Objective
The aim of this study was to examine if maternal plasma concentrations of soluble vascular endothelial growth factor receptor (sVEGFR)-2 change prior to the diagnosis of preeclampsia.
Study Design
A longitudinal study was conducted in normal pregnant women (n = 160) and patients with preeclampsia (n = 40). Blood samples were collected at 7 gestational age intervals from 6 weeks to term. Plasma concentrations of sVEGFR-2 were determined by enzyme-linked immunosorbent assay. Analysis was performed with cross-sectional and longitudinal (mixed effects model) approaches.
Results
Mothers destined to develop preeclampsia have lower plasma sVEGFR-2 concentrations than those who will have a normal pregnancy (longitudinal approach; P < .05). Cross-sectional analysis suggested that the median plasma sVEGFR-2 concentration in women destined to develop preeclampsia was significantly lower than that in normal pregnant women from 28-31 weeks of gestation ( P = .001) or 6-10 weeks prior to the diagnosis ( P < .001).
Conclusion
A lower maternal plasma sVEGFR-2 concentration precedes the development of preeclampsia, both term and preterm.
Preeclampsia is one of the leading causes of perinatal and maternal mortality. Despite several decades of research, the pathophysiology of this syndrome is still unclear. Maynard et al proposed that preeclampsia is an antiangiogenic state characterized by an increased plasma concentration of soluble vascular endothelial growth factor (VEGF) receptor (sVEGFR)-1 and a decrease in plasma concentration of placental growth factors (PlGF) as well as VEGF. Since then, studies of angiogenic and antiangiogenic factors in preeclampsia have gained increasing attention.
VEGF is an endothelial cell-specific growth factor with potent angiogenic properties. Its function is to promote endothelial cell proliferation, migration, and prevent endothelial cell apoptosis. While VEGFR-1 is considered a decoy receptor, VEGFR-2 is the major mediator of the mitogenic, angiogenic, permeability-enhancing, and endothelial survival effects of VEGF. The contribution of sVEGFR-1 to the maternal syndrome of preeclampsia is thought to be, at least in part, related to its inhibition of VEGF stimulation of the endothelium-dependent nitric oxide system. Plasma sVEGFR-1 concentration has been found to be elevated in preeclampsia both prior to and at the time of the diagnosis of preeclampsia.
The natural form of sVEGFR-2 has recently been detected in human plasma, although the use of a recombinant form or adenovirus encoding for sVEGFR-2 gene in cancer therapy has been under investigation for quite some time. Under experimental conditions, this protein can bind to VEGF and its recombinant form has antiangiogenic activity. The role of sVEGFR-2 in human health and diseases is unclear. However, recent studies have evaluated its potential as a surrogate biomarker for tumor progression in malignant melanoma, myelodysplastic syndrome, and acute leukemia. In nonmalignant conditions, plasma sVEGFR-2 concentration is lower in patients with systemic lupus erythematosus disease and dengue hemorrhagic fever compared to healthy controls. Similarly, patients with preeclampsia or those with isolated small-for-gestational-age fetuses at the time of clinical diagnosis have lower plasma concentrations of sVEGFR-2 than healthy pregnant women. The objective of this study was to examine if the maternal plasma concentrations of sVEGFR-2 change prior to the clinical diagnosis of preeclampsia.
Materials and Methods
Study design
A longitudinal nested case-control study was conducted by searching our clinical database and bank of biologic samples from 2002 through 2006. Patients with preeclampsia (n = 40) and healthy pregnant women (n = 160) were included. Exclusion criteria were: (1) chronic hypertension; (2) known major fetal or chromosomal anomaly; and (3) multiple gestations. All women were enrolled in the prenatal clinic at the Sotero del Rio Hospital, Santiago, Chile, and followed up until delivery.
Subjects were included only if they had plasma samples available at least once before and after 24 weeks of gestation (unless delivered <28 weeks). All patients had a minimum of 3 samples during pregnancy (ranging from 3-7 samples). Plasma samples were selected once from each patient at the following 7 intervals: 6-14; 15-19; 20-24; 25-27; 28-31; 32-36; and ≥37 weeks of gestation. The earliest sample for each interval is used. Samples collected after the clinical diagnosis of preeclampsia were not included.
Clinical definition
Preeclampsia was defined as hypertension (systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg on at least 2 occasions, 4 hours-1 week apart) and proteinuria. Severe preeclampsia was defined as previously described. Early-onset preeclampsia was defined as a diagnosis ≤34 weeks of gestation. Pregnant women were considered normal if they had no medical, obstetric, or surgical complications, and delivered a healthy term (≥37 weeks) infant whose birthweight was appropriate for gestational age (10th-90th percentile).
The collection and utilization of the samples was approved by both the Human Investigation Committee of the Sotero del Rio Hospital, Santiago, Chile (a major affiliate of the Catholic University of Santiago) and the institutional review board of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (National Institutes of Health, Department of Health and Human Services). Many of these samples were used in previous studies.
Sample collection and immunoassays
Venipunctures were performed and blood was collected into tubes containing EDTA. Samples were centrifuged and stored at −70°C. Maternal plasma concentrations of sVEGFR-2 were determined by immunoassays (R&D Systems, Minneapolis, MN) as previously described. The interassay and intraassay coefficients of variation were 2% and 4%, respectively. The sensitivity was 19 pg/mL.
Statistical analysis
Cross-sectional analysis
Kruskal-Wallis and post hoc Mann-Whitney U tests were utilized to determine the differences of the median among and between groups. Fisher’s exact and χ 2 tests were employed for comparisons of proportions. Logistic regression was applied to examine the association between low plasma concentrations of sVEGFR-2 (defined as plasma sVEGFR-2 concentrations below the first quartile of normal pregnancy) and the development of preeclampsia in samples obtained prior to the clinical diagnosis after adjusting for potential confounders. The statistics package used was SPSS V.15 (SPSS Inc, Chicago, IL). A P value of < .05 was considered significant.
Longitudinal analysis
Changes in the plasma concentrations of sVEGFR-2 over time and between groups were examined using a linear mixed effects model (fixed effects + random effects). The fixed effects were the diagnosis (a factor with 2 levels: normal pregnancy and preeclampsia), the linear and quadratic effects of gestational age, the interaction term between the diagnosis and gestational age, plus several covariates including: maternal age, body mass index, smoking, nulliparity, previous preeclampsia, and sample storage time. The random effects were the patient identification numbers, therefore allowing examination of the deviation of each individual from the average profile of each diagnostic group and accounting for the unknown variability among patients. The model was fitted to the transformed plasma concentration (log10 of [1 + concentration]) of the analyte. This logarithmic transformation was employed to improve normality of the data and stabilize variance across the entire range of gestational age. Statistical significance of fixed effects was assessed using t scores, and a P value < .05 was considered significant. The analysis was performed using the nonlinear mixed effect model package of the R statistical environment ( www.r-project.org ).
To identify the gestational age at which the difference in the median concentration between groups became significant, a moving window approach was used. Unlike in the cross-sectional study, where the limits of the intervals were predetermined, in the moving window approach, the number of data points was fixed to 200, 250, and 300 for each window. All observations were sorted as a function of the gestational age in ascending order. A set of 200-300 data points was chosen starting with the smallest gestation age and moving up the list. A Wilcoxon test was used to determine if there was significant difference between the 2 groups in each window of gestational age. The procedure was repeated until the point when the difference between groups remained significant (either for P < .05 or P < .01) in the current window and all consecutive ones. The median gestational age in the current window recorded.
Results
Clinical characteristics of the study population are displayed in Table 1 . The gestational age at which preeclampsia was diagnosed varied. Three patients had hypertension and proteinuria at 25-27 weeks, 4 at 28-31 weeks, 8 at 32-36 weeks, and 25 at term (≥37 weeks). There were no significant differences in the median gestational age at which venipuncture was performed by the interval window between the group of patients who eventually developed preeclampsia and the control group except in 1 gestational age interval (32-36 weeks, P = .04) ( Table 2 ).
| Characteristic | Normal pregnancy | Preeclampsia | P |
|---|---|---|---|
| n = 160 | n = 40 | ||
| Age, y | 25 (16–47) | 22 (17–38) | .02 a |
| Body mass index, kg/m 2 | 23.8 (16.1–37.0) | 25.7 (17.8–36.3) | .002 a |
| Nulliparity | 71 (44%) | 26 (65%) | .02 a |
| Smoking | 23 (14%) | 2 (5%) | .1 |
| Previous preeclampsia | 2 (1.3%) | 5 (12.5%) | .004 a |
| GA at delivery, wk | 40 (37.1–41.9) | 38.0 (25–41.0) | < .001 a |
| Birthweight, g | 3415 (2540–4090) | 2980 (500–4580) | < .001 a |
| Birthweight <10th percentile | – | 11 (27%) | < .001 a |
| Severe preeclampsia | – | 26 (65%) | |
| Early-onset preeclampsia | – | 9 (22%) |
| Variable | Normal pregnancy | P | Preclinical samples preeclampsia | P | Clinical samples preeclampsia | P β |
|---|---|---|---|---|---|---|
| 1st blood sampling (6-14.9 wk) | ||||||
| sVEGFR-2, ng/mL | 10.2 (4.7–14.8) | .3 | 9.9 (6.5–14.7) | |||
| Gestational age, wk | 12.2 | .2 | 11.7 | |||
| Range | 6.6–14.9 | 7.6–14.9 | ||||
| n = 160 | n = 40 | |||||
| 2nd blood sampling (15-19.9 wk) | ||||||
| sVEGFR-2, ng/mL | 10.8 (6.9–19.2) | .01 a | 9.9 (4.9–13.8) | |||
| Gestational age, wk | 17.4 | .3 | 17.8 | |||
| Range | 15.0–19.9 | 15.7–19.9 | ||||
| n = 160 | n = 38 | |||||
| 3rd blood sampling (20-24.9 wk) | ||||||
| sVEGFR-2, ng/mL | 10.8 (2.8–15.7) | .06 | 10.3 (3.6–15.4) | |||
| Gestational age, wk | 22.0 | .05 | 22.5 | |||
| Range | 20.0–24.6 | 20.0–24.9 | ||||
| n = 158 | n = 40 | |||||
| 4th blood sampling (25-27.9 wk) | ||||||
| sVEGFR-2, ng/mL | 10.7 (5.6–17.1) | .3 | 10.8 (6.9–14.6) | .03 a | 6.1 (5.3–6.9) | .02 a |
| Gestational age, wk | 26.4 | .3 | 26.7 | .5 | 27.0 | .3 |
| Range | 25.0–27.9 | 25.0–27.9 | 26.4–27.6 | |||
| n = 160 | n = 20 | n = 2 | ||||
| 5th blood sampling (28-31.9 wk) | ||||||
| sVEGFR-2, ng/mL | 10.7 (6.0–17.7) | .001 a | 9.7 (6.3–13.5) | .02 a | 4.6 (3.7–5.6) | .02 a |
| Gestational age, wk | 30.3 | .9 | 30.3 | .5 | 29.9 | .5 |
| Range | 28.0–31.9 | 28.0–31.9 | 29.1–30.6 | |||
| n = 160 | n = 32 | n = 2 | ||||
| 6th blood sampling (32-36.9 wk) | ||||||
| sVEGFR-2, ng/mL | 9.7 (5.7–15.0) | < .001 a | 8.6 (4.6–14.6) | .04 a | 6.2 (4.3–8.4) | .002 a |
| Gestational age, wk | 35.3 | .04 a | 34.7 | .2 | 33.7 | .07 |
| Range | 32.6–36.7 | 32.7–36.9 | 32.7–35.9 | |||
| n = 160 | n = 24 | n = 4 | ||||
| 7th blood sampling (>37 wk) | ||||||
| sVEGFR-2, ng/mL | 8.6 (4.1–13.5) | 7.7 (5.1–11.3) | .009 a | |||
| Gestational age, wk | 39.7 | 39.0 | .043 a | |||
| Range | 37.1–41.9 | 37.3–40.9 | ||||
| n = 102 | n = 15 |
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