.67), versus 19.79% (95% CI 17.36–22.23) and 17.58% (95% CI 15.03–20.12), respectively (, ).
Figure 1. (a) – Eng expression in endotheliocytes and cytotrophoblast of chorionic villi; (b) – placental growth factor (PlGF) expression in the syncytiotrophoblast of chorionic villi; (с) – Lep expression in the syncytiotrophoblast and cytotrophoblast of chorionic villi; (d)- HIF-1αexpression is verified in the syncytiotrophoblast and cytotrophoblast of chorionic villi. Immune histochemical method, х 400.
Table 2. Expression of Eng, PlGF, Lep, HIF-1αin the study groups.
Discussion
Endoglin inhibits NO activity which leads to generalized vasoconstriction as an anti-angiogenic factor [Citation2]. The largest amount of cells with an expression of Eng was predictably detected in the PE group. Our data are in line with several studies [Citation6,Citation7]. Sela et al. reported that endoglin expression increases in syncytiotrophoblast in PE cases with simultaneous sEng growth in maternal serum [Citation6]. We also noticed high Eng expression in the T1DM (not planned), T2DM, and GDM groups receiving insulin therapy. In their large-scale study, Holmes et al. established that women with T1DM who developed pre-eclampsia showed higher sEng levels later than women without pre-eclampsia [Citation7].
In the meantime, the PlGF expression had a reverse trend and its decrease was typical of patients with DM and pre-eclampsia. The lowest PlGF levels were observed in the T1DM group without preconception care, T2DM, GDM receiving insulin, and PE group. In addition, the DM with severe pre-eclampsia showed the lowest PlGF level. Cohen et al. identified that subsequent pre-eclampsia in women with pre-gestational DM was associated with less PlGF values than that in the control [Citation8]. Cawyer et al. concluded that hyperglycemia distorted the cytotrophoblast invasive profile by suppressing the PlGF and boosting sEng activity [Citation9]. Nuzzo et al. showed that PlGF placental gene expression was significantly decreased in GDM, PE, and GDM-PE cases comparing to the control [Citation10].
The level of placental expression of leptin within each DM group grew in patients with unplanned pregnancy and receiving insulin. The lowest Lep expression was observed in the PE group while the maximum expression – in the T1DM group (not planned). In the T2DM and GDM with insulin therapy, the Lep expression area was higher in patients receiving insulin. Other studies discovered that placental expression of Lep and its receptor increases in cases of GDM [Citation11]. Another data contradicts our findings; thus, placental leptin expression grew in PE [Citation12]. It was shown, that leptin concentration was higher in late-onset PE, while early-onset PE was associated with insignificant Lep expression growth [Citation12].
The increased HIF-1α expression was seen in all studied groups comparing to the control, but the highest value was noted in T1DM (not planned) and PE groups. T2DM and GDM groups treated with insulin showed significantly higher HIF-1α expression comparing to the diet group. It was proved that increased placental HIF-1α level after the first trimester was associated with pre-eclampsia in pregnant women [Citation4]. The existing studies demonstrated that persistently increased HIF-1α placental levels are typical of women with PE [Citation13]. Li et al. noticed increased HIF-1α expression in the placenta and blood plasma in experimental female rats with GDM [Citation14]. The key regulatory role of this factor is to initiate cell pro-oxidant status as well as stimulate indirectly new vessel growth and cause increased placenta vascularization in DM [Citation5]. However, we see absence of studies on estimation of HIF-1α placental expression in DM types 1 and 2.
The obtained data shows that DM and pre-eclampsia are associated with changes in angiogenic and metabolic placental factor expressions. The degree of changes depends on preconception care and the control of glycemia level during pregnancy.
| Abbreviations | ||
| Eng | = | endoglin |
| sEng | = | soluble endoglin |
| DM | = | diabetes mellitus |
| T1DM | = | type-1 diabetes mellitus |
| T2DM | = | type-2 diabetes mellitus |
| GDM | = | gestationaldiabetes mellitus |
| GH | = | gestationalhypertension |
| HIF-1α | = | hypoxia-inducible factor 1-alpha |
| PE | = | preeclampsia |
| PlGF | = | placental growth factor |
| Lep | = | leptin |
| SGA | = | small for gestational age |
| IUGR | = | intrauterine growth restriction |
| ANOVA | = | analysis of variance |
| BMI | = | body mass index |
| HbA1c | = | glycated hemoglobin A1c |
| IADPSG | = | International Association of Diabetes and Pregnancy Study Groups |
| eNOS | = | endothelial nitric oxide synthase |
Disclosure statement
No potential conflict of interest was reported by the authors.
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References
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