Background
Pre-eclampsia (PE) is a potentially life-threatening pregnancy related vasculopathy characterized by hypertension and proteinuria. PE results in high morbi-mortality for both mother and her unborn child. Between five and 10 % of pregnancies are complicated by hypertensive disorders and worldwide the incidence of PE has increased by 25% in the past two decades [
1].
In normal pregnancy vascular remodelling of the maternal uterine spiral arteries occurs. Trophoblast cells invade the spiral arterioles within the first 12 weeks of pregnancy and replace the muscular wall of the vessels converting them into wide bore, low resistance, large capacity vessels, a process normally completed by 20 weeks gestation [
2]. The free radical nitric oxide (•NO) is an important mediator of the placentation process. •NO is an endogenous endothelium-derived relaxing factor influencing endothelial function. Under physiologic conditions, endothelial release of •NO in the placental circulation dilates the fetal placental vascular bed, ensuring feto-maternal exchange [
3]. •NO is formed out of L-Arginine by NOS (Nitric Oxide Synthase). This reaction is regulated by VEGF (Vascular endothelial growth factor), an endothelial mitogen that has an important function in the proliferation of endothelial cells and in angiogenesis. VEGF stimulates eNOS (endothelial NOS) and induces therefore •NO production [
4]. In an oxidative environment, the lack of NOS-stabilizing factors results in NOS-uncoupling. NOS-coupling causes a shift from •NO production to superoxide (O
2•
−) production which maintains an oxidative setting.
The pathogenesis of generalized endothelial dysfunction is well known in PE and is subdivided into two phases. The first phase exists of a poor trophoblast invasion of the spiral arteries during the placentation process, causing failure to transform the placental bed arteries from high to low resistance vessels. This results in local ischemia, reperfusion damage and oxidative stress (OS). The local damage activates the second phase where disturbed production of angiogenic and anti-angiogenic factors (placental growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFlt-1), respectively) results in systemic inflammation, endothelial activation, systemic OS and altered endothelial •NO production [
5,
6]. When this vascular endothelial activation and dysfunction occurs at the level of liver, kidney, brain and placenta, the clinical presentation of PE arises [
7].
In the past, PE has been divided in two different entities; angiogenic or placental PE (formerly called early onset, before 34 weeks) and non-angiogenic or maternal PE (formerly called late onset, after 34 weeks) [
8]. Impaired placentation and endothelial dysfunction have been described in placental PE, whereas pre-existing maternal cardiovascular risk factors (essential hypertension, high BMI, diabetes, renal disease,..) usually precede maternal PE. This description however oversimplifies and overstates recent existing findings. Maternal risk factors can precede early onset PE as well as abnormal concentrations of placental angiogenic factors are found in late onset PE [
6]. Fetal growth restriction and endothelial dysfunction caused by systemic inflammation are usually described in placental PE, nevertheless they are common in late onset PE. It is therefore more accurate to state that both maternal and placental factors contribute to PE and research should focus on classifications based on pathophysiologic processes, for instance endothelial and vascular dysfunction and amount of systemic inflammation and OS [
9,
10].
In normal pregnancy, placental OS is present during all three trimesters and is necessary to obtain normal cell function, including activation of redox-sensitive transcription factors and activation of protein kinases [
11‐
15]. Although OS is a common necessary feature of normal pregnancy, persistent OS gives rise to different disease-states, such as PE [
13,
15‐
17]. Although considerable research has been devoted to OS in PE [
13‐
15], less attention has been paid to the evolution of OS during the course of normal pregnancy. Little research has described an increase in •NO concentration with gestational age, suggesting an important role for •NO in the cardiovascular changes of normal pregnancy [
3].
Recent literature has elucidated that PE is an important risk factor for cardiovascular disease in later life. Bellamy et al. and McDonald et al. describe a 3-fold risk for hypertension and a 2-fold risk of ischemic heart disease and stroke in women with a history of PE [
18‐
21]. Women with hypertensive disorders during pregnancy also have a greater risk of chronic kidney disease and end-stage renal disease [
22,
23]. With a view to detecting those women at risk, objectifying endothelial function and vascular function after healthy pregnancy and PE can help to establish reference values for disturbed post-pregnancy vascular function.
Methods/design
Study hypothesis
Endothelial and vascular function improve during healthy pregnancy to answer the higher hemodynamic demands. Due to the deficient placentation in PE, disturbed production of (anti-) angiogenic and inflammatory factors results in arterial stiffness and endothelial dysfunction. After PE, this vascular dysfunction continues in patients at risk for developing cardiovascular events later in life.
A certain amount of OS is necessary in healthy pregnancy. The deficient placental oxygenation in PE causes excessive local formation of reactive oxygen and nitrogen species (O2•− and •NO respectively). When the balance between pro-oxidant species and the antioxidants is disturbed, OS arises. We hypothesize that in PE, there is a higher amount of O2•− in the maternal circulation and a lower concentration of •NO and eNOS measurable in the placenta.
Endothelial and vascular dysfunction is correlated to the amount of OS present in the circulation.
Objectives
Primary study objective
To evaluate OS during pregnancy.
Measurement of •NO in placental tissue and O2•− in maternal blood using EPR.
Measurement of eNOS in placental tissue using immuno-histochemical staining.
Prospective longitudinal study: To evaluate the OS profile in normal pregnancies.
Matched case-control study: To compare the OS profile in normal versus PE pregnancies.
Secondary study objectives
To evaluate endothelial function during pregnancy (using peripheral arterial tonometry (PAT) and flow mediated dilatation (FMD) techniques) and to relate endothelial function to •NO, O2•− and eNOS concentration.
Prospective longitudinal study: To evaluate endothelial function in normal pregnancies.
Matched case-control study: To compare endothelial function in normal versus complicated pregnancies.
To evaluate arterial stiffness during pregnancy (Pulse wave velocity, pulse wave analysis using Sphygmocor ®) and to relate arterial stiffness to •NO, O2•− and eNOS concentration.
Prospective longitudinal study: To evaluate arterial stiffness in normal pregnancies.
Matched case-control study: To compare arterial stiffness in normal versus complicated pregnancies.
Methodology
Description of investigations (scheme of investigations, Fig. 1)
Statistical methods
Sample size calculations
As sample size determining factor, we took the RHI since this is an important variable in our study, there is normal distribution and the population standard deviation is known.
Descriptive statistics and data analysis
Discussion
PE is responsible for 11.5% of maternal deaths in Flanders. Ten percent of early neonatal deaths are caused by maternal hypertensive disorders [
39]. This project will contribute to the knowledge of PE with the ultimate goal of reducing maternal morbidity and mortality.
As stated before, the gold standard for non-invasive assessment of endothelial function is FMD [
26]. Previous literature suggests that during normal pregnancy, there is a steady increase in FMD until week 32, with a stabilization or even decline at week 36 [
40]. In PE, a significant reduction in FMD is suggested [
41]. Data using PAT during pregnancy and PE are limited, based on small studies and they show opposing results [
38,
42]. Due to measurement of peripheral microcirculatory function, PAT is less NO-dependent than FMD. As such, FMD and PAT assess different aspects of vascular function [
43‐
45]. Arterial stiffness has been evaluated in pregnancy, using applanation tonometry (AP) [
2]. During normal pregnancy AIx falls during mid pregnancy and rises at the end of pregnancy. In PE AIx is significantly increased and a significant role of first trimester AIx in the early screening of PE has been proposed [
2]. Arterial stiffness is independently associated with cardiovascular risk and may, therefore, provide a potential marker to select women who will develop cardiovascular events later in life after PE [
2,
46]. Concerning endothelial function and arterial stiffness in normal pregnancy and PE there is still no consent in literature and further research is warranted.
Both in normal pregnancy and PE are inflammatory effects present, which can be objectified by higher neutrophil to lymphocyte ratio (NLR) and higher mean platelet volume (MPV) [
47]. Increase in NLR and MPV are described to be more prominent in PE, and these factors have been proposed as predictive biomarkers for PE [
31,
48]. Increased systemic low grade inflammation possibly contributes to alterations in endothelial function.
Previous research has demonstrated that markers of OS, like O
2•
−, hydrogen peroxide (H
2O
2), hydroxyl radical (•OH), nitric oxide (•NO), and peroxynitrite (ONOO
−) are involved in the pathophysiology of placental pregnancy disorders. OS at the site of the placenta causes placental damage and this ischemic placenta releases cytotoxic, anti-angiogenic and inflammatory markers in the circulation resulting in systemic endothelial dysfunction and peripheral organ damage [
3]. O
2•
−, the most abundant free radical, encloses an important role in the beneficial effects of OS. Studies measuring O
2•
− concentration longitudinally in normal pregnancy and during PE are lacking, creating a gap in the knowledge of OS in pregnancy and PE. Evidence for OS at the placenta and in the maternal circulation in PE has led to the suggestion that anti-oxidant therapy can improve or even prevent PE. Vitamins E and C, L-arginine (precursor of •NO) and •NO donors have been proposed to limit both endothelial injury and for the prevention of PE with conflicting results [
49‐
55]. Substantial knowledge of the evolution of OS in healthy and PE pregnancies might influence introduction of these therapies in regular medical practice.
ENDOPREG is the first clinical study comparing in vivo measurements of endothelial function with in vitro markers of endothelial dysfunction in a longitudinal and case-control setting. To our knowledge, serial changes in maternal endothelial function have not been evaluated previously in pregnancy using two different methods, i.e. PAT/FMD and EPR, simultaneously. The main strength of our study is the longitudinal design. Little studies determined maternal •NO or reactive oxygen species concentration at the moment of diagnosis [
56] and only a few performed a longitudinal approach. Studying endothelial function and OS profile in and after normal pregnancies and PE, will give a better insight in the pathophysiology of this pregnancy complication and will help with the detection of patients a risk of developing cardiovascular events later in life. Within the ENDOPREG study, research groups from biomedical and pharmaceutical sciences will collaborate to unravel yet another step in the pathophysiology of the disease of many theories, PE.