**2. Endothelial dysfunction**

*Prediction of Maternal and Fetal Syndrome of Preeclampsia*

can reach up to 10% of pregnancies in developing countries [1].

cure. Pre-eclampsia complicates 2–5% of pregnancies in Europe and America and

dysfunction that leads to the main clinical symptoms of pre-eclampsia [1].

pre-eclampsia might be more than a single condition [2].

women with T1D and 10–14% in women with T2DM [8].

that leads to diabetic complications years after pregnancy [8].

subsequent increased blood glucose levels [4].

for both mother and child [8].

This disorder can have an early onset (before 34 weeks of gestation) or a late onset (after 34 weeks of gestation), with the placentas of women with early onset pre-eclampsia presenting hypoplasia (small placental size) and a significantly higher number of placental vascular lesions compared to those with late onset PE, which present hyperplasia (increased placental size) and histological evidence of placental inflammation, with absence of vascular insufficiency, suggesting that

Gestational diabetes mellitus (GDM) is defined as hyperglycemia that is first diagnosed during pregnancy. This definition of GDM does not preclude the possible existence of unrecognised pre-pregnancy diabetes. The prevalence of GDM ranges from 2 to 10% of all pregnancies in developed countries [3] and is associated with birth complications, including macrosomia and operative delivery. GDM develops from a dysfunction of the pancreatic Beta cells such that the insulin supply is inadequate to meet tissue demands for normal blood glucose regulation. This insulin resistance leads to increased levels of glucose production and free fatty acids, with

All forms of diabetes (GDM, type 1 diabetes - T1D and type 2 diabetes mellitus - T2DM) increase the risk of pre-eclampsia, with GDM being an independent risk factor for the development of pre-eclampsia [5, 6], and pre-existing diabetes being a risk factor for both early- and late-onset pre-eclampsia [7]. The incidence of preeclampsia increases from 2–7% of pregnancies in non-diabetic women to 15–20% in

Pre-eclampsia and GDM share a number of risk factors, including advanced maternal age, nulliparity, multifetal pregnancies, non-white ethnicity, and prepregnancy obesity [5, 9]. Both pre-eclampsia and GDM also have long-term health implications, with pre-eclampsia increasing the risk of future cardiovascular disease, stroke, kidney disease, ophthalmic disease and development of T2DM (even without GDM), while GDM increases the risk of cardiovascular disease and T2DM

Although the exact pathophysiology is still unknown, it would seem that a combination of maternal risk factors contribute to the similar biochemical dysregulation present in both pre-eclampsia and GDM, compared to healthy pregnancies, including endothelial dysfunction, angiogenic imbalance, insulin resistance, oxidative stress, inflammation and dyslipidemia [8] suggests shared etiological pathways underlying these conditions. Such biochemical changes might result from a common aetiology, have a common trigger (such as insulin resistance during pregnancy [10]) or be similar responses to different underlying disease processes that existed prior to pregnancy [11]. Similarly, genetic and/or environmental factors that contribute to pre-eclampsia could also increase the risk of diabetic complications later in life or it could be just as possible that pre-eclampsia causes lasting damage

Pre-eclampsia is characterised by a first, asymptomatic stage involving impaired trophoblastic penetration of the decidua (both into the superficial myometrium at 14–16 weeks and into the deep myometrium at 18–20 weeks), limiting the remodelling of the maternal uterine spiral arteries for uteroplacental blood perfusion and producing local placental hypoxia and oxidative stress, which consequently leads to insufficient blood perfusion, inflammation, apoptosis, and structural damage. In the second stage, placental factors released into the maternal circulation from the poorly perfused placenta, together with the aberrant expression of pro-inflammatory, anti-angiogenic, and angiogenic factors, eventually cause the endothelial

**104**

Within the placenta, limited remodelling of the maternal uterine spiral arteries may cause hypoxia [12] or repeated ischemia–reperfusion injury [13], such that the damaged placenta then releases factors into the maternal circulation that contribute to vascular dysfunction [12]. These include the anti-angiogenic proteins soluble vascular endothelial growth factor receptor 1 (sVEGFR-1; or more commonly known as soluble fms-like tyrosine kinase 1 - sFlt-1) and soluble endoglin (sEng) [14, 15]. Excess of these anti-angiogenic proteins contributes to systemic maternal endothelial dysfunction in women with pre-eclampsia [16].

The sFlt-1 protein is a truncated form of VEGF receptor 1, composed of six immunoglobulin-like domains from the ligand-binding, extracellular domain [1]. Once secreted, sFlt-1 binds to the pro-angiogenic ligands vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), acting as a as a non-signalling decoy, reducing their bio-availability and enhancing endothelial dysfunction [16–18].

The sEng protein is composed of the extracellular domain of Endoglin, following proteolytic cleavage by metalloproteinase (MMP)-14. It binds to Transforming Growth Factor-b1 (TGF-β1), inhibiting binding to Endoglin (a TGF-β1 co-receptor), preventing the activation of endothelial Nitric Oxide Synthase (NOS) and subsequent vasodilation [15].

The levels of sFlt-1 and sEng were found to be proportional to the severity of preeclampsia [19–21], with maternal plasma concentrations of sFlt-1 and sEng increasing before pre-eclampsia was diagnosed, making them potential biomarkers for the disease [1, 22–29]. Concomitantly, the increase of sFlt-1 brings about a decrease in maternal plasma concentration of PlGF [18, 30–32]. However, the relative change in sFlt1 and sEng concentrations between two consecutive visits (first and second trimester) seems more useful as a predictive marker for developing pre-eclampsia among both low- and high-risk women that the absolute concentrations [30, 33].

The relationship between anti-angiogenic factors and pre-eclampsia in women with GDM has been explored only in a handful of studies. Women with GDM were found to have higher serum sFlt-1, Placental Protein 13 (PP13), Pentraxin 3 (PTX3), myostatin and follistatin levels early in the second trimester, with sFlt-1 and PTX3 having potential predictive value [34]. Quantitative proteomics of syncytiotrophoblasts from women with GDM and pre-eclampsia identified 11 upregulated and 12 downregulated proteins including increased Flt-1 [35]. Moreover, high sEng, high sFlt-1, low PlGF and high sFlt-1/PlGF ratio increased the odds of developing pre-eclampsia among women with pre-existing diabetes [30].

Further vascular dysfunction results from the inhibition of NOS. Asymmetric dimethylarginine (ADMA) is an analogue of L-arginine and endogenous competitive inhibitor of NOS, resulting in reduced NO synthesis from L-arginine and higher superoxide generation. NO is important in maintaining endothelial homeostasis and elevated ADMA levels are associated with inflammation, insulin resistance, dyslipidemia, obesity, and cardiovascular disease. Numerous studies have measured ADMA levels in women with pre-eclampsia and normotensive women but discrepant findings have been observed. Nevertheless, some reported elevated ADMA levels prior to the development of clinical symptoms of PE, which suggests that ADMA may contribute to the pathophysiology of pre-eclampsia [1, 29].

Poor placentation, oxidative stress, endothelial cell dysfunction and altered glucose metabolism among others generate Damage-Associated Molecular Patterns (DAMPs) including Heat Shock Proteins (HSPs), TNF-α, fetal DNA, hyaluronan, oxidised low-density lipoprotein (LDL) and long pentraxin-3 [36]. HSP70 (and its post-translational modifications) has been shown to be elevated in the placentas and sera of women with PE, reflecting systemic inflammation and oxidative stress, with

HSP70 initially protecting against placental oxidative stress but its overexpression may lead to intervillous endothelial dysfunction and may play a role in the pathogenesis of pre-eclampsia [1, 29]. TLR-4 protein expression, which recognises such DAMPs at the feto-maternal interface, is increased in women with pre-eclampsia [37].

## **3. Insulin resistance**

Insulin resistance or hyperinsulinemia is an impaired response to insulin, characteristic of normal pregnancy, which results in increased insulin secretion by the pancreatic β-cells or relative insulin deficiency due to the pancreatic β-cell deterioration. Insulin resistance is due to an overall decreased expression of the insulin receptor substrate (IRS)-1/2 protein, decreased IRS-1/2 tyrosine phosphorylation and increased IRS-1/2 serine phosphorylation, resulting in reduced glucose transport activity, which was found to be even more pronounced in women with pre-eclampsia and GDM, which might also underlie the future risk for developing T2DM [38].

Insulin resistance via the inhibition of IRS1/2 results in impaired activation of the phosphoinositide 3-kinase (PI3K) and Ak strain transforming (Akt)-dependent signalling pathway, and increased activity of the mammalian target of rapamycin (mTOR) resulting from lower activity of the mitogen activated protein kinase (MAPK) pathway. The reduced Akt activity leads to a decreased production of nitric oxide (NO) (a vasodilator) and increase of endothelin (ET)-1 (a vasoconstrictor) [39], linking endothelial dysfunction and increased risk of pre-eclampsia with GDM.

Compared to normotesive women, women who develop pre-eclampsia are more insulin resistant prior to pregnancy [40], in the first and second trimesters [41], and years after pregnancy [42], and in fact a number of pre-eclampsia risk factors are also associated with insulin resistance [40, 41]. The same was found in women that developed GDM, presenting chronic insulin resistance and chronic β-cell function prior to pregnancy [4, 43]. Women with GDM are then unable to increase insulin production to compensate for the increased insulin resistance and destruction, as happens in normal pregnancy [44]. The metabolic changes observed in GDM are the same as those found in the pre-diabetic stages of T2DM, where pre-diabetes may include patients with metabolic syndrome, GDM, and impaired glucose tolerance.

### **4. Oxidative stress and mitochondrial dysfunction**

During normal pregnancy generation of reactive oxygen species (ROS) is known to be increased and necessary for proper physiology [45]. However, both pre-eclampsia and GDM present a reduced antioxidant status when compared to normal pregnancies, with increased levels of protein and lipid oxidation products [46]. Free radicals react with nucleic acids, proteins and lipids, bring about post-translational modification of proteins [47] and cause structural and functional damage [46]. The changes in a wide variety of oxidative stress metabolites (such as NO, superoxide and peroxynitrile) as well as antioxidant enzymes and compounds (such as catalase, superoxide dismutase (SOD) and vitamin E) have been analysed in relation to pre-eclampsia and GDM compared to normal pregnancies but there is still no consensus since their levels were found to be variable (the same, higher or lower) depending on the cohort studied [48–50]. Although supplementation with antioxidants such as vitamin C, vitamin E or n-acetylcysteine have been found to be ineffective in reducing the risk of pre-eclampsia, calcium and vitamin D supplementation could lower risk of pre-eclampsia [50, 51].

In the case of hyperglycemia, it is known to stimulate ROS production by four major sources, namely glucose auto-oxidation, mitochondrial superoxide

**107**

*Biochemical Dysregulation of Pre-Eclampsia and Gestational Diabetes Mellitus*

production, endothelial NOS uncoupling and advanced glycation end product (AGE)-dependent NADPH oxidase activation, with glucose auto-oxidation and mitochondrial superoxide likely being the initial contributors to ROS-mediated dysfunction caused by hyperglycemia [52, 53]. Advanced glycation end products are of particular interest as these were found to be able to promote TNF-α mRNA expression and secretion as well as bringing about a significant decrease in eNOS

mRNA expression and protein levels via serine phosphorylation [54, 55].

the activation of a RAGE/NADPH oxidase dependent pathway [59].

In women with PE, oxidative markers were significantly higher, while antioxidative markers were significantly lower, indicating gradual oxidative damage of the placenta, even before the onset of clinical symptoms [60]. Similarly, women with GDM had higher serum malondialdehyde levels and significantly lower serum glutathione peroxidase activity in the first trimester, with negative correlation in the

Looking directly at the mitochondria, women with early-onset pre-eclampsia showed increased mitochondrial activation, with up-regulation of optic atrophy, type 1 (OPA-1), increased placental mitochondrial DNA copy number, and mitochondrial transcription factor A down-regulation, while both early- and late-onset pre-eclampsia were associated with an elevated phosphate/oxygen ratio [62]. Moreover, a comparative proteomics analysis of placental mitochondria in women with pre-eclampsia compared to healthy pregnancies identified up-regulation of 4 proteins and down-regulation of 22 proteins involved in ROS generation, apoptosis, fatty acid oxidation, respiratory chain function, and the tricarboxylic acid cycle [63].

After ischemia and reperfusion injury, together with oxidative stress, the placenta mounts an inflammatory response releasing cytokines and other inflammatory factors such as Tumour Necrosis Factor-alpha (TNF-α), Interleukin (IL)-6, and C-reactive protein (CRP), and damaging levels of ROS, which are a characteristic of pre-eclampsia [64] and the altered levels of inflammatory cytokines in both early and late-onset pre-eclampsia correlated with the type of histopathologic changes in the placenta [65]. The proposed mechanism linking insulin resistance and inflammatory pathways involves a reduction in Akt activity, which also reduces NO generation. Reduced Akt activity and reduced plasma level of adiponectin reduce adenosine monophosphate protein kinase (AMPK) activity, such that mTOR activation is facilitated. The increased mTOR-activated signalling and increased extracellular level of leptin and TNF-α result in c-Jun N-terminal kinase (JNK) activation, inhibiting IRS1/2 and reducing insulin signalling. Thus hyperinsulinemia activates a feedback loop of

In women who later developed GDM, increased leukocyte counts were observed

since the first trimester, indicating that inflammation is associated with the

increased vascular inflammation and insulin resistance [39].

The serum levels of AGEs were higher in women with both early- and late-onset pre-eclampsia and in women with severe pre-eclampsia positively correlated with serum levels of TNF-α and VCAM-1, indicating AGEs are important mediators in regulating the inflammatory pathways of pre-eclampsia [56–58]. Furthermore, treatment with AGEs increased intracellular ROS generation and over-expression of sFlt-1 in an extravillous trophoblast cell line, suggesting that AGEs may be important mediators in the regulation of angiogenic pathways, with accumulation of AGEs possibly contributing to pre-eclampsia by promoting sFlt-1 production via

*DOI: http://dx.doi.org/10.5772/intechopen.85843*

second and third trimester [61].

**5. Inflammation**

**5.1 Cytokines**

#### *Biochemical Dysregulation of Pre-Eclampsia and Gestational Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.85843*

production, endothelial NOS uncoupling and advanced glycation end product (AGE)-dependent NADPH oxidase activation, with glucose auto-oxidation and mitochondrial superoxide likely being the initial contributors to ROS-mediated dysfunction caused by hyperglycemia [52, 53]. Advanced glycation end products are of particular interest as these were found to be able to promote TNF-α mRNA expression and secretion as well as bringing about a significant decrease in eNOS mRNA expression and protein levels via serine phosphorylation [54, 55].

The serum levels of AGEs were higher in women with both early- and late-onset pre-eclampsia and in women with severe pre-eclampsia positively correlated with serum levels of TNF-α and VCAM-1, indicating AGEs are important mediators in regulating the inflammatory pathways of pre-eclampsia [56–58]. Furthermore, treatment with AGEs increased intracellular ROS generation and over-expression of sFlt-1 in an extravillous trophoblast cell line, suggesting that AGEs may be important mediators in the regulation of angiogenic pathways, with accumulation of AGEs possibly contributing to pre-eclampsia by promoting sFlt-1 production via the activation of a RAGE/NADPH oxidase dependent pathway [59].

In women with PE, oxidative markers were significantly higher, while antioxidative markers were significantly lower, indicating gradual oxidative damage of the placenta, even before the onset of clinical symptoms [60]. Similarly, women with GDM had higher serum malondialdehyde levels and significantly lower serum glutathione peroxidase activity in the first trimester, with negative correlation in the second and third trimester [61].

Looking directly at the mitochondria, women with early-onset pre-eclampsia showed increased mitochondrial activation, with up-regulation of optic atrophy, type 1 (OPA-1), increased placental mitochondrial DNA copy number, and mitochondrial transcription factor A down-regulation, while both early- and late-onset pre-eclampsia were associated with an elevated phosphate/oxygen ratio [62]. Moreover, a comparative proteomics analysis of placental mitochondria in women with pre-eclampsia compared to healthy pregnancies identified up-regulation of 4 proteins and down-regulation of 22 proteins involved in ROS generation, apoptosis, fatty acid oxidation, respiratory chain function, and the tricarboxylic acid cycle [63].
