Screening test for preeclampsia through assessment of uteroplacental blood flow and biochemical markers of oxidative stress and endothelial dysfunction

American Journal of Obstetrics and Gynecology (2005) 193, 1486–91

www.ajog.org

Screening test for preeclampsia through assessment of uteroplacental blood flow and biochemical markers of oxidative stress and endothelial dysfunction Mauro Parra, MD,a,* Ramo´n Rodrigo, MSc,b Pilar Barja,b Cleofina Bosco,b Virginia Ferna´ndez, PhD,b Herna´n Mun˜oz, MD,a Emiliano Soto-Chaco´n, MDa Fetal Medicine Unit, Hospital Clı´nico Universidad de Chile,a Institute of Biomedical Sciences, Faculty of Medicine, University of Chile,b Santiago, Chile Received for publication September 23, 2004; revised February 19, 2005; accepted February 22, 2005

KEY WORDS Preeclampsia Doppler Screening Biochemical markers

Objective: This study was undertaken to evaluate whether screening through a uterine artery (UtA) Doppler and biochemical markers of oxidative stress and endothelial dysfunction predict preeclampsia. Study design: UtA Doppler was performed at 11 to 14 and 22 to 25 weeks on 1447 asymptomatic pregnant women. Oxidative stress, endothelial dysfunction, and antiangiogenic state were assessed in women who later developed preeclampsia and normotensive controls. Results: There was a significantly increased of UtA pulsatility index (PI), plasma levels of soluble fms-like tyrosine kinase 1 (sFlt1), PAI-1/PAI-2 ratio, and F-2 isoprostane in women who subsequently developed preeclampsia compared with control pregnancies. Multivariate logistic regression showed that increased UtA PI performed at 23 weeks was the best predictor for preeclampsia. Conclusion: This study demonstrates early changes in markers of impaired placentation, antiangiogenic state, oxidative stress, and endothelial dysfunction suggesting that these derangements may play a role in the pathogenesis of preeclampsia. Our data point to UtA as the best test to predict preeclampsia at 23 weeks of gestation. Ó 2005 Mosby, Inc. All rights reserved.

Preeclampsia (PE) is a systemic disorder of pregnancy characterized by maternal hypertension, proteinuria, edema, causing fetal growth restriction, and premature delivery.1 Supported by the Fondo Nacional de Ciencia y Tecnologı´ a (FONDECYT), grant number 1020080. * Reprint requests: Mauro Parra, MD, Unidad Medicina Fetal, Hospital Clı´ nico Universidad de Chile, Santos Dumont 999, Independencia, Santiago, Chile. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2005 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2005.02.109

Although during the last years there has been a significant improvement in clarifying the cause of PE, the underlying pathogenic mechanisms of this disease remain elusive. In normal pregnancy, progressive trophoblast invasion transforms the high resistance uteroplacental spiral arteries into a low-resistance circulation. Histologic studies have shown that the incomplete process of spiral artery vascular transformation occurring in pregnancies affected by PE results in elevated resistance to flow in

Parra et al the maternal uterine arteries,2 which can be detected noninvasively by the use of Doppler ultrasound.3 PE is therefore associated with relative placental underperfusion, which may produce unknown substance(s) leading to endothelial dysfunction.4 Several candidates have been proposed as the elusive substance synthesized by the placenta. Accordingly, markers of lipid peroxidation, including malondialdehyde and 8-epiprostaglandinF2a are increased in the plasma of women with PE5 and the low concentrations of water-soluble and lipidsoluble antioxidants in the plasma and the placenta further suggest a state of oxidative stress.6 Thus, free radicals have emerged as likely promoters of maternal vascular malfunction.7 Reactive oxygen species, particularly super oxide anions, evoke endothelial-cell activation through many pathways.8 Recently, it has been shown that sera from patients who later develop PE have increased levels of soluble fms-like tyrosine kinase 1 (sFlt-1),9 a circulating antiangiogenic protein adhering to the receptor-binding domains of placental growth factor (PlGF) and vascular endothelial growth factor (VEGF), thereby leading to endothelial dysfunction.10 However, further prospective studies are still lacking. Although most of the clinical pathologic findings of PE may be attributed to functional impairment of plasma factors that regulate the endothelial function,11 it is not clear whether endothelial dysfunction is a cause or consequence of the disease.12 The aim of this study was to evaluate whether systematic screening of pregnant women through a uterine artery (UtA) Doppler and maternal serum biochemical markers of antiangiogenic processes, oxidative stress, and endothelial dysfunction predict efficiently PE and placental dysfunction and thereby to gain insight into the mechanism of the disease.

Material and methods Participants and study design This is a nested case-control study within a Chileanfunded project for screening and prevention of PE using antioxidant vitamins, and involved 1447 asymptomatic pregnant women scanned routinely during the first or second trimester of pregnancy from April 2002 to June 2003 with local ethics committee approval from the University of Chile Hospital. Blood samples were drawn from 82% of patients and outcome was available in 80% of them, so we end up with 949 asymptomatic pregnant women. To select the subject and control groups for UtA Doppler and biochemical study we excluded 77 who had missing data on maternal or perinatal outcome, 4 whose pregnancy ended before 20 weeks, 5 who had stillbirth, 21 pregnancy-induced hypertension, 23 with major fetal

1487 abnormalities, 50 with small for gestational age (SGA) infants, 13 women with chronic hypertension, 13 with other placental or chronic diseases, leaving 743 women. Of these women, 33 women had PE, whereas 710 remained normotensive throughout pregnancy. From the latter, we randomly selected the following 4 women after the index case either at the first or second trimester scans, being 137 women and becoming the control group for the biochemical and UtA Doppler assessments.

Procedures Transvaginal UtA color Doppler was performed at 11 to 14 weeks’ and 22 to 25 weeks’ gestation as indicated in previous publications.13,14 The mean pulsatility index (PI) was calculated from 3 consecutive waveform readings. We also recorded maternal age, ethnic group, smoking status, parity, and body mass index. Normal pregnancy was defined as a pregnancy in which the mother had normal blood pressure (% 140/90 mm Hg), absence of proteinuria, and no medical complications. Mild PE was defined as a maternal blood pressure of 140/90 mm Hg or greater with proteinuria (300 mg/24 hours). Severe PE was defined as a maternal blood pressure of 160/110 mm Hg or greater with proteinuria (5 g/24 hours), and resolution of hypertension and proteinuria after delivery. SGA was defined as neonatal weight below 10th percentile for our population.15 UtA Doppler mean PI above 95th percentile for 11 to 14 weeks and 22 to 25 weeks of gestation was defined on the basis of our own data (not published), being 2.43 (n = 2357) and 1.54 (n = 4007), respectively. Healthy, normotensive pregnant women were included as controls. Blood samples were collected at the time of routine ultrasound scan during the first and/or second trimester of pregnancies into plastic tubes containing either 5% EDTA or 0.106 mol/L citrate as anticoagulants for the measurement of endothelial dysfunction or oxidative stress parameters, respectively. After centrifugation, plasma was quickly stored in deep freezer (–84(C). For the purposes of F2-isoprostanes sampling, the plastic tubes were previously treated with butylated hydroxytoluene (final concentration 1 mmol/L) as antioxidant.

Assessment of oxidative stress The total antioxidant capacity of plasma was analyzed with respect to their ability to reduce the ferric iron (FRAP, ferric reducing ability of plasma).16 Also, plasma uric acid levels were determined to assess its contribution to the reducing ability of plasma.

Lipid peroxidation and protein carbonylation The assay for products of lipid peroxides was performed on plasma. Free F2-isoprostane, products of non enzymatic

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Table I Baseline characteristics of women who later develop PE, control group, and all population at enrollment and characteristics of their infants Characteristics

All population (n = 922)

Control (n = 137)

PE (n = 33)

Maternal age (y) Smokers (n %) Body mass index (kg/m2, IQR) Primigravida (n %) Gestational age at enrollment (wk, IQR) Gestational age at delivery (wk, IQR) Newborn weight (g, IQR) Delivery at !37 wk (n %) SGA !10th percentile (n %)

29.2 G 0.2 39 (4.3) 24.0 (22.0-27.0) 331 (35.9) 22.0 (13.0-23.0) 39.0 (38.0-40.0) 3360 (3050-3700) 69 (7.3) 60 (6.5)

30.6 G 0.6 7 (5.1) 24.0 (22.0-27.0) 37 (27) 22 (14.0-23.0) 39.0 (38.0-40.0) 3410 (3188-3732) 7 (5.1) (-)

29.8 G 1.1 1 (3) 24.2 (22.5-25.9) 17 (51.5)* 23 (22.0-23.2) 36.6 (29.1-38.7)* 2320 (1035-3152)* 15 (45.5)* 20 (60.1)*

IQR, Interquartile range (25-75). * P ! .05 compared with control and/or all population. Plus-minus values are mean G SEM.

peroxidation of arachidonic acid, were measured in plasma by enzyme immunoassay (EIA)17 by using 8isoprostane kits (Cayman, Ann Arbor, MI) and a Sunrise microplate reader (Tekan, Salzburg, Austria). The assessment of protein oxidation was performed in plasma by the spectrophotometric determination of carbonyl content, based on the reaction of 2,4dinitrophenylhydrazine with protein carbonyls.18

Antioxidant enzymes Homogenates of the red blood cells in 0.25 mol/L sucrose were used for the determination of the antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), and gluthatione peroxidase (GSH-Px), according to procedures published elsewhere.19

Assessment of endothelial dysfunction Plasma von Willebrand factor (vWF), plasminogen activator inhibitor type-1 (PAI-1), PAI-2 and thrombomodulin (TM) were determined by the use of IMUBIND enzyme-linked immunosorbent assay (ELISA) kit (American Diagnostica Inc, Stamford, Conn).

Assessment of angiogenesis-related factors The determination of circulating sFlt1, human PlGF, and human VEGF were performed with the use of ELISAs commercial kits (R&D Systems, Milford, Iowa).10

Statistical analyses Shapiro-Wilk test was used to assess the normality of continuous data. Results are given as means G SEM, or median (25-75 percentile intervals) when the data were not normally distributed. Student t test or MannWhitney test was used if the population was normally or not normally distributed, respectively. Categorical variables were compared with a c2 test. Univariate,

linear, and multivariate regression analyses were performed if appropriate. A difference was considered statistically significant when P ! .05.

Results Characteristics of patients Both groups were similar regarding gestational age, body mass index and maternal age, but there was a significant different in newborn weight, primiparous, gestational age at delivery, preterm birth, and SGA less than the 10th percentile as we expected to see on PE patients compared with normotensive group (Table I). Of the 33 women with PE, 15 were associated with fetal growth restriction (FGR) and 33% had severe PE, including 3 with HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome.

Role of UtA Doppler during the first and second trimester of pregnancy Mean PI of the UtA Doppler that was measured either at first or second trimester was significantly higher in women who subsequently developed PE compared to controls (Figure 1).

Assessment of biochemical markers 11 to 14 weeks’ gestation None of the biochemical markers measured during the first trimester was significantly different in patients who subsequently developed PE from the control group (Table II). 22 to 25 weeks’ gestation Endothelial dysfunction: Table III shows a comparison of biochemical markers of endothelial dysfunction (PAI-1/ PAI-2 ratio, thrombomodulin [TM], and von Willebrand factor [vWF]) between control group and

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1489 Table III Endothelial dysfunction and oxidative stressrelated parameters in plasma and erythrocytes of women enrolled at 22 to 25 weeks who subsequently develop PE and controls Control (n = 100)

Figure 1 UtA mean PI at the first and second trimester of pregnancy in women who subsequently develop PE and control group. Values are means G SEM. *P ! .05 vs control. PE groups at each gestation were compared with normotensive control group.

Table II Biochemical markers at 11 to 14 weeks of gestation in women who later develop PE and control

F2-isoprostanes (pg/mL) FRAP (mmol/L) PAI-1/PAI-2 ratio sFlt1 (pg/mL) PlGF (pg/mL)

Control (n = 37)

PE (n = 8)

17.7 (13.5-25.1) 338.7 G 19.3 1.54 (1.04-1.79) 546.2 G 45.1 45.7 G 5.2

16.1 (10.5-20.6) 325.4 G 22.7 1.05 (0.74-2.09) 379.6 G 80.5 42.5 G 3.2

Oxidative stress parameters: FRAP (mmol/L) 305.9 G 9.2 Uric acid (mg/dL) 3.08 (2.7-3.8) Carbonyl 0.68 (nmoles/mg prot) (0.45-1.19) F2-isoprostane 24.9 G 1.2 (pg/mL) Endothelial dysfunction parameters: PAI-1/PAI-2 ratio 1.36 (1.17-1.69) TM (ng/mL) 35.5 G 1.7 vWF (IU/mL) 1.84 (1.57-1.99) Antioxidant enzymes in erythrocytes: CAT activity 125.1 (k/mg Hb) (97.9-139.5) SOD activity 719.2 G 25.4 (k/mg Hb) GSH-Px activity 1690.1 G 99.8 (k/mg Hb)

PE (n = 26) 296.5 G 25.7 3.43 (2.8-4.3) 0.58 (0.42-0.83) 32.5 G 3.2*

1.65 (1.33-2.05)* 37.3 G 2.6 1.88 (1.74-1.96) 122.2 (107.3-145.8) 748.5 G 46.3 1833.9 G 158.3

Values are means G SEM and median (IQR 25-75). * P ! .05.

in the production of protein carbonylation, FRAP and uric acid plasma levels. Also, there were no significant changes in the activity of CAT, SOD, and GSH-Px of erythrocytes (Table III).

UtA Doppler and biochemical indexes as predictors of PE

Values are mean G SEM and median G IQR (25-75).

preeclamptic women assessed at 23 weeks’ gestation. PAI1/PAI-2 ratio of preeclamptic patients was significantly higher than control values (P ! .05), but there were no significant differences between TM and vWF levels.

sFlt1 and free PlGF plasma levels In preeclamptic pregnancies there were higher plasma levels of sFlt1 and lower levels of free PlGF than control groups (Figure 2). Furthermore, in preeclamptic patients sFlt1 was negatively correlated with PlGF (R2= 0.61; P ! .001), gestational age at delivery (R2= 0.53, P ! .05), and newborn size (R2= 0.21, P ! .05).

Oxidative stress assessment Compared with controls, F2-isoprostane levels of preeclamptic patients were 22.8% higher, without changes

The OR (CI G 95) for the prediction of PE using univariate analyzes showed 4 parameters during the second trimester of pregnancies that were identified as potential predictors. Those parameters were the UtA mean PI (110.8 [15.1-817.5], P ! .001), PAI-1/PAI-2 ratio (2.51 [1.11-5.68], P ! .03), F2-isoprostane (1.04 [1.003-1.08], P ! .034), and free PlGF (0.98 [0.96-0.99], P ! .045). sFlt-1 (1.03 [1.00- 1.01], P = .082) was just marginally not significantly different between the groups. Furthermore, none of the parameters assessed during the first trimester was significantly associated with PE, although UtA Doppler at that stage was almost able to predict this condition (5.48 [0.97-31.15], P ! .055). A multivariate analysis shows that UtA Doppler is the best predictor of PE and none of biochemical markers significantly improved its capacity to screen for this condition. With the use of a 95th percentile of the mean PI as a threshold value during the first and

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Parra et al Table IV Screening characteristics of a mean PI above 95th percentile at 12 and 23 weeks of gestation Characteristic

Sensitivity (%)

All preeclamptic patients Doppler 25 11-14 wk Doppler 48.4 22-25 wk

Specificity (%)

PPV (%)

NPV (%)

LR (C)

95.2

10.3

98.3

5.3

95.8

24.6

98.5

11.5

6.8

99.8

13.5

18.0

99.8

18.8

Preeclamptic patients delivered !35 wk Doppler 66.6 95.1 11-14 wk Doppler 84.6 95.5 22-25 wk

All population was used to calculate these parameters; prevalence of PE is 3.7%. PPV, Positive predictive value; NPV, negative predictive value; LR, likelihood ratio.

Figure 2 Concentration of sFlt1 and PlGF in plasma from women enrolled at 22 to 25 weeks who later had PE and control group. Values are means G SEM. *P ! .05 vs control.

second trimester, the sensitivity, specificity, positive and negative predictive value, and likelihood ratio for PE were determined (Table IV).

Comment The current study confirms some interesting findings and adds new insight to the etiopathogenesis and prediction of PE. First, our data confirm that impaired placentation and endothelial dysfunction are the main features of preeclamptic syndromes. Second, women who later developed PE not only had increased UtA mean PI during the first and second trimester but they were also associated with increased PAI-1/PAI-2 ratio, sFlt-1, F2-isoprostane, and reduced free PlGF at 23 weeks of gestation. These data suggest that impaired placentation, antiangiogenic mediators, endothelial dysfunction, and oxidative stress predated the development of PE by several weeks. And third, although the biochemical markers were significantly increased during the second trimester, they were not during the first trimester, indicating that UtA Doppler remains the best predictor of PE, especially in the severe cases. The very high likelihood ratio of PE in the screenpositive group confirms the reliability of UtA Doppler in diagnosing impaired placentation, and the findings are compatible with those of other previous screening studies during the first13 and second trimester.14,20,21 The maternal plasma concentration of PAI-1 and PAI-2 increases with gestation in normal pregnancy,

although in preeclamptic women PAI-1/PAI-2 ratio increases because of maternal endothelial cell activation and placental insufficiency.22 Our finding that PAI-1/ PAI-2 ratio is significantly increased in women who later develop PE is in agreement with other authors23 and with the hypothesis that endothelial dysfunction is the main feature of this condition and predates its onset.4,11 Increased plasma concentration of 8-epi-prostaglandin F2a supports the hypothesis that poor uteroplacental perfusion predisposes to an increase in placental free-radical synthesis and, thereby, to maternal oxidative stress. Our results are in agreement with Chappel et al23 who showed that this marker of lipid peroxidation was increased in women who later had PE. Furthermore, the increased circulating antiangiogenic factor (sFlt1) and reduced free PlGF in women who subsequently develop PE observed in our study are in agreement with a previous publication.9,10 As also shown by our data, Levine et al9 has demonstrated that alterations of the sFlt1 appeared to be greater in women who had early-onset PE and in women with PE who delivered a SGA infant, suggesting that defective angiogenesis may be especially important in these cases. In summary, this study has shown early and selective changes in markers of impaired placentation, oxidative stress, and endothelial dysfunction, which suggest that these may play a role in the mechanism of PE. However, UtA Doppler was shown to be the best predictor test of PE, and other abnormal biochemical profiles were also evident several weeks before the clinical onset of PE. Further prospective investigations are now required in a large cohort study to determine whether these biochemical variables may be applied with equal efficacy to all pregnant women, independent of previous risk assessment.

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Acknowledgments We thank all the staff in the Obstetric and Gynecology Department and the Fetal Medicine Unit at the University Hospital of Chile for help with the patient identification, drawn blood samples, and recruitment.

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