**3. Maternal and umbilical cord blood modifications**

PE is a disorder involving maternal and placental changes. It involves fetal complications, such as, prematurity and IUGR, which are the most representative. Newborns, whose mothers develop PE, usually present a lower birth weight than infants born from mothers with a normal pregnancy (Catarino et al., 2008a). Moreover, newborns small for gestational age, as well as newborns with Apgar score below 7, are frequently observed in PE (Catarino et al., 2008a).

There are several studies concerning maternal modifications in PE, but there are few studies carried out in umbilical cord blood. We will address the effect of some modifications usually observed in PEc women, such as, in lipid profile, in hematologic profile, inflammatory and antioxidant markers, angiogenic/anti-angiogenic factors and hemostatic disturbances, in umbilical cord blood.

#### **3.1 Angiogenic/anti-angiogenic factors**

A normal placental development is essential for adequate feto-maternal nutrients and gas exchanges. In addition, placenta is an important endocrine organ that synthesizes various hormones, cytokines and angiogenic growth factors. These factors are released into the maternal circulation, and may contribute to changes in endothelial function. It is

factors. It has been shown that PEc placentas present an increased expression of sFlt1 (Gu et al., 2008), and a decreased expression of PlGF (Gu et al., 2008). These placental factors and cytokines appear to be released in maternal circulation, resulting in a generalized endothelial dysfunction, causing the multisystem complications of a PEc pregnancy

(Rusterholz et al., 2007; Maynard et al., 2008) (Fig. 1).

Fig. 1. Possible mechanisms involved in the pathogenesis of preeclampsia

PE is a disorder involving maternal and placental changes. It involves fetal complications, such as, prematurity and IUGR, which are the most representative. Newborns, whose mothers develop PE, usually present a lower birth weight than infants born from mothers with a normal pregnancy (Catarino et al., 2008a). Moreover, newborns small for gestational age, as well as newborns with Apgar score below 7, are frequently observed in PE (Catarino

There are several studies concerning maternal modifications in PE, but there are few studies carried out in umbilical cord blood. We will address the effect of some modifications usually observed in PEc women, such as, in lipid profile, in hematologic profile, inflammatory and antioxidant markers, angiogenic/anti-angiogenic factors and hemostatic disturbances, in

A normal placental development is essential for adequate feto-maternal nutrients and gas exchanges. In addition, placenta is an important endocrine organ that synthesizes various hormones, cytokines and angiogenic growth factors. These factors are released into the maternal circulation, and may contribute to changes in endothelial function. It is

**3. Maternal and umbilical cord blood modifications** 

et al., 2008a).

umbilical cord blood.

**3.1 Angiogenic/anti-angiogenic factors**

recognized that placenta has an important role in the protection and development of the fetus, promoting feto-maternal exchanges which are essential to a normal pregnancy. On the other hand, it is also recognized that in PE there are changes in placental development (Pijnenborg et al., 2006; Young et al., 2010), which can compromise the feto-maternal exchange, limiting fetal development, and trigger a maternal and fetal response to adapt to these changes.

Angiogenic factors have been subject of intensive research in recent years, as they appear to be involved in the aetiology of PE. Several studies show a decrease in the concentration of PlGF (Polliotti et al., 2003; Levine et al., 2004) and an increase in circulating levels of sVEGFR-1 (McKeeman et al., 2004; Levine et al., 2004) in PEc women. Nevertheless, there is some controversy regarding the concentration of VEGF in pregnant women with PE (Simmons et al., 2000; McKeeman et al., 2004). Since sVEGFR-1 is an antagonist of PlGF and VEGF, there is a reduction of the effects of these factors, i.e., a change in angiogenesis and in endothelial function (Luttun & Carmeliet, 2003). The anti-angiogenic effect in pregnant women with PE seems to disappear after delivery (Maynard et al., 2008; Myatt & Webster, 2009), strengthening the involvement of placental factors in PE. VEGF plays an important role in vascular development, especially at placenta level, but also induces NO and prostaglandins synthesis, which are mediators of vasodilation (Myatt & Webster, 2009).

The processes of vasculogenesis/angiogenesis are crucial for the development of uteroplacental circulation and placenta. In a study performed by our group (Catarino et al., 2009a), we observed a disturbance of the angiogenic/anti-angiogenic factors in the maternal circulation in PE. PEc women had significantly higher levels of sVEGFR-1 (Fig. 2B) and VEGF values and significantly lower PlGF levels (Fig. 2A). This disruption in angiogenesis factors seems to be associated with placental dysfunction, since these factors are mainly produced by the placenta (Kaufmann et al., 2004), during pregnancy. 

We also observed a positive correlation between levels of PlGF and maternal placental weight, in PEc pregnancy, which highlights the importance of PlGF in placental development (Catarino et al., 2009a). It is worthy to note that sVEGFR-1, an inhibitor of PlGF and VEGF, was significantly correlated with the amount of proteinuria, a marker of PE severity, suggesting an important role of sVEGFR-1 in the pathogenesis of PE (Catarino et al., 2009a). The abnormal development of the placenta in PE, reduces the placenta perfusion and may also contribute to the observed increase in the amounts of sVEGFR-1 and VEGF, since the tissue hypoxia regulates its production, stimulating it.

In cord blood samples of PEc cases, we observed a significant decrease in PlGF (Fig. 2C) and VEGF concentrations and a significant increase in the levels of sVEGFR-1 (Fig. 2D) (Catarino et al., 2009a). This disruption, particularly at values of PlGF and sVEGFR-1, seems to be an indirect indicator of the involvement of placental dysfunction in PE, since both are produced primarily in the placenta.

The observed correlation between mother and child for sVEGFR-1 seems to indicate that the release of these factors by placenta, occurs both for the maternal and fetal circulation. However, the levels of sVEGFR-1 are higher in the blood of mothers, when compared with cord blood levels, which allows us to suppose that decidua, since it is able to secrete sVEGFR-1 (Lockwood et al., 2007), may also contribute for maternal levels of sVEGFR-1.

Umbilical Cord Blood Changes in Neonates from a Preeclamptic Pregnancy 277

The hemostatic system is altered in PE, which may increase the incidence of maternal and fetal complications. In PEc women, a decrease in the number of platelets compared with normal pregnancy is frequently described (Howarth et al., 1999; Osmanağaoğlu et al., 2005), and thrombocytopenia may occur. Maternal thrombocytopenia is usually associated with more severe pathology, including the HELLP syndrome. Harlow et al. (2002) stated that the decrease in the platelets value is the result of an increase in consumption, as they observed intense platelet activation in patients with PE compared with other pregnancy hypertensive diseases and normal pregnancy. On the other hand, the increase in the mean platelet volume (increased younger platelets, with larger size) (Howarth et al., 1999) and the higher thrombopoietin circulating levels (specific growth factor that promotes thrombocytopoiesis) in women with PE (Johnson et al., 2001), reflect an increase in platelet production. Thus, the

The fibrinolytic system is usually disturbed in PE, due to increased plasminogen activator inhibitor (PAI)-1 and PAI-2. Several studies describe a significant increase in plasma levels of PAI-1 in cases of PE compared to normal pregnancy (Belo et al., 2002a; Tanjung et al., 2005; Sartori et al., 2008; Hunt et al., 2009). The expression of PAI-1 is also elevated in placentas from PEc pregnant women, and PAI-1 plasma levels seem to be positively correlated with the severity of placental damage (Estelles et al., 1998). PAI-2 can be considered a marker of placental function, as it is mainly synthesized at trophoblastic tissue. In PE a decrease in plasma PAI-2 seems to occur (Roes et al., 2002; Tanjung et al., 2005; Sartori et al., 2008), suggesting a modification of placental function. Considering the increased PAI-1 and the decrease of PAI-2, some authors suggest that the rise in the ratio PAI-1/PAI-2 in maternal plasma may be considered a marker of PE (Chappell et al., 2002; Hunt et al., 2009). Several authors report that tissue plasminogen activator (tPA), one of the physiological plasminogen activators, is increased in PE (Belo et al., 2002a; Tanjung et al., 2005; Hunt et al., 2009). Increased levels of D-dimers (fragments of fibrin degradation products) were also reported in PE (Belo et al., 2002a; Hunt et al., 2009), reflecting an increase in the activation process of coagulation and fibrinolysis. The production of Ddimer depends on the formation of thrombin, resulting from activation of the coagulation and fibrinolytic systems. At hemostatic level changes in other parameters, such as a decrease in antithrombin III (Osmanağaoğlu et al., 2005; Tanjung et al., 2005) and an increase in thrombin-antithrombin complex (TAT) in PE (Hunt et al., 2009) were

In order to clarify the involvement of hemostatic abnormalities in PE, we assessed fibrinolytic markers, in particular, tPA and PAI antigens and fibrin degradation products (D-dimers). We found that the values of tPA (Fig. 3A) and PAI-1 were significantly higher in PE, without changes in D-dimers (Catarino et al., 2008b). The high levels of tPA and PAI-1 suggest endothelial dysfunction in this syndrome, as both substances are produced by the endothelial cell and exert antagonic roles in the fibrinolytic process. In addition, both PAI-1 and tPA present positive correlations with proteinuria, suggesting that the severity of PE is associated with increased activation/endothelial dysfunction (Catarino et al., 2008b). Furthermore, the maternal endothelial (dys)function, appears to be related to placental (dys)function, considering the positive correlation that we observed between tPA

and sVEGFR-1 values in normal and PEc pregnancy (Catarino et al., 2009a).

reduction of platelet count in PE seems to be due to a higher consumption.

**3.3 Hemostasis**

described.

Fig. 2. Concentrations of PlGF in maternal blood (A) and cord blood (C), and sVEGFR-1 levels in maternal blood (B) and cord blood (D) from women with preeclampsia (PE) or with normal pregnancy (N). (Adapted from Catarino et al., 2009a)

#### **3.2 Endothelial dysfunction**

Several investigators support the hypothesis that in PE endothelial dysfunction occurs, what contributes to the onset of maternal clinical manifestations. Furthermore, this dysfunction seems to be the link between changes in placental and multisystem complications (Roberts & Gammil, 2005; Young et al., 2010).

In literature, a large number of studies describing changes in endothelial function markers in PE can be found, in particular, an increase of plasma endothelin-1 (Baksu et al., 2005a; Bernardi et al., 2008a), soluble vascular cell adhesion molecule (sVCAM), plasma fibronectin (Aydin et al., 2006; Dane et al., 2009) and tissue fibronectin (Powers et al., 2008), a decrease of plasma and urinary nitric oxide (NO) levels (Baksu et al., 2005a; Mao et al., 2009).

PE is thus associated with a decrease in vasodilatory mediators such as NO and prostacyclin, and an increase in vasoconstrictive mediators such as endothelin-1, angiotensin II and thromboxane A2 (Myatt & Webster, 2009). There are also markers related with hemostasis that are altered in PE and can also be considered as markers of endothelial dysfunction (see Hemostasis section). as

In PE, there are several entities that may induce or contribute to injury and/or endothelial dysfunction. These include changes at the level of angiogenic factors/anti-angiogenic, the presence of oxidative stress, exacerbation of the inflammatory process, changes in lipid profile and also a hypoxic environment (Myatt & Webster, 2009).

#### **3.3 Hemostasis**

276 From Preconception to Postpartum

Fig. 2. Concentrations of PlGF in maternal blood (A) and cord blood (C), and sVEGFR-1 levels in maternal blood (B) and cord blood (D) from women with preeclampsia (PE) or with

Several investigators support the hypothesis that in PE endothelial dysfunction occurs, what contributes to the onset of maternal clinical manifestations. Furthermore, this dysfunction seems to be the link between changes in placental and multisystem complications (Roberts

In literature, a large number of studies describing changes in endothelial function markers in PE can be found, in particular, an increase of plasma endothelin-1 (Baksu et al., 2005a; Bernardi et al., 2008a), soluble vascular cell adhesion molecule (sVCAM), plasma fibronectin (Aydin et al., 2006; Dane et al., 2009) and tissue fibronectin (Powers et al., 2008), a decrease

PE is thus associated with a decrease in vasodilatory mediators such as NO and prostacyclin, and an increase in vasoconstrictive mediators such as endothelin-1, angiotensin II and thromboxane A2 (Myatt & Webster, 2009). There are also markers related with hemostasis that are altered in PE and can also be considered as markers of endothelial

In PE, there are several entities that may induce or contribute to injury and/or endothelial dysfunction. These include changes at the level of angiogenic factors/anti-angiogenic, the presence of oxidative stress, exacerbation of the inflammatory process, changes in lipid

factors/anti-angiogenic,

of plasma and urinary nitric oxide (NO) levels (Baksu et al., 2005a; Mao et al., 2009).

normal pregnancy (N). (Adapted from Catarino et al., 2009a)

profile and also a hypoxic environment (Myatt & Webster, 2009).

**3.2 Endothelial dysfunction** 

& Gammil, 2005; Young et al., 2010).

dysfunction (see Hemostasis section).

The hemostatic system is altered in PE, which may increase the incidence of maternal and fetal complications. In PEc women, a decrease in the number of platelets compared with normal pregnancy is frequently described (Howarth et al., 1999; Osmanağaoğlu et al., 2005), and thrombocytopenia may occur. Maternal thrombocytopenia is usually associated with more severe pathology, including the HELLP syndrome. Harlow et al. (2002) stated that the decrease in the platelets value is the result of an increase in consumption, as they observed intense platelet activation in patients with PE compared with other pregnancy hypertensive diseases and normal pregnancy. On the other hand, the increase in the mean platelet volume (increased younger platelets, with larger size) (Howarth et al., 1999) and the higher thrombopoietin circulating levels (specific growth factor that promotes thrombocytopoiesis) in women with PE (Johnson et al., 2001), reflect an increase in platelet production. Thus, the reduction of platelet count in PE seems to be due to a higher consumption.

The fibrinolytic system is usually disturbed in PE, due to increased plasminogen activator inhibitor (PAI)-1 and PAI-2. Several studies describe a significant increase in plasma levels of PAI-1 in cases of PE compared to normal pregnancy (Belo et al., 2002a; Tanjung et al., 2005; Sartori et al., 2008; Hunt et al., 2009). The expression of PAI-1 is also elevated in placentas from PEc pregnant women, and PAI-1 plasma levels seem to be positively correlated with the severity of placental damage (Estelles et al., 1998). PAI-2 can be considered a marker of placental function, as it is mainly synthesized at trophoblastic tissue. In PE a decrease in plasma PAI-2 seems to occur (Roes et al., 2002; Tanjung et al., 2005; Sartori et al., 2008), suggesting a modification of placental function. Considering the increased PAI-1 and the decrease of PAI-2, some authors suggest that the rise in the ratio PAI-1/PAI-2 in maternal plasma may be considered a marker of PE (Chappell et al., 2002; Hunt et al., 2009). Several authors report that tissue plasminogen activator (tPA), one of the physiological plasminogen activators, is increased in PE (Belo et al., 2002a; Tanjung et al., 2005; Hunt et al., 2009). Increased levels of D-dimers (fragments of fibrin degradation products) were also reported in PE (Belo et al., 2002a; Hunt et al., 2009), reflecting an increase in the activation process of coagulation and fibrinolysis. The production of Ddimer depends on the formation of thrombin, resulting from activation of the coagulation and fibrinolytic systems. At hemostatic level changes in other parameters, such as a decrease in antithrombin III (Osmanağaoğlu et al., 2005; Tanjung et al., 2005) and an increase in thrombin-antithrombin complex (TAT) in PE (Hunt et al., 2009) were described.

In order to clarify the involvement of hemostatic abnormalities in PE, we assessed fibrinolytic markers, in particular, tPA and PAI antigens and fibrin degradation products (D-dimers). We found that the values of tPA (Fig. 3A) and PAI-1 were significantly higher in PE, without changes in D-dimers (Catarino et al., 2008b). The high levels of tPA and PAI-1 suggest endothelial dysfunction in this syndrome, as both substances are produced by the endothelial cell and exert antagonic roles in the fibrinolytic process. In addition, both PAI-1 and tPA present positive correlations with proteinuria, suggesting that the severity of PE is associated with increased activation/endothelial dysfunction (Catarino et al., 2008b). Furthermore, the maternal endothelial (dys)function, appears to be related to placental (dys)function, considering the positive correlation that we observed between tPA and sVEGFR-1 values in normal and PEc pregnancy (Catarino et al., 2009a).

Umbilical Cord Blood Changes in Neonates from a Preeclamptic Pregnancy 279

increase of lipid peroxidation (MDA), followed by decreased total antioxidant capacity and ascorbic acid levels in newborns whose mothers developed PE (Mehendale et al., 2008; Howlader et al., 2009), while other studies report no difference in lipid peroxidation

Experimental studies (Poston et al., 2006; Rahimi et al., 2009; Xu et al., 2010), performed in pregnant women at risk, showed no beneficial effect on the antioxidant consumption such as vitamins E and C, in preventing the development of PE, despite the evidence for the involvement of a state of oxidative stress in PE. In fact, according to these trials, antioxidant therapy does not appear to be sufficient to prevent the development of the PE, further increasing the risk of neonatal complications, including increased rate of newborns with low

There are various mechanisms by which oxidative stress is linked to the inflammatory process. During the inflammatory response production and release of reactive oxygen metabolites occurs leading to oxidative stress. On the other hand, products of oxidative stress, such as those resulting from lipid peroxidation, are considered pro-inflammatory.

PE may be considered as an exacerbated inflammatory condition compared with physiological pregnancy, which appears to contribute to endothelial dysfunction. Initially, there is a localized inflammatory response within the placenta, while in a second phase predominates a systemic inflammatory response. Numerous studies have reported an increase of pro-inflammatory cytokines such as IL-6 and TNF-α (Bernardi et al., 2008b; Guven et al., 2009; Ouyang et al., 2009) in pregnant women with PE, when compared with

Concerning acute phase proteins, C-reactive protein (CRP) is probably the most studied one, mainly due to its sensibility in detecting inflammation, with a significant increase being observed in PEc pregnancy (Belo et al., 2003; Tjoa et al., 2003; Guven et al., 2009). Tjoa et al. reported an increase in plasma concentration of CRP between 10 and 14 weeks of gestation in pregnant women who subsequently developed PE and gave birth to newborns with

Cell adhesion molecules (CAM), necessary for the adhesion of leukocytes to vascular endothelium, are also altered in PE. Despite some contradictory results, plasma levels of (sICAM)-1, soluble vascular cell adhesion molecule (sVCAM)-1, soluble platelet endothelial cell adhesion molecule (sPECAM)-1 and soluble E-selectin are raised in PEc pregnant

The neutrophil activation also seems to be associated with PE, as some studies mentioned an increase in circulating levels of myeloperoxidase (Mellembakken et al., 2001; Gandley et al., 2008) and elastase (Belo et al., 2003; Gupta et al., 2006), both released during the

The increase in inflammatory markers in the maternal circulation could result from the release of substances from the placenta (local inflammation), then triggering a systemically inflammatory response. Some authors have suggested that tissue hypoxia resulting from

(Tastekin et al., 2005; Braekke et al., 2006).

**3.5 Inflammation** 

normotensive pregnant women.

growth restriction (Tjoa et al., 2003).

women (Kim et al., 2004; Chavarrνa et al., 2008).

degranulation of neutrophils in the inflammatory process.

birth weight (Poston et al., 2006; Rahimi et al., 2009).

As already mentioned, different hemostatic modifications are recognized in normal and in PEc pregnancy; however, the exact pattern of these changes in the fetus is still poorly understood. Some authors have reported a decrease in fibrinogen levels, but there were no differences in tPA, PAI-1 and D-dimer (Zanardo et al., 2005), while others reported an increase in PAI-1, however with no differences in tPA values (Roes et al., 2002). Higgins et al. (2000) suggest that infants are somehow protected, at hemostatic system level, since no differences in D-dimers were observed in newborns whose mothers developed PE (Higgins et al., 2000). In a study performed by our group (Catarino et al., 2008b), we also observed similar values of D-dimers. However, and similarly to what we observed in PEc mothers, significantly increased tPA values were found in the newborns of these women (Fig. 3B). Our results suggest that these changes do not arise in response to activation of coagulation, but as a result of endothelial cell dysfunction. Furthermore, we observed a relationship between placental dysfunction and endothelial dysfunction in fetal circulation in PE, suggested by the positive significant correlation that we identified between the levels of tPA and sVEGFR-1 in umbilical cord blood (Catarino et al., 2009a).

Fig. 3. Maternal (A) and fetal (B) tPA levels in PE and normal pregnancy. (Adapted from Catarino et al., 2008b)

#### **3.4 Oxidative stress**

In PE there is evidence of oxidative stress that results from an increased production of oxidizing agents that is not counteracted by antioxidant activity. In fact, an increase in reactive oxygen species, namely in superoxide anion, and a decrease in superoxide dismutase (SOD) activity is observed in trophoblast cells from PEc women (Wang & Walsh, 2001). Different studies demonstrate the development of oxidative stress in PE (Raijmakers et al., 2004; Roberts & Gammil, 2005); either by decreased concentrations of antioxidants, or indirectly by increased lipid peroxidation in maternal circulation, that results from the action of oxygen metabolites, provided by leukocyte activation and/or increased cellular metabolism. The oxidative stress can also take place at the placenta, as a result of the hypoxia/reoxygenation mechanism (intermittent placental perfusion) observed in placentas of pregnant women with PE (Hung & Burton, 2006).

The preterm infants have a lower antioxidant capacity and are therefore more susceptible to oxidative stress triggered at birth, which appears to be associated with complications, including retinopathy and bronchopulmonary dysplasia (Saugstad, 2003). However, there are conflicting results considering oxidative stress in newborns. Some studies describe an increase of lipid peroxidation (MDA), followed by decreased total antioxidant capacity and ascorbic acid levels in newborns whose mothers developed PE (Mehendale et al., 2008; Howlader et al., 2009), while other studies report no difference in lipid peroxidation (Tastekin et al., 2005; Braekke et al., 2006).

Experimental studies (Poston et al., 2006; Rahimi et al., 2009; Xu et al., 2010), performed in pregnant women at risk, showed no beneficial effect on the antioxidant consumption such as vitamins E and C, in preventing the development of PE, despite the evidence for the involvement of a state of oxidative stress in PE. In fact, according to these trials, antioxidant therapy does not appear to be sufficient to prevent the development of the PE, further increasing the risk of neonatal complications, including increased rate of newborns with low birth weight (Poston et al., 2006; Rahimi et al., 2009).
