Oxidative Stress and Dyslipidemia as Indicators of Pathogenesis of Preeclampsia in Pregnant Sudanese Women

Background: Preeclampsia (PE) still ranks as one of obstetrics major problems and is still a serious threat, mainly in underdeveloped countries where its incidence and mortality rates are higher and is a major cause of preterm birth and intra-uterine growth restriction. Aim: To evaluate the role of oxidative stress and dyslipidemia as indicators of pathogenesis and risks of preeclampsia in pregnant Sudanese women attending Wad-Medani Obstetrics and Gynecology Teaching Hospital. Material and Method: This was a cross-sectional (case-control) study carried out in preeclamptic pregnant Sudanese women attending Wad-Medani Obstetrics and Gynecology Teaching Hospital. A total of 208 pregnant women were enrolled in the study, 111 patients and 97 women with normal pregnancy as controls; pregnant women suffering from any systemic or endocrine disorder were excluded. We compared the serum levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, nitric oxide, and total antioxidant capacity between preeclamptic and controls. Result and Conclusion: Pregnant women with increase in BMI have a higher chance of developing PE. Low level of high-density lipoprotein cholesterol and high level of low-density lipoprotein cholesterol define that dyslipidemia increases the risk of PE. Decreased levels of NO and TAC might reflect the oxidative stress and likely contribute to the pathophysiological mechanisms of PE.

PE is one of the most common complications of human pregnancy with an overall incidence 2% -12% [10] and the syndrome results in more than 63,000 maternal deaths worldwide annually, among which 75% are experienced in a mild form, and 25% in a severe form [11]. Prevalence of preeclampsia has increased by up to 30% over the last decade [12].
The exact cause of PE remains uncertain. Therefore, PE is still a disease of theories [13]. Although the cause remains largely unknown, the pathogenesis is thought to occur in two main phases. The first stage is asymptomatic, characterized by abnormal placental development during the first trimester resulting in placental insufficiency and the release of excessive amounts of placental materials into the maternal circulation. This in turn leads to the second, symptomatic stage, where in the pregnant woman develops characteristic hypertension, renal impairment, and proteinuria and is at risk for the hemolysis, elevated liver enzymes and low platelet account (HELLP) syndrome, eclampsia, and other end organ damage [14]. At the cellular level, preeclampsia is associated with the release of free radicals by the placenta and therefore generation of oxidative stress. Placenta-borne oxidative and stresses are even sometimes considered as the major molecular determinants of the maternal disease [15].
As PE is considered as a leading cause of maternal mortality and morbidity, measurement of some biochemical markers can enhance the screening and prediction of PE, and therefore the early detection and intervention can limit its progression and harmful effects.
Research works on the PE and particularly the indicators of pathogenesis are lacking in Sudan. Consequently, this study was conducted to clarify some indicators of pathogenesis of PE in Sudanese preeclamptic women.
Objectives of this study are to estimate the serum lipid profile (TC, TG, LDL, HDL) for evaluating the dyslipidemia in preeclamptic women and to test the association of serum nitric oxide (NO) and total antioxidant capacity (TAC) with the development of preeclampsia.

Material and Methods
This was a cross-sectional (case-control) study conducted to evaluate the role of oxidative stress and dyslipidemia as indicators of pathogenesis of preeclampsia in pregnant Sudanese women attending Wad-Medani Obstetrics and Gynecology Teaching Hospital from January 2015 to January 2016.
A total of two hundred and eight pregnant women were included in this study. They were selected from the Wards of the Hospital at admission before starting treatment. Informed consent was obtained from each participant before been recruited in to the study. The pregnant women were two groups; patients group of 111 preeclamptic pregnant women, and control group of 97normal pregnant women.

Inclusion Criteria
Preeclamptic women with blood pressure ≥ 140/90 mmHg, and proteinuria ≥300 mg/24 hrs or ≥1 + dipstick) were included. The controls were healthy pregnant women with no past history or family history of PE.

Exclusion Criteria
For both patients and controls: pregnant women with pre-gestational cardiac, hepatic or renal disorders, diabetes mellitus, primary or secondary lipid disorders, severe anemia, those who suffer from any other systemic or endocrine disorder were excluded.
A questionnaire was designed to obtain personal data, anthropometric measurements, social and medical information, and clinical investigations.

Results
The body mass index of preeclamptic women was significantly higher than that of normal pregnant women (25.33 ± 0.16 versus 24.65 ± 0.18, p = 0.005) ( Figure   1). The systolic blood pressure/diastolic blood pressure of the preeclamptic women showed significant elevation compared to normal pregnant women  The biochemical parameters of cases and controls were presented as mean ± SEM. All the measured parameters were differed significantly between the two study groups ( Table 2). The TC, LDL and TG were significantly higher in preeclamptic women than controls (p-values were 0.001, <0.001 and 0.007 respectively). The HDL level was significantly decreased in preeclamptic patients when compared with control group (p < 0.001). Levels of NO and TAC were significantly lower (p-values ≤ 0.001) in preeclamptic women than control group. Pregnant women that have high level of TC, LDL, and low level of HDL were    Preeclamptic women tended to develop oxidative stress by 7.6 times for NO and 2.6 fold for TAC which in turn leads to development of PE (Table 3).

Multiple Linear Regression Analysis of the PE-Associated Risk and Complication Factors
Multiple linear regression analysis with forward elimination was conducted to determine among the significant PE-associated risk factors what were the most significant factors that actually contribute to the development of PE as indicated by SBP and DBP. Because the hypertension is the main clinical manifestation of PE, SBP and DBP were taken as dependent variable with PE-associated risk factors (BMI, lipid profile, TAC and NO) as independent variables of risk estimate. SBP and DBP were taken in two different models.  Table 6.

Systolic Blood Pressure Model
These results indicate that for an increase of one unit in standard deviations of BMI and LDL the expected increase in standard deviation of SBP was 0.112 and 0.277 respectively. A decrease of one unit in standard deviations of HDL, TAC and NO the expected increase in standard deviation of SBP was 0.328, 0.121 and 0.318 respectively.
The regression lines between SBP and BMI, LDL, HDL, NO, TAC and CK are shown in Figure 3. Although TC and TG were significant PE-associated risk factors, they were eliminated from the multiple linear regression analysis model.

Discussion
Preeclampsia is a multisystem disorder that remains a major cause of maternal   and fetal morbidity and mortality. Yet, no preventive measures are known to preclude the occurrence of the disease. Free radical damage and several metabolic derangement have been implicated in the pathophysiology of this condition [16]. This study aimed to evaluate the role of oxidative stress, dyslipidemia and markers of tissue damage as indicators of pathogenesis and risk of preeclampsia in pregnant Sudanese women.
In the present study, the ages of the two study groups were not statistically different, while the body mass index was significantly higher in preeclamptic women than control group.
Pregnancy is associated with physiologic hyperlipidemia, and in normal pregnancy this feature is not atherogenic, and attributed to hormonal changes [17]. Pregnancy-related disorders such as PE are associated with a dysregulation of lipid metabolism manifesting in adverse maternal blood lipid levels [18] [19]; in consistency, the present study showed marked increased levels of TC, TG and LDL in preeclamptic women than controls, while preeclamptic women revealed significant decrease in HDL level as compared to controls.
These findings agree with other studies. It was reported that there was a significant increase in TC, LDL and TG with significant decrease in HDL in preeclamptic group compared to normotensive group and stated that dyslipidemia plays an important role in the pathogenesis of PE [20]- [25].
The mechanisms underlying dyslipidemia in PE are implicit. One possibility; inconsequential gestational insulin resistance in PE probably increases the mobilization of fatty acid from visceral adipocytes, leading to over production of VLDL by the liver, and suppresses the activity of lipoprotein lipase, culminating in elevated serum TG concentration and reduced serum HDL level which is major risk factor for vascular dysfunction in PE [26]. Alternatively, PE is a state of hypoestrogenemia, which leads to decreased expression of VLDL/apo E receptors resulting in reduced transport of VLDL to fetal compartment and therefore occurrence of maternal hypertriglyceridemia [3] [27]. Further LDL taken up by the fetus is decreased due to reduced fetoplacental perfusion leading to increased LDL [28]. The elevated TG result in increased atherogenic small dense LDL and reduced HDL levels [29]. For the dyslipidemia and PE association, two mechanismsare suggested: firstly, dyslipidemia may induce endothelial dysfunction secondary to oxidative stress and hence occurrence of PE [30]. Secondly, dyslipidemia may impair trophoblast invasion by the deposition in predisposed vessels, such as the uterine spiral arteries and contributes to the endothelial dysfunction, therefore to a cascade of pathophysiological events of the development of PE [31]. Lipid excess and oxidative stress can provoke endothelial dysfunction. Alterations of the endothelial dysfunction may underlie the hypertension of PE [32]. In Examination of the placental transcriptome, recognized pathways affected by inflammation, lipotoxicity, and oxidative stress were amplified significantly in placenta from obese women. Besides, RNA-seq analysis recognized pronounced decrease in genes related to angiogenesis and hormone activity, in- ing to increased ROS production, oxidative stress and cellular dysfunction [33].
Oxidatively stressed placenta releases a number of trophoblast-derived antiangiogenic (e.g., soluble fms-like tyrosine kinase-1, soluble vascular endothelial growth factor, and soluble endoglin), and proangiogenic (e.g., placenta growth factor) factors that contribute to an exaggerated maternal inflammatory response with generalized endothelial dysfunction [34]. caused by increased levels of ROS [35]. Vascular function modulated by the interference of ROS and NO. Increased ROS production seems to suppress the expression and function of endothelial NO synthase [36].
NO exhibits multiple and complex biological functions and many of its effects can be mostly attributed to its strong oxidant capacity. Thus, NO is an important mediator of immune homeostasis and host defense, and changes in its generation or action can contribute to pathologic states [37].
In this study, maternal serum NO levels were significantly low in the preeclamptic women compared to controls. NO was negatively and significantly correlated with SBP, DBP, TC, LDL, TG, LDH, AST, and CK, While, HDL and TAC showed positive and significant correlation. Several reports ascertain the decreased levels of NO in PE, and its negative correlation with body weight, systolic blood pressure and diastolic blood pressure [38] [39] [40] [41]. In contrast, Norris et al. (1999) reported that the production of NO was significantly higher in the uteroplacental, feto-placental, and peripheral circulation in PE compared to normotensive pregnancies. They attributed the marked increase in NO levels to compensatory mechanism to the pathological effect of PE [42].
In normal pregnancy, NO derived from endothelial nitric oxide synthase (eNOS) contributes to the maintenance of vascular tone to increase uterine blood flow [43]. An up-regulation of eNOS, resulting in increased NO production has been shown to contribute to increases in uteroplacental blood flow via changes in vascular tone [44]. In addition, there is evidence that genetic eNOS polymorphisms may affect the susceptibility to hypertensive disorders of pregnancy [45]. to modulate PE susceptibility [46]. Vascular tone is altered by the interference of ROS and NO, increased ROS production seems to suppress the expression of eNOS and hence reduced production of NO [47]. In fact, several studies have shown that impaired vascular relaxation in PE has been attributed to reduced bioavailability of NO produced via endothelial NOS [48]. A reduction in the bioavailability of NO and the imbalance between ROS and antioxidant activity seem to play a critical role in endothelial dysfunction contributing to raised in blood pressure and hence pathogenesis of PE [49] [50]. NO is also a potent inhibitor of platelet aggregation and activation by both cGMP-dependent and -independent mechanisms [51]. Drugs that target various components of the nitric oxide soluble guanylylcyclase pathway can help to increase NO bioavailability, and the delivery of exogenous NO is an attractive therapeutic option [52]. who reported that the serum level of TAC was significantly low in preeclamptic cases signifying that the decrease in TAC leads to an imbalance between prooxidants and antioxidants in those women that go on to develop PE [55]. Oxidative stress reflects an imbalance between the formation of oxidative substances and the innate antioxidants that make up the endogenous defense system [56]. During normal pregnancy there is a slight increase in oxidative stress, even in the presence of antioxidant systems [57]. In PE, the abnormal vascular development of the blood vessels in the placenta leads to reduced placental perfusion and induce hypoxia which is by itself a potent stimulus for ROS formation [16].
A genome-wide transcriptomic view identified genes involved in lipid metabolism, angiogenesis, hormone activity, and inflammation to be significantly altered in placenta from obese women. These studies provide evidence for increased lipids and decreased TAC in placenta from obese women, and pinpointed key signaling pathways (increased JNK/FoxO4 signaling) and downstream mediators (HIF-1α and VEGF-A) that provide a link between maternal-obesity, placental inflammation/oxidative stress, and altered angiogenic factors. Obesity provokes cellular stress, which may in turn adversely affect placental development and function [33]. Antioxidants may be utilized to greater extent to counteract and scavenging free radicals, resulting in the reduction of H. A. Hobiel, H. Y. Z. Tadros Journal of Biosciences and Medicines their levels [58].
The limitations of this study include the relatively small sample size. There were no follow up criteria set. Even with this limitation, the findings of this study serve as ground for further studies to better understanding the associations of total antioxidant capacity and nitric oxide levels with preeclampsia.

Conclusions
Women with excessive BMI change have a higher chance of developing PE. Low