Background: Preeclampsia (PE) and gestational hypertension (GH) are associated with increased morbidity and mortality among mothers and neonates worldwide. Angiogenesis and oxidative stress (OS) are essential processes for normal pregnancy (NP) but can serve as a risk factor when levels are above normal. Normal pregnancy as well as hypertensive disorders of pregnancy may trigger release of reactive oxygen species (ROS) which damage lipid, proteins in the mothers’ placenta. This study assessed levels of angiogenic factors and oxidative stress biomarkers in newly diagnosed gestational hypertensive (GH) and preeclamptic (PE) women from prenatal to postpartum.

Methods: In a prospective study, a cohort of 150 pregnant women (50 PE, 50 GH and 50 normal pregnant women) who has registered to attend antenatal at the Komfo Anokye Teaching Hospital Obstetrics and Gynaecology department were followed. The levels of their placental growth factor (PlGF), soluble fms-like tyrosine kinase 1 (sFlt-1), 8-epi-prostaglandin F2alpha (8-epi-PGF2α) were estimated by ELISA and their total antioxidant capacity (T-AOC) were measured spectrophometrically at 22-26week, 27-31week, 32-36 week, 37-40week and 48 hours postpartum.

Results: Levels of sFlt-1, 8-epi-PGF2α and sFlt-1/PlGF increased from 22-26week, 27-31week to 32-36 week and reduced from 37-40week of gestation in all study group. A significant positive correlation (PIGF vs T-AOC; and sFlt-1 vs 8-epi-PGF2α) and a negative correlation (PlGF vs sFlt-1, sFlt-1 vs T-AOC, PlGF vs 8-epi-PGF2α, and T-AOC vs 8-epi-PGF2α) was observed in PE (p<0.0001) before and 48 hrs postpartum.

Conclusions: Imbalance in the levels of angiogenic factors and oxidative biomarkers was prominent among PE than GH and NP subjects. Therapeutic supplementation with proangiogenic and antioxidant molecules could ameliorate the effect of the imbalance and improve management outcomes among GH and PE subjects.

Owiredu WKBA, Sakyi SA, Anto EO, Turpin CA, Fondjo LA, et al. (2016) Interplay Between Angiogenic Factors and Oxidative Stress Biomark-ers in Normal Pregnancy, Gestational Hypertension and Preeclampsia Med J Obstet Gynecol 4(3): 1086.

•    Angiogenic factors
•    Oxidative stress
•    Preeclampsia
•    Gestational hypertension
•    Postpartum

Preeclampsia (PE) and gestational hypertension (GH) are associated with increased morbidity and mortality among mothers and neonates worldwide [1, 2]. The pathogenesis of PE is still unknown. However, endothelial dysfunction originating from a reduced placental perfusion has been implicated [3, 4], as well as increased inflammatory response, alterations in the renin-angiotensin-aldosterone axis, and cardiovascular diseases[5-7].

Angiogenesis and oxidative stress (OS) are essential processes for normal pregnancy (NP) but can serve as a risk factor when levels are above normal [8]. Soluble fms-like tyrosine kinase 1 (sFlt-1) has been implicated in preeclamptic pregnancy [4, 9]. Increased concentration of sFlt-1 is suggested to be linked with decreased placental growth factor (PlGF) in preeclamptic pregnancies [9-11]. Normal pregnancy as well as hypertensive disorders of pregnancy may trigger release of reactive oxygen species (ROS) which damage lipid, proteins and DNA in the mothers placenta, kidney and brains and later compromise the endothelium integrity [12, 13].

The dynamics and characterization of pregnancy varies immensely and the actual developmental stage where normal becomes abnormal remains uncertain. Changes in angiogenic factors and oxidative stress markers has been explored in normal pregnant women [8, 14] but little or no attention have been given to changes among women presenting with PE and GH from prenatal to post partum. Moreover, there is no data on the combination of angiogenic factors and oxidative stress biomarkers among normal pregnant women, GH and PE women in Ghana. Furthermore, postpartum levels of angiogenic factors and oxidative stress markers have shown conflicting results [10] and most of these studies did not adjust for confounding factors such as maternal age, early gestation body mass index, and parity. It is against this background that we hypothesized that pregnancy-related imbalance in angiogenesis and OS compromise the integrity of endothelium and cause it to malfunction. This study thus recruited normal pregnant women and newly diagnosed GH and PE subjects, measured their angiogenic and oxidative stress markers periodically from 22nd week to the 40th week and 48 hours postpartum for the first time in Ghana.

Study design/ setting

This prospective cohort study was carried out from April 2013 to November, 2014 at the Obstetrics and Gynaecology (O & G) department of the Komfo Anokye Teaching Hospital (KATH) in the Ashanti Region of Ghana. The Ashanti region has an average population of 4,780,380 (Ghana Statistical service, 2012). KATH is the second largest tertiary hospital in Ghana with a thousand (1000) bed capacity and serves as a major referral centre for the middle belt and northern part of Ghana.

Selection of participants

A total of 150 pregnant women (50 PE, 50 GH and 50 NP) who patronize antenatal services at the O&G department of KATH were followed. During their periodic visits, samples were collected at 22-26week, 27-31week, 32-36 week, 37- 40week and 48 hours postpartum period. During the 37-40th week, 9 subjects (6 NP, 1 GH, 2 PE) were lost to follow ups. The diagnosis of hypertensive disorders of pregnancy was done by qualified Obstetrician/Gynaecologist using the National High Blood Pressure Education Program Working Group diagnostic criteria (NBPEPWG, 2000). Information relating to obstetric and demographic characteristics were obtained from record reviews of hospital database and structured closed ended questionnaires. Preeclampsia was defined as hypertension (>140/90 mmHg) and proteinuria (> + 0.3 g/l) noticeable after 20th week of gestation. Gestational hypertension was defined as hypertension (>140/90 mmHg) occurring after 20 weeks of gestation without proteinuria.

Inclusion criteria and Exclusion criteria

Nulliparous and multiparous pregnant women aged 18-40 years, within the gestational age of ≥ 20 - 40 weeks with singleton pregnancies were included for this study. Participants previously diagnosed with chronic hypertension, heart disease, diabetes mellitus, renal disease, and are on antihypertensive prior to recruitment as well as those who were unable to give informed consent were excluded from the study.

BP measurements

Trained personnel used a mercury sphygmomanometer (Accoson, England) and a stethoscope to measure the blood pressure of participants in accordance with recommendations of the NBPEPWG, 2000. The procedure was duplicated for each patient with 5-10 minutes resting interval. Mean values of duplicate measurements were recorded as the blood pressure to the nearest 2.0 mmHg.

Urine sample collection and estimation of proteinuria

Participants provided 10-20 ml of freshly voided urine in clean leak proof containers. Proteinuria was measured using semi-quantitative colour scale on the urine reagent dipstick (URIT 2VPG Medical electronic Co., Ltd. China). Proteinuria was defined as the presence of urinary protein in concentrations ≥ 0.3g/l or 2+ on urine dipstick.

Blood sample collection and biochemical assay

10mls of venous blood sample was collected from each participant. Blood was dispensed into serum separator tubes and centrifuged (Nüve NF 200, Germany) at 7000 rpm for 15 min. Serum was aliquoted under sterile conditions and stored at −80°C (Thermo Scientific™ Revco™ UxF −Ultra-Low Temperature Freezers, USA) until assay.

Serum levels of sFlt-1, PIGF and 8-epi-PGF2α were measured in duplicate using commercially available ELISA kits from R&D System Inc. (Minneapolis, MN USA). The optical density was measured at 450 nm using microplate ELISA reader (Mindray MR-96A; Shenzhen Mindray Bio-medical electronics Co., Ltd, China). The plasma levels of each factor were calculated using standard curves derived from a known concentration of the respective recombinant factors.

Total antioxidant capacity (TAOC) reagents was obtained from Green stone Swiss Co., Ltd, China and serum levels were estimated spectrophotometrically (Mindray BA-88A; Shenzhen Bio-medical electronics Co., Ltd, China) at 593nm.This assay was measured based on the ferric reducing ability of plasma (FRAP) method as described by Benzie and Strain [15]. All samples were analyzed in triplicate.

Ethical consideration

Ethical approval for this study was granted by the Committee on Human Research, Publications and Ethics (CHRPE) (CHRPE/ AP/365/14), School of Medical Science, Kwame Nkrumah University of Science & Technology (KNUST) and the Research and Development Committee of the KATH. All procedures were also duly approved by the committee. Written informed consent in the form of signature or fingerprint was obtained from all the participants prior to enrolment.

Statistical analysis

Statistical analysis was performed using Graphpad Prism® version 5.0 (Graph Pad Software Inc., Los Angeles) for windows. Kruskal Wallis test followed by Dunnet test was used to compare more than two groups of non-parametric variables. Association between categorical variable were tested using Chi-square for trend. Spearman correlation was employed to test an association between angiogenic factors and oxidative biomarkers while partial correlation was used to assess the correlational effect after adjusting for maternal age, BMI, gestational age and parity. Statistical significance was accepted at p<0.05 for all comparisons.

Table I shows sociodemographic, obstetrics and clinical characteristics of study participants. The mean age of the general study participant was 29.78 years. Greater proportion (83.3 %) of the pregnant women were married whilst 16.7 % were singles. The percentage of married participants with PE (80.0 %) and GH (76.0 %) were significantly lower compared to the normal pregnant women (94.0 %) (p = 0.0179). Among the 55.3 % of the participant who had completed primary education, 72.0 % developed PE and 58.0 % had GH compared to 36.0 % among normal pregnant women (p = 0.0052). A higher percentage (42.7 %) of the participants were nulliparous whilst 35.3 and 22.0 % were multiparous and primiparous respectively. Majority of the participants were multigravida 59/150 (39.3 %) of which 52.0 % developed GH and 24.0 % had PE. Significantly higher proportion of preeclamptic participants than GH and NP had spontaneous abortion (64.0 % vs 44.0 % vs 42.0 %; p = 0.0288), family history of hypertension (34.0 % vs 8.0 % vs 2.0; p < 0.0001) and previous caesarean section (48.0 % vs 18.0 % vs 12.0 %; p < 0.0001). Participants with PE and GH had a significantly higher mean levels proteinuria (p<0.0001) and early-gestational BMI (p=0.0001) compared to normotensive pregnant women. PE women delivery at preterm compared to GH and NP (p=0.0151) (Table 1).

Figure 1 depicts the prospective changes in angiogenic factors at various gestational age of pregnancy. Within each group (NP, GH and PE) median levels of PlGF and PlGF/sFlt-1 increased up to 22-26 week decreased from 27-31 to 32-36 weeks and transiently increased from 37-40 week of gestation to 48 hour postpartum. Conversely, sFlt-1 and sFlt-1/PlGF ratio levels were significantly decreased up to 22-26 week but began to increases from 27-31week to 32-36 week and decreased from 37-40 week until 48 hours postpartum (PP). There was significant difference in median levels across the study groups (p<0.0001) (Figure 1).

Graphs are presented a scatter line plot of median (interquartile range). Throughout pregnancy women who developed PE showed significantly (p<0.0001) lower levels of PlGF and PlGF/sFlt-1 ratio and higher levels of sFlt-1 and sFlt-1/ PlGF ratio compared to GH and NP. Levels of PlGF and PlGF/sFlt-1 ratio increased at 48hrs PT while sFlt-1 and sFlt-1/PlGF ratio decreased at 48hr PP (NP>GH>PE). PP: Postpartum; NP: Normal pregnancy; GH: Gestational hypertension; PE: Preeclampsia. PIGF: Placental growth factor (PlGF), sFlt-1: soluble fms-like tyrosine kinase 1.

Figure 2 depicts the prospective changes in oxidative stress biomarkers at various gestational age of pregnancy. Within each group (NP, GH and PE) median levels T-AOC increased up to 22- 26 week decreased from 27-31 to 32-36 weeks and transiently increased from 37-40 week of gestation to 48 hour postpartum. Meanwhile, levels of 8-epi-PGF2α significantly decreased up to 22-26 week, increased from 27-31week to 32-36 week and decreased again from 37-40 week until 48 hours PP. There was significant difference in median levels across the study groups (Figure 2).

Graphs are presented scatter line plots of median (interquartile range). Throughout pregnancy women who developed PE showed significantly (pGH>PE) (p<0.0001). PP: Postpartum; NP: Normal pregnancy; GH: Gestational hypertension; PE: Preeclampsia. 8-epi-PGF2α: 8-epi-prostaglandin F2alpha; T-AOC: total antioxidant capacity

Correlation between angiogenic factors and oxidative stress markers before delivery are shown in (Table 2). A significant positive correlation was observed between PIGF and T-AOC (r=0.802; p<0.0001) and sFlt-1 and 8-epi-PGF2α (r=0.858; p<0.0001) in PE patients before delivery. Negative correlation was observed between PlGF and sFlt-1 (r= -0.804; p<0.0001), sFlt-1 and T-AOC (r= -0.844; p<0.0001), PlGF and 8-epi-PGF2α (r= -0.760; p<0.0001), T-AOC and 8-epi-PGF2α (r= -0.960; p<0.0001) in PE patients before delivery. Correlation between angiogenic factors and oxidative stress markers was significant after adjusting for maternal age, gestational age, early pregnancy BMI and parity (p<0.0001) (Table 2).

Correlation between angiogenic factors and oxidative stress markers 48hrs after delivery are shown in (Table 3). A significant positive correlation was observed between PIGF and T-AOC (r= 0.553; p=0.026) and sFlt-1 and 8-epi-PGF2α (r= 0.661; p=0.013) in postpartum PE patients. Negative correlation was observed between PlGF and sFlt-1 (r= -0.628; p=0.012), sFlt-1 and T-AOC (r= -0.517; p=0.021), PlGF and 8-epi-PGF2α (r= -0.593; p=0.017), T-AOC and 8-epi-PGF2α (r= -0.749; p=0.003) in postpartum PE patients. Correlation between angiogenic factors and oxidative stress markers was significant after adjusting for maternal age, gestational age, early pregnancy BMI and parity (p<0.05) (Table 3).

Table 1: Sociodemographic, obstetric and clinical characteristics of study participants.

This study evaluated levels of angiogenic factors and oxidative stress markers among Ghanaian women presenting with GH and PE from prenatal to postpartum. Findings of the present study showed a significantly lower level of PlGF and PIGF/sFlt-1 ratio and a corresponding increased concentration of sFlt-1 and sFlt-1/PlGF ratio in PE than in GH compared to normal pregnant women across the prospective gestational age (Figure 1). This in agreement with prospective studies by Yelumalai et al., [10] among Malaysian women and Noori et al., [9] among women visiting Chelsea and Westminster Hospital. Results of this study showed that increased and shift of the balance in favour of sFlt-1 were significantly associated with 32-36 week of gestation in all study group though PE were the high risk population. This findings are consistent with a prospective study by Yelumalai et al., [10] among Malaysia women who observed a significantly elevated sFlt-1 and reduced PlGF at 32-36 week of gestation. Hertig and colleagues in previous prospective study also found that levels of sFlt-1 peaks significantly in third trimester [16]. However, in another study by Palm et al., [8] among normal pregnant women in Sweden indicated that sFlt-1 peak from the onset of PE until term, which is not consistent with this current study finding. This disparity could be buttressed by the fact that levels of angiogenic factors markers fluctuate throughout pregnancy making it difficult to identify the actual gestational age associated with increased or decreased [8]. However, the increased levels of sFlt-1 and sFlt-1/PlGF ratio observed in PE and GH could be explained by the increased evidence of hypoxic placenta due to shallow trophoblasts invasion and reduced placental perfusion [17]. The probable explanation to the reduced levels of PlGF and PlGF/sFlt-1 ratio is due to the increased sFlt-1 which may have antagonized the action of PlGF by adhering to the receptor binding domains of PIGF and thus prevent it interaction with the endothelial cell surface receptors [10].

This study also observed that 48 hrs postpartum levels sFlt-1 were significantly reduced and with correspondingly elevated levels of PIGF in favour of normal pregnancies than in GH compared to PE is consistent with prospective study by Noori et al., [9] and Yelumalai et al., [10] . The reduced postpartum levels of sFlt-1 may be compensatory as the syncytiotrophoblast may be producing more PlGF and less sFlt-1 and thus enabling its binding to ligand receptors on the endothelium [8]. In this study postpartum levels of sFlt-1 and PIGF were statistically significant different compared to the prospective gestational levels indicating that postpartum levels did to return to normal. This finding agree with Noori et al., [9] but not Yelumalai et al., [10]. The disparity in findings could be due the different stages of postpartum sampling. As Yelumalai and colleagues measured 6 week postpartum levels of angiogenic factors, this current study measured 48 hrs postpartum levels. Our finding support the hypothesis that endothelial dysfunction can last for days after the episode of complicated hypertensive pregnancy especially preeclampsia [18].

Again this study observed an increased lipid peroxidation activity as depicted by an increased serum level of 8-epi-PGF2α and a reduced anti-oxidant system (T-AOC) with significant effect associated with PE than in GH compared to NP. This is in agreement with earlier studies [12, 14]. Most studies [19, 20] measured malondialdehyde (MDA) which is unstable and affected by several factors. The current study used 8-epi-PGF2α which is considered a potent and stable lipid peroxidation marker responsible for abnormalities such as hypertension, endothelial cell dysfunction, renal vasoconstriction, placental vasoconstriction, and cerebral vasospasm of eclampsia [12, 21] and thus could be responsible for the elevated levels observed in hypertensive pregnancies. The significantly reduced levels of T-AOC in PE is a further impetus to the increased lipid peroxidation which indicates that preeclamptics pregnancies suffer a huge compromised antioxidant system subsequent to a widespread endothelial dysfunction compared to GH and NP.

Despite the scarcity of prospective data on oxidative stress biomarkers in hypertensive pregnancies, we observed a transient rise in oxidative stress from the 22-26 week to 27-31week and a peak rise at the 32-36 week of gestation in the pattern of PE>GH>NP (Figure 2). This finding is consistent with prospective study by Hung et al., [22] among Chinese pregnant women who reported an increased imbalance in OS at third trimester of pregnancy. Clinical onset of PE usually arise in the third trimester of pregnancy long after initiation of the underlying process could explain this findings [16]. Although delivery of the placenta and the baby remains the curative remedy to hypertensive pregnancies, levels of T-AOC did not return to normal reference limit in normal pregnancies and this was worsen in GH and PE. This latter finding could be explained that phagocytosis of placental debris by endothelial cells after delivery may have activated the endothelium and cause release of reactive oxygen species and thus the persistent postpartum OS [23].

Another finding of this study is the significantly negative correlation between sFlt-1 and PIGF which have been previously studied by some authors [24, 25]. However, this study is the first to report a significant association of 8-epiPGF2α with sFlt-1, and PlGF and that of TOAC with PlGF and sFlt-1 (Table 2). The positive correlation between sFlt-1 and 8-epi-PGF2α and that between TAOC and PlGF buttresses the earlier findings (Figure 1 and Figure 2) which indicated that significantly elevated levels of both sFlt-1 and 8-epiPGF2α is associated with a subsequent reduced concentration of PlGF and TAOC. This indicates that anti-angiogenic factors and lipid peroxidation markers play a synergistic role in the pathogenesis of hypertensive pregnancy. A further strength of this association was observed after adjusting for the confounding effects of maternal age, early-pregnancy BMI, gestational age and parity indicating that the role angiogenic factors and oxidative stress biomarkers in the pathogenesis of hypertensive pregnancy cannot be excluded. In this study correlation picture between angiogenic factors and oxidative stress biomarkers before delivery was similar to 48 hrs postpartum in PE (Table 3). The probable explanation to postpartum imbalance could be persistent PE. The PE after delivery did not resolve totally and this might be a picture of postpartum PE. The major limitation was that this study did not evaluate levels of angiogenic factors and oxidative stress biomarkers in first trimester of pregnancy with the possibility of identifying women who are destined to develop PE, GH and adverse pregnancy outcomes. Further prospective study is needed to explore the predictive accuracy of these markers in the first trimester pregnancy.

Table 2: Spearman rho and partial correlation between angiogenic and oxidative stress biomarkers at all gestational cohort of study participants before delivery.

Table 3: Spearman rho and partial correlation between angiogenic and oxidative stress biomarkers at all gestational cohort of study participants 48 hrs after delivery

Imbalances in angiogenic factors and oxidative stress biomarkers increases the progression of PE with clinical effect in the third trimester of pregnancy. The role of reactive oxygen specie signalling in pregnancy is synergic to angiogenesis in the placental development. Pharmacologic remedies of exogenous proangiogenic molecules and antioxidant supplements and or inhibiting the action of anti-angiogenic molecules could provide inventive approaches to the management of GH and PE and potentially alleviate the adverse complications suffered by these patients.

The authors would like to acknowledge the Department of O&G, KATH and Department of Molecular medicine for endorsing this study. We also wish to thank the Midwife for their immense support during sample collection. We also would like to thank Mr. Peter Brenyah and Mr. Albert Dompere at the Medilab diagnostic centre and Immunology department KATH, Kumasi respectively, for their support during the storage of samples and biochemical analysis. We wish to thank the study participants.

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