Early Prediction of Preeclampsia in High-Risk Women

Abstract

Background:

The purpose of this study was to determine the role of the combined use of uterine artery Doppler velocimetry (UADV) and maternal serum placental growth factor (PlGF), vascular endothelial growth factor receptor-1 soluble fms-like tyrosine kinase-1 also called soluble (sVEGFR-1), and nitric oxide (NO) products concentrations for the prediction of preeclampsia in high-risk women and to compare these parameters between patients with mild and severe preeclampsia.

Methods:

Patients at risk of preeclampsia (n=112) were subclassified as having either severe (n=38), mild (n=17), or no preeclampsia (n=57). Blood samples were obtained between 22 and 26 weeks of gestation. Doppler ultrasound of the uterine arteries was done at the time of blood sampling. Maternal serum PlGF and sVEGFR-1 concentrations were determined with enzyme-linked immunosorbent assay (ELISA). Nitric oxide colorimetric assay was used also to measure NO products in the maternal blood.

Results:

Among patients with abnormal UADV, maternal serum sVEGFR-1, PlGF, and NO product concentrations contributed significantly in the identification of patients destined to develop mild and severe preeclampsia. sVEGFR-1 (pg/mL) concentration followed by NO product concentration (μmol/L) were found to be the best predictors for preeclampsia, with high sensitivity and specificity, followed by PlGF (pg/mL).

Conclusions:

Abnormal UADV and high concentrations of sVEGFR1 combined with low concentrations of PlGF and NO products may be used to predict the development of preeclampsia.

Introduction

Preeclampsia, a pregnancy-specific syndrome characterized by new-onset hypertension and proteinuria, is a considerable obstetrical problem and a significant source of maternal and neonatal morbidity and mortality.¹ It has been recognized that women who endure preeclampsia are at a greater risk than nonpreeclamptic women for cardiovascular disease (CVD).² Although the pathophysiology of preeclampsia remains undefined, placental ischemia/hypoxia is widely regarded as a key factor.³ Inadequate trophoblast invasion leading to incomplete remodeling of the uterine spiral arteries is considered to be a primary cause of placental ischemia.⁴ The poorly perfused and hypoxic placenta is thought to synthesize and release increased amounts of such vasoactive factors as soluble fms-like tyrosine kinase 1 (sFlt-1) (also called soluble vascular endothelial growth factor receptor-(sVEGFR-1)), cytokines, and possibly the angiotensin II (ANG II) type 1 receptor autoantibodies (AT₁-AA).^4
–6

Several lines of evidence support the hypothesis that the ischemic placenta contributes to endothelial cell dysfunction in the maternal vasculature by inducing an alteration in the balance of circulating levels of angiogenic/antiangiogenic factors, such as vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and sVEGFR-1.^7

–10 Data suggest that circulating sVEGFR-1concentrations may presage the clinical onset of preeclamptic symptoms.^9,11,12 VEGF is primarily recognized for its potent angiogenic and mitogenic effects on endothelial cells. It exerts its actions mainly by two receptors, VEGFR-1 and VEGFR-2 (also known as Flt-1) and the kinase domain region (Flk/KDR), respectively.¹³ A soluble and endogenously secreted form of VEGFR-1 is produced mainly in the placenta by alternative splicing and contains the extracellular ligand-binding domain but not the transmembrane and cytoplasmic portions.¹⁴

sVEGFR-1, a circulating antiangiogenic protein that sequesters the proangiogenic proteins PlGF and VEGF, is increased before the onset of clinical disease in the circulation of women with preeclampsia.¹⁵ sVEGFR-1 disrupts VEGF signaling either by binding VEGF and PlGF or by forming heterodimers with the KDR receptor.¹⁶ Although sVEGFR-1 is not a vasoconstrictor, it does significantly inhibit the dilatory actions of both VEGF and PlGF in vitro, and chronic elevations in circulating concentrations cause increased blood pressure.^5,17 Preeclampsia is strongly linked to an imbalance between proangiogenic (VEGF and PlGF) and antiangiogenic (sVEGFR-1) factors in the maternal circulation.^5,9,10,18 It has been reported that increased sVEGFR-1 may have a predictive value in diagnosing preeclampsia because concentrations seem to increase before manifestation of overt symptoms (e.g., hypertension and proteinuria).^9,15

Substantial evidence indicates that nitric oxide (NO) production is elevated in normal pregnancy and that this increase appears to play an important role in the renal vasodilatation of pregnancy.¹⁹ NO synthase (NOS) inhibition in pregnant rats produces hypertension associated with renal vasoconstriction, proteinuria, intrauterine growth restriction, and increased fetal morbidity.^20,21

Abnormal uterine artery doppler velocimetry (UADV) between 22 and 25 weeks of gestation has been called best test for identification of patients destined to develop preeclampsia, compared with biochemical indicators in the maternal serum. Abnormal UADV results and abnormal maternal serum concentrations of proangiogenic and antiangiogenic factors are risk factors for the subsequent development of preeclampsia.^22,23

Currently, there is no widely accepted screening test for prediction of preeclampsia in individual women. The development of an accurate biomarker for preeclampsia in high-risk women has the potential to substantially improve care by allowing closer prenatal monitoring, recognition of preeclampsia earlier in the disease course, expeditious administration of steroids for fetal lung maturity, and appropriate antihypertensive therapy. The aim of this study was to determine the utility of maternal serum concentrations of the angiogenic factor PlGF and the antiangiogenic factor sVEGFR-1, in combination with UADV, for prediction of preeclampsia in the midtrimester of pregnancy.

Subjects and Methods

A prospective cohort study was conducted between April 2008 and January 2010. Patients were recruited from those attending the gynecology clinic at Kasr El Aini hospital, Cairo University. Inclusion criteria were pregnancy of <24 weeks' gestation at enrollment and at least one of the following risk factors for preeclampsia: pregestational diabetes mellitus, maternal age ≤18 years, systemic lupus erythematosus (SLE), or prior history of preeclampsia.

Preeclampsia was diagnosed and subdivided into either severe or mild according to published guidelines.²⁴ UADV was performed at the time of blood sampling. The presence of an early diastolic notch in the uterine arteries was determined according to the criteria proposed by Bower et al.²⁵ An abnormal UADV was defined as the presence of bilateral uterine artery notches or a mean pulsatility index of >95th percentile for the gestational age or both. The mean pulsatility index was calculated by measuring the pulsatility index of the right and left uterine arteries. Primary pregnancy outcome was the diagnosis of early onset preeclampsia or severe preeclampsia, and secondary outcomes included small for gestational age (SGA), premature delivery (PTD), and intrauterine growth retardation (IUGR).

Human PlGF and sVEGFR-1 assays

The concentrations of sVEGFR-1 were measured in all sera with an enzyme-linked immunosorbent assay (ELISA) (R&D Systems). The inter and intraassay coefficients of variation (CV) were 4.8% and 6.9%, respectively. The sensitivity of the assay was 3.5 pg/mL. A specific and sensitive ELISA was used to determine concentrations of PlGF in maternal serum (R&D Systems). The calculated interassay and intraassay CVs were 4.6% and 2.27%, respectively. The minimum detectable dose of PlGF is <7 pg/mL.

Nitrite assay

The nitric oxide colorimetric assay kit provides a convenient measure of total nitrate/nitrite in a simple two-step process. The first step converts nitrate to nitrite using nitrate reductase. The second step uses Griess reagents to convert nitrite to a deep purple azo compound. The amount of the azo chromophore accurately reflects the NO amount in samples. The detection limit of the assay is approximately 0.1 nmole nitrite/well, or 1 μM (Abcam).

Statistical analysis

The data were coded and entered using the statistical package SPSS version 12. The data were summarized using descriptive statistics: mean, standard deviation (SD), minimal and maximum values for continuous variables, and number and percentage for categorical values. Statistical differences between groups were tested using the chi-square test for categorical variables, analysis of variance (ANOVA) for continuous normally distributed variables, and the nonparametric Mann-Whitney test and Kruskal-Wallis test for not normally distributed continuous variables. Correlations were done to test for linear relations between continuous variables. Logistic regression analysis was done to test for significant predictors for preeclampsia. Receiver operator characteristic (ROC) curves were constructed to evaluate the predictive potential of each biomarker for preeclampsia occurrence and severity. p Values ≤0.05 were considered statistically significant.

Results

One hundred twelve pregnant women were enrolled in the study; 57 did not develop preeclampsia, and 55 developed preeclampsia (17 mild preeclampsia and 38 severe preeclampsia). All cases of preeclampsia had abnormal UADV.

Baseline characteristics and pregnancy outcomes are shown in Table 1. Subjects with mild and severe preeclampsia had a higher body mass index (BMI) (p1, p2<0.001) and increase in gestational systolic blood pressure (SBP) and diastolic blood pressure (DBP) (p1, p2<0.001) compared with those who did not develop preeclampsia (normotensive group). Also, gestational SBP and DBP were significantly higher in the severe group than in the mild group (p3<0.001). Patients with severe preeclampsia delivered at an earlier gestational age, had a higher mean arterial pressure, and delivered smaller infants than subjects in the control and mild groups. A significant difference in all other pregnancy outcome parameters (birth weight, placental weight, gestational age at delivery, mean pulsatile index, SGA, and IUGR) was found between the studied groups.

Table 1.

Characteristics and Pregnancy Outcomes of All Subjects

Characteristic	Normotensive subjects ( n=57)	Mild preeclampsia (n=17)	Severe preeclampsia (n=38)	p1 ^a	p2	p3
Maternal age	27.8±9.6^b	29.4±7.3	27.3±8.4	>0.05	>0.05	>0.05
Parity	1.77±0.88	1.76±0.83	1.73±0.89	>0.05	>0.05	>0.05
BMI (kg/m²)	25.0±5	31±4	29.0±4.0	<0.001^*	<0.001^*	>0.05
Gestational SBP (mm Hg)	104±13	143±6	170±16	<0.001^*	<0.001^*	<0.001^*
Gestational DBP(mm Hg)	66±9	94±4	109±13	<0.001^*	<0.001^*	<0.001^*
Birth weight (kg)	3.54±0.24	3.08±0.29	2.20±0.25	<0.001^*	<0.001^*	<0.001^*
Placental weight (g)	567.98±54.63	486.47±38.88	422.37±45.83	<0.001^*	<0.001^*	<0.001^*
Gestational age at delivery (weeks)	39.54±0.56	37.65±0.99	33.66±0.62	<0.001^*	<0.001^*	<0.001^*
Mean pulstile index	2.73±0.08	2.60±0.40	1.25±0.42	>0.05	<0.001^*	<0.001^*
SGA	0 (0%)^c	0 (0%)	6 (15.8%)	>0.05	<0.001^*	<0.001^*
IUGR	0 (0%)	1 (5.9%)	24 (63.2%)	<0.001^*	<0.001^*	<0.001^*

p1, between normotensive and mild preeclampsia groups; p2, between normotensive and severe preeclampsia groups; p3, between mild preeclampsia and severe preeclampsia groups.

mean±standard deviation (SD).

number (%).

Significant.

BMI, body mass index; DBP, diastolic blood pressure; IUGR, intrauterine growth retardation; SBP, systolic blood pressure; SGA, small for gestational age.

Table 2 shows a significant increase in mean sVEGFR-1 levels in subjects who developed mild and severe preeclampsia compared with those who did not develop preeclampsia (p1, p2<0.001), whereas no significant difference was found between the mild and severe groups (p3>0.05). The mean serum PlGF levels were significantly lower in the severe group compared with those without preeclampsia and the mild group (p2, p3<0.001). The mean levels of serum NO products were significantly decreased in both the mild and severe groups compared with the normotensive group (p1, p2<0.001), but no significant difference was found between the mild and severe groups (p3>0.05).

Table 2.

Measured Parameters Estimating Extent of Preeclampsia in All Studied Groups

Parameter/group	Normotensive	Mild preeclampsia	Severe preeclampsia	p value ^a
PlGF (pg/mL)	407.55±149.82^b	260.73±112.37	174.97±116.48	p1>0.05 p2<0.001^* p3<0.001^*
sVEGFR-1 (pg/mL)	1101.70±157.86	4707.05±2134.32	5184.21±1652.60	p1<0.001^* p2<0.001^* p3>0.05
NO products (μmol/L)	117.17±28.42	43.25±23.84	38.70±12.53	p1<0.001^* p2<0.001^* p3>0.05

p1, between normotensive and mild preeclampsia groups; p2, between normotensive and severe preeclampsia groups; p3, between mild preeclampsia and severe preeclampsia groups.

Mean±SD.

Significant.

NO, nitric oxide; PlGF, placental growth factor; sVEGFR-1, soluble vascular endothelial growth factor receptor-1.

We determined the ROC curve to examine the diagnostic performance of maternal serum PlGF, sVEGFR-1, and NO product concentrations in identifying patients destined to develop mild or severe preeclampsia. sVEGFR-1 (pg/mL) followed by NO product (μmol/L) concentration were found to be best for diagnosis of preeclampsia, with high sensitivity and specificity, followed by PlGF (pg/mL) concentrations (Tables 3 and 4). A significant correlation was observed between concentrations of sVEGFR-1, PlGF, and NO products and pregnancy outcome characteristics (Table 5).

Table 3.

Receiver Operator Characteristic Curves of Maternal Plasma Concentration of Placental Growth Factor, Soluble Vascular Endothelial Growth Factor Receptor-1, and Nitric Oxide Products for Identification of Patients Destined to Develop Mild Preeclampsia

Test result variable	Cutoff value	Sensitivity (%)	Specificity (%)	Area under the curve
PlGF (pg/mL)	≤286.32	80.00	80.70	0.854
sVEGFR1 (pg/mL)	≥2005.00	98.20	100.00	0.982
NO products (μmol/L)	≤50.90	90.90	100.00	0.985

Table 4.

Receiver Operator Characteristic Curves of the Maternal Plasma Concentration of Placental Growth Factor, Soluble Vascular Endothelial Growth Factor Receptor-1, and Nitric Oxide Products for Identification of Patients Destined to Develop Severe Preeclampsia

Test result variable	Cutoff value	Sensitivity (%)	Specificity (%)	Area under the curve
PLGF (pg/mL)	≤234.56	81.60	85.10	0.867
sVEGFR-1 (pg/mL)	≥2900.00	100.00	81.10	0.901
NO products (μmol/L)	≤54.00	81.60	79.70	0.878

Table 5.

Correlations Between Estimated Maternal Plasma Placental Growth Factor, Soluble Vascular Endothelial Growth Factor Receptor-1, and Nitric Oxide Products Versus Other Variables Among Studied Groups

Variable	PlGF(pg/mL)	sVEGFR-1 (pg/mL)	NO products (mmol/L)
BMI kg/m²	r=−0.24	r=0.30	r=−0.44
	p=0.009	p=0.001	p<0.001
SBP	r=−0.54	r=0.79	r=−0.76
	p≤0.001	p<0.001	p<0.001
DBP	r=−0.56	r=0.79	r=−0.72
	p≤0.001	p<0.001	p<0.001
Birth weight (kg)	r=0.54	r=−0.39	r=0.70
	p<0.001	p<0.001	p<0.001
Placental weight (g)	r=0.45	r=−0.27	r=0.68
	p<0.001	p=0.003	p<0.001
Gestational age at delivery (weeks)	r=0.58	r=−0.36	r=0.75
	p<0.001	p<0.001	p<0.001
Mean pulstile index	r=0.50	r=−0.41	r=0.62
	p<0.001	p<0.001	p<0.001
PlGF (pg/mL)		r=−0.47	r=0.47
		p<0.001	p<0.001
sVEGFR-1 (pg/mL)	r=−0.47		r=−0.73
	p<0.001		p<0.001
NO products (μmol/L)	r=0.47	r=−0.73
	p<0.001	p<0.001

Discussion

Considerable clinical evidence has accumulated that preeclampsia is strongly linked to an imbalance between proangiogenic (VEGF and PlGF) and antiangiogenic (sVEGFR-1) factors in the maternal circulation.⁹ Recent studies have reported that increased sVEGFR-1 may have a predictive value in diagnosing preeclampsia, as concentrations seem to increase before manifestation of overt symptoms (e.g., hypertension and proteinuria).²⁶

The present study showed a significant increase in mean maternal sVEGFR-1 levels in subjects who developed mild and severe preeclampsia compared with those who did not develop preeclampsia. The mean serum levels of maternal PlGF were significantly decreased. Our sVEGFR-1 and PlGF findings were consistent with results of the study by Moore Simas et al.²⁷ They found that mean sVEGFR-1 levels were significantly higher in subjects who developed preeclampsia before 34 weeks compared with those without preeclampsia. At the same time, they found that the mean PlGF levels tended to be lower for subjects who developed preeclampsia compared with those without preeclampsia. They studied the sVEGFR-1/PlGF ratio as an index of antiangiogenic activity that reflects changes in the balance between sVEGFR-1 and PlGF. They suggested that this ratio has been shown to be more strongly associated with preeclampsia than either measure alone in healthy women. They concluded that in high-risk women, serum sVEGFR-1 and the sVEGFR-1/PlGF ratio are altered before preeclampsia onset and may be predictive of preeclampsia.

In this study, ROC curves were constructed to describe the relationship between sensitivity and the false positive rate of serum PlGF, sVEGFR-1, and NO products in identifying patients destined to develop preeclampsia. sVEGFR-1 (pg/mL) and NO products (μmol/L) were found to be the best predictors for preeclampsia, with high sensitivity and specificity, followed by PlGF (pg/mL), whereas in severe preeclampsia, sVEGFR-1 was the best predictor, followed by NO products, then PlGF. Logistic regression analysis indicated that maternal serum concentrations of sVEGFR-1>2005 pg/mL, NO products<50.9 μmol/L and PLGF<286.3 pg/mL were independent variables for the occurrence of preeclampsia. Espinoza et al.²⁸ agree with these results in their study of identification of patients at risk for early onset or severe preeclampsia using UADV and PlGF. They stated that a maternal serum concentration of PlGF<280 pg/mL was an independent explanatory variable for the occurrence of preeclampsia, early onset preeclampsia, and severe preeclampsia. In contrast, maternal serum sVEGFR-1 concentration was of limited value in the prediction of early onset and severe preeclampsia.

These results are also consistent with a previous report by Stepan et al.,²³ indicating that a low maternal serum concentration of PlGF in the first or second trimester of pregnancy and abnormal UADV results between 23 and 25 weeks of gestation are risk factors for the development of preeclampsia. Muller et al.²⁹ differ with those results, however, indicating a lack of association between abnormal UADV and low PlGF. Differences in sample size, gestational age at ultrasound, and study outcomes may account for these discrepancies. Muy-Rivera et al.³⁰ examined the relationship of maternal serum VEGF, sVEGFR-1, and PlGF levels to the risk of preeclampsia among Zimbabwean women. They noted a strong positive association between preeclampsia risk and sVEGFR-1 concentrations. Meanwhile, there was no clear evidence of a linear relation in risk of preeclampsia with PlGF concentrations (maternal serum PlGF concentrations were similar in both cases and controls).

Our results of high concentrations of sVEGFR-1 in the serum of preeclamptic women are consistent with those reported by others.^15,31 It has also been shown that placentas from preeclamptic women produce higher concentrations of sVEGFR-1 in vitro as compared to controls.^32,33 Interestingly, the increase in sVEGFR-1 corresponds to a decrease in free VEGF and PlGF in the serum of patients with preeclampsia causing endothelial dysfunction.¹⁵ sVEGFR-1 is a major contributor to the pathogenesis of preeclampsia. It has been shown in animal models that administration of sVEGFR-1 induces hypertension, proteinuria, and glomerular endotheliosis in pregnant rats.⁵

Many researchers have spent years trying to find the cause of preeclampsia, but the mechanism remains elusive. It is possible that diminished maternal serum levels of PlGF and increased levels of sVEGFR-1 may contribute to increased vascular permeability. However, it is just as likely these serum proteins are markers for the disease and have no role in the mechanism. Christopher et al.³⁴ evaluated PlGF and sVEGFR-1 levels in mild and severe preeclampsia and stated that the serum PlGF level is lower in patients with severe preeclampsia compared with mild preeclampsia. Meanwhile, sVEGFR-1 levels are higher in patients with severe preeclampsia, although not statistically significantly, compared with women with mild preeclampsia. To determine if PlGF serum levels decrease and sVEGFR-1 levels increase as the disease progresses, an ideal study design would be to follow patients longitudinally. PlGF serum concentrations peak at 26–30 weeks and then decline as term approaches.³⁵ sVEGFR-1 has a stable concentration until 33–36 weeks and then increases about 145 pg/mL per week.¹⁵

This study showed significantly decreased serum levels of maternal NO products in mild and severe cases of preeclampsia, suggesting that it is a predictor for the occurrence of mild and severe preeclampsia, which is consistent with the results of Dikensoy et al.³⁶ They concluded that the serum level of NO is decreased and the malondialdehyde (MDA) level is increased in subjects with preeclampsia. They concluded that the previous parameters might contribute to the pathophysiology of preeclampsia through the lack of a paracrine vasodilatory effect on uteroplacental blood flow. Also, Seligman et al.³⁷ stated that circulating levels of nitrite are decreased in patients with preeclampsia. These data support the concept that diminished NO synthesis contributes to the pathophysiological changes seen in preeclampsia. Tranquilli et al.¹² assessed whether amniotic fluid concentrations of NO and VEGF in early pregnancy correlate with subsequent preeclampsia. They found their concentrations to be significantly lower than in healthy controls and concluded that low concentrations of VEGF and NO in the second trimester may represent an impaired stimulus to vascular formation and endothelial regulation that induce placental disease and preeclampsia. All these findings are consistent with the finding of the current study that low NO concentrations in the maternal blood are implicated in the pathogenesis of preeclampsia and can be considered a potential predictor marker for the disease. This is the first work to study the role of NO in the prediction of preeclampsia.

The results of the current study suggest that identification of high concentrations of sVEGFR-1 combined with low concentrations of PlGF and NO products along with an abnormal UADV may be used to predict the development of preeclampsia. This may be beneficial in identifying patients at high risk for the mild or severe form of preeclampsia in whom prophylactic interventions are more likely to reduce the morbidity and mortality rates associated with this obstetrical syndrome.

Footnotes

Disclosure Statement

The authors have no conflicts of interest to report.

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