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Maternal plasma concentrations of angiogenic/antiangiogenic factors in the third trimester of pregnancy to identify the patient at risk for stillbirth at or near term and severe late preeclampsia
Correspondence to: Roberto Romero, MD, DMedSci, and Tinnakorn Chaiworapongsa, MD, Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Wayne State University/Hutzel Women's Hospital, 3990 John R, Box 4, Detroit, MI 48201
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University, Detroit, MI
Correspondence to: Roberto Romero, MD, DMedSci, and Tinnakorn Chaiworapongsa, MD, Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Wayne State University/Hutzel Women's Hospital, 3990 John R, Box 4, Detroit, MI 48201
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University, Detroit, MI
Department of Obstetrics and Gynecology, Pontificia Universidad Católica de Chile, Center for Research and Innovation in Maternal-Fetal Medicine (CIMAF), Sótero del Río Hospital, Santiago, Chile
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University, Detroit, MI
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University, Detroit, MI
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University, Detroit, MI
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University, Detroit, MI
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University, Detroit, MI
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI
Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University, Detroit, MI
To determine whether maternal plasma concentrations of placental growth factor (PlGF), soluble endoglin (sEng), and soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) at 30-34 weeks of gestation can identify patients at risk for stillbirth, late preeclampsia, and delivery of small-for-gestational-age (SGA) neonates.
Study Design
A prospective cohort study included 1269 singleton pregnant women from whom blood samples were obtained at 30-34 weeks of gestation and who delivered at >34 weeks of gestation. Plasma concentrations of PlGF, sEng, and sVEGFR-1 were determined by enzyme-linked immunosorbent assay.
Results
The prevalence of late (>34 weeks of gestation) preeclampsia, severe late preeclampsia, stillbirth, and SGA was 3.2% (n = 40), 1.8% (n = 23), 0.4% (n = 5), and 8.5% (n = 108), respectively. A plasma concentration of PlGF/sEng <0.3 MoM was associated with severe late preeclampsia (adjusted odds ratio, 16); the addition of PlGF/sEng to clinical risk factors increased the area under the receiver-operating characteristic curve from 0.76 to 0.88 (P = .03). The ratio of PlGF/sEng or PlGF/sVEGFR-1 in the third trimester outperformed those obtained in the first or second trimester and uterine artery Doppler velocimetry at 20-25 weeks of gestation for the prediction of severe late preeclampsia (comparison of areas under the receiver-operating characteristic curve; each P ≤ .02). Both PlGF/sEng and PlGF/sVEGFR-1 ratios achieved a sensitivity of 74% with a fixed false-positive rate of 15% for the identification of severe late preeclampsia. A plasma concentration of PlGF/sVEGFR-1 <0.12 MoM at 30-34 weeks of gestation had a sensitivity of 80%, a specificity of 94%, and a likelihood ratio of a positive test of 14 for the identification of subsequent stillbirth. Similar findings (sensitivity 80%; specificity 93%) were observed in a separate case-control study.
Conclusion
Risk assessment for stillbirth and severe late preeclampsia in the third trimester is possible with the determination of maternal plasma concentrations of angiogenic and antiangiogenic factors at 30-34 weeks of gestation.
A novel bridge between oxidative stress and immunity: the interaction between hydrogen peroxide and human leukocyte antigen G in placental trophoblasts during preeclampsia.
Evidence supporting a role for blockade of the vascular endothelial growth factor system in the pathophysiology of preeclampsia: Young Investigator Award.
Preeclampsia may be classified as “early” or “late” according to gestational age at diagnosis or delivery. The gestational age cutoff most frequently used is 34 weeks.
In contrast, preeclampsia at term is associated with better neonatal outcomes than preterm preeclampsia. Although emphasis has focused on early preeclampsia, most cases of preeclampsia occur at or near term.
In high-income countries, stillbirth is associated with fetal growth restriction or placental insufficiency; however, in nearly one-half of the cases, the cause is unknown.
Placental angiogenic growth factors and uterine artery Doppler findings for characterization of different subsets in preeclampsia and in isolated intrauterine growth restriction.
Maternal plasma concentrations of angiogenic/anti-angiogenic factors are of prognostic value in patients presenting to the obstetrical triage area with the suspicion of preeclampsia.
Circulating angiogenic factors determined by electrochemiluminescence immunoassay in relation to the clinical features and laboratory parameters in women with pre-eclampsia.
The maternal plasma soluble vascular endothelial growth factor receptor-1 concentration is elevated in SGA and the magnitude of the increase relates to Doppler abnormalities in the maternal and fetal circulation.
Fetal death: a condition with a dissociation in the concentrations of soluble vascular endothelial growth factor receptor-2 between the maternal and fetal compartments.
Changes in the concentrations of the angiogenic factor (placental growth factor [PlGF]) and antiangiogenic factors (soluble vascular endothelial growth factor receptor-1 [sVEGFR-1; also known as soluble fms-like tyrosine kinase-1] and soluble endoglin [sEng]) in maternal circulation precede the clinical diagnosis of preeclampsia,
The change in concentrations of angiogenic and anti-angiogenic factors in maternal plasma between the first and second trimesters in risk assessment for the subsequent development of preeclampsia and small-for-gestational age.
A longitudinal study of angiogenic (placental growth factor) and anti-angiogenic (soluble endoglin and soluble vascular endothelial growth factor receptor-1) factors in normal pregnancy and patients destined to develop preeclampsia and deliver a small for gestational age neonate.
An elevated maternal plasma, but not amniotic fluid, soluble fms-like tyrosine kinase-1 (sFlt-1) at the time of mid-trimester genetic amniocentesis is a risk factor for preeclampsia.
Circulating angiogenic factors in early pregnancy and the risk of preeclampsia, intrauterine growth restriction, spontaneous preterm birth, and stillbirth.
Prediction of early and late pre-eclampsia from maternal characteristics, uterine artery Doppler and markers of vasculogenesis during the first trimester of pregnancy.
Ultrasound Obstet Gynecol.2012; (Epub ahead of print)
An elevated maternal plasma, but not amniotic fluid, soluble fms-like tyrosine kinase-1 (sFlt-1) at the time of mid-trimester genetic amniocentesis is a risk factor for preeclampsia.
The results of such studies largely suggest that an imbalance between angiogenic and antiangiogenic factors increases the likelihood of preterm preeclampsia at a higher magnitude than that of term preeclampsia.
Evidence supporting a role for blockade of the vascular endothelial growth factor system in the pathophysiology of preeclampsia: Young Investigator Award.
Prediction of early and late pre-eclampsia from maternal characteristics, uterine artery Doppler and markers of vasculogenesis during the first trimester of pregnancy.
Ultrasound Obstet Gynecol.2012; (Epub ahead of print)
A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
Alterations in placental growth factor levels before and after the onset of preeclampsia are more pronounced in women with early onset severe preeclampsia.
Accuracy of circulating placental growth factor, vascular endothelial growth factor, soluble fms-like tyrosine kinase 1 and soluble endoglin in the prediction of pre-eclampsia: a systematic review and meta-analysis.
Thus far, no cohort study has evaluated the predictive performance of these biomarkers in the third trimester for the identification of patients at risk for stillbirth at or near term or late-onset preeclampsia.
Recently, a new approach in screening for adverse pregnancy outcomes proposes to focus on the prevention of pregnancy complications at term. Such an approach would identify the more prevalent disease (eg, preeclampsia at term), and predictive models could be applied to low-income settings, where most maternal and perinatal deaths occur.
The objective of this study was to determine whether maternal plasma concentrations of PlGF, sEng, sVEGFR-1, and their ratios at 30-34 weeks of gestation could be used to identify patients at risk for stillbirth, late preeclampsia, severe late preeclampsia, or delivery of SGA neonates.
Methods
Study design
We first designed a cohort study of women who had a venipuncture at 30-34 weeks of gestation and outcome data to examine the value of PlGF, sVEGFR-1, and sEng in the identification of patients who subsequently experienced late preeclampsia, severe late preeclampsia, stillbirth, and SGA. Subsequent to this cohort study, a case-control study was performed to determine whether these biomarkers and their ratios could identify patients at risk for stillbirth at or near term in a different population.
Cohort study
A prospective longitudinal cohort study was conducted between November 2003 and August 2006 to identify biological markers for the prediction of preeclampsia, SGA neonates, and stillbirth. Patients were enrolled in the prenatal clinic of the Sotero del Rio Hospital, a tertiary care center in Santiago, Chile, and followed until delivery. Inclusion criteria were singleton gestation and 6-22 weeks of gestation. Exclusion criteria were (1) preterm labor, preterm prelabor rupture of membranes, preeclampsia, or impaired fetal growth at the time of recruitment; (2) known major fetal anomaly or fetal death; (3) active vaginal bleeding; and (4) serious medical illness (renal insufficiency, congestive heart disease, chronic respiratory insufficiency, or active hepatitis). At enrollment and each subsequent visit, patients underwent a venipuncture for the collection of maternal blood. The protocol consisted of collecting samples every 4 weeks until 24 weeks of gestation and every 2 weeks thereafter until delivery.
We previously reported the predictive performance of angiogenic/antiangiogenic factors at 6-15 and 20-25 weeks of gestation and uterine artery Doppler velocimetry (UADV) at 20-25 weeks of gestation for preeclampsia in this cohort.
A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
In summary, 2998 consecutive women were enrolled during the study period; 2495 women had a plasma sample collected in early pregnancy. Of those, an additional plasma sample was obtained in the mid trimester from 1917 women. Subsequently, an additional 204 patients without results of UADV in the second trimester were excluded. Ninety-one patients were lost to follow up; the remaining 1622 patients had been included in a previous article that examined the role of angiogenic/antiangiogenic factors at 6-15 and 20-25 weeks of gestation.
A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
The current study involved a subset of this cohort that excluded patients who delivered at ≤34 weeks of gestation (n = 27) and those who did not have a plasma sample collected at 30-34 weeks of gestation (n = 326) to examine the role of angiogenic/antiangiogenic factors at 30-34 weeks for the identification of adverse pregnancy outcomes at >34 weeks of gestation.
All women provided written informed consent before participating in the study. The use of clinical and ultrasound data and the collection and use of maternal blood for research purposes was approved by the institutional review boards of the Sotero del Rio Hospital, Santiago, Chile (an affiliate of the Pontificia Catholic University of Santiago, Chile), and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services.
Outcomes of the study
The outcomes of the study included late preeclampsia, severe late preeclampsia, SGA without preeclampsia, and stillbirth. Preeclampsia was defined as new-onset hypertension that developed at >20 weeks of gestation and proteinuria. Hypertension was defined as systolic ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg that was measured at 2 occasions, 4 hours to 1 week apart. Proteinuria was defined as a urine protein of ≥300 mg in a 24-hour urine collection or 2 random urine specimens that were obtained 4 hours to 1 week apart and showed ≥1+ by dipstick or 1 dipstick that demonstrated ≥2+ protein. Late preeclampsia was defined as patients with preeclampsia who delivered at >34 weeks of gestation.
Stillbirth was defined as death of a fetus before delivery that was not a consequence of an induced termination of pregnancy (including intrapartum and antepartum stillbirth).
Fetal Medicine Foundation Second-Trimester Screening Group Prediction of pre-eclampsia by uterine artery Doppler imaging: relationship to gestational age at delivery and small-for-gestational age.
Customized case report forms and a perinatal database were generated. Data were extracted from medical records by trained research nurses. To account for misclassification, abstracters were trained, the data collection methods were verified, and data logic was monitored. Cases of uncertainty were resolved by iterative discussion among 3 of the authors. Gestational age at venipuncture and at delivery was based on best obstetrical estimates with the use of the last menstrual period and the earliest fetal biometric parameters, which were performed at ≤20 weeks of gestation in 98.2% of cases.
Sample collection and immunoassays
Blood was obtained by venipuncture and collected into tubes that contained ethylenediaminetetraacetic acid. Samples were centrifuged and stored at −70°C. Maternal plasma concentrations of sVEGFR-1, PlGF, and sEng were determined by sensitive and specific immunoassays (R&D Systems, Minneapolis, MN) as previously described.
A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
The inter- and intra-assay coefficients of variation (CV) were 1.4% and 3.9% for sVEGFR-1, 2.3% and 4.6% for sEng, and 6.02% and 4.8% for PlGF. The sensitivity of the assays was 16.97 pg/mL for sVEGFR-1, 0.08 ng/mL for sEng, and 9.52 pg/mL for PlGF. The laboratory personnel who performed the assays were blinded to the clinical information.
Statistical analysis
Differences in distributions of dichotomous and categorical variables were tested with the χ2 test or Fisher exact test where appropriate; continuous parameters were compared by analysis of variance or Friedman's 2-way nonparametric analysis of variance test with Bonferroni correction for multiple comparisons, depending on the distribution of data. Normality was assessed with the Kolmogorov-Smirnov test and visual plot inspection.
Quantile regression was used to calculate median analyte ratio concentrations (PlGF/sVEGFR-1, PlGF/sEng) that were conditional on gestational age among uncomplicated pregnancies (n = 886).
Multiple of the median (MoM) values were calculated for both analyte ratios for each patient. MoM cutoffs were determined based on inspection of receiver operating characteristic curves that were calculated for each outcome (stillbirth, late preeclampsia, severe late preeclampsia, and SGA without preeclampsia). Prognostic logistic regression models were constructed for each outcome. Covariables included in adjusted models were selected on the basis of clinical knowledge. Model reduction was performed additionally based on the plausibility of regression coefficients, association with independent variables, and the magnitude of change in the main-effect parameter estimates.
To account for potential model over-fitting, when van Houwelingen and le Cessie's heuristic shrinkage estimator was below 0.85 (indicator of instability), bootstrap estimated linear shrinkage factors and Firth's penalized maximum likelihood estimation were used to calculate conservative estimates that were less likely to be affected by over-fitting.
Predictive performance metrics were also calculated for each outcome. Paired sample nonparametric statistical techniques were used to compare area under the receiver operating characteristic curves (AUC) of models that were constructed with logistic regression for the identification of selected pregnancy outcomes.
A McNemar's test was also used to test for differences in sensitivity at a fixed false-positive rate of 15%. A 5% threshold for type I error was used to determine statistical significance. Statistical analyses were performed with SAS software (version 9.3; Cary, NC).
Case-control study for stillbirth
Participants were identified from a cohort of 5828 singleton pregnancies who were either enrolled in a similar longitudinal protocol to that used in the Chilean cohort or another cross-sectional protocol from 2007-2009 at Hutzel Women's Hospital, Detroit, MI. Stillbirth was defined as death of a fetus before delivery (which was not a consequence of an induced termination of pregnancy).
In the longitudinal study, plasma samples were obtained from the first or early second trimester and at the time of each prenatal visit (scheduled every 4 weeks until 24 weeks and every 2 weeks thereafter until delivery). In the cross-sectional study, patients were enrolled when they presented to the labor and delivery unit with a suspicion of spontaneous preterm labor or medically-indicated preterm birth. Among 31 cases of stillbirth at ≥34 weeks of gestation, 5 cases were included because they had a plasma sample collected at 30-34 weeks of gestation. Control subjects were identified from uncomplicated pregnancies who both delivered an appropriate weight for gestational age neonate at term and had a plasma sample collected at 30-34 weeks of gestation. Control subjects were matched to cases at a ratio of 6 to 1 on gestational age at venipuncture, parity, ethnicity, tobacco use, and body mass index. Maternal plasma concentrations of sVEGFR-1, sEng, and PlGF were determined by sensitive and specific immunoassays (R&D Systems) similar to those used in the Chilean cohort, as described earlier.
All women provided written informed consent before participating in the study. The use of clinical and ultrasound data and collection and utilization of maternal blood for research purposes was approved by the institutional review boards of the Wayne State University, Detroit, MI, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services.
Differences among cases and control subjects were tested with the use of χ2, Fisher exact, or Mann-Whitney U tests where appropriate. AUC was calculated, and sensitivities and specificities were determined with the use of absolute value thresholds for each biomarker ratio that were derived from the inspection of receiver operating characteristic curves.
Results
The cohort study included 1269 pregnant women (Figure 1). The prevalence of late preeclampsia, severe late preeclampsia, stillbirth, and SGA without preeclampsia was 3.2% (n = 40), 1.8% (n = 23); 0.4% (n = 5), and 8.5% (n = 108), respectively. Among 23 patients who received a diagnosis of severe preeclampsia, 6 experienced severe high blood pressure and severe proteinuria; 4 had severe high blood pressure; 4 had severe high blood pressure with severe proteinuria with SGA fetuses; 3 had SGA fetuses; 2 had severe headache with severe proteinuria; 2 had severe proteinuria, 1 had severe high blood pressure, and 1 had severe proteinuria and pulmonary edema. Table 1 displays the demographic and obstetrical characteristics of patients with SGA neonates, preeclampsia, stillbirth, other complications and of patients without any of these complications (uncomplicated pregnancy). There were no significant differences in the mean gestational age at venous sampling or mean duration of sample storage among the 4 groups. The distribution of baseline characteristics did not significantly differ between patients included in the current study compared with the overall cohort (data not shown). Similarly, there were no significant differences in the risk of stillbirth or SGA between the entire cohort and subcohort. However, by design, participants in the subcohort were more likely to deliver at >34 weeks of gestation. Patients in this subcohort had a lower frequency of preeclampsia than those in the entire cohort (3.2% vs 4.8%; P = .03). Three patients who had a diagnosis of gestational hypertension before venipuncture at 30-34 weeks of gestation; however, subsequent preeclampsia did not develop. The median MoM plasma concentration of PlGF/sVEGFR-1 and PlGF/sEng was significantly lower in patients with subsequent stillbirth, preeclampsia, and SGA than in patients without these conditions (P < .05 for each comparison; Table 1).
FIGURE 1Flow diagram of patients enrolled in the study
Other complications included spontaneous preterm delivery (3%; n = 38), chronic hypertension (2.2%; n = 28), gestational hypertension (6.8%; n = 86), gestational and pregestational diabetes mellitus (4.6%; n = 58), placental abruption (0.4%; n = 5), cholestasis of pregnancy (0.9%; n = 12), and placenta previa (0.2%; n = 3).
Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.
a Data are given as mean ± SD;
b Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
c Data are given as median (interquartile range) that were calculated among uncomplicated pregnancies (n = 886) by quantile regression:
d Median = 1.8863+ (−0.0508 × gestational week);
e Median = 354.3280 + (−8.9791 × gestational week).
Table 2 displays the magnitude of association between abnormal biomarker profiles and late preeclampsia (overall and severe), delivery of SGA neonates (birthweight: <10%, <3%), and stillbirth. Patients with plasma PlGF/sEng or PlGF/sVEGFR-1 ratio concentrations of <0.3 MoM were significantly more likely to experience late preeclampsia (adjusted odds ratio [aOR], 7.1; 95% confidence interval [CI], 3.6–13.8; and 6.1; 95% CI, 3.1–11.8, respectively) and severe late preeclampsia (aOR, 16.1; 95% CI, 5.8–44.6 and 12.2; 95% CI, 4.6–32, respectively) than those with MoMs at or above the threshold (Table 2). The likelihood ratio of a positive test and sensitivity for either PlGF/sEng or PlGF/sVEGFR-1 ranged from 4.5–4.8 and 74–78%, respectively; both had a specificity of 84% for the identification of patients with severe late preeclampsia (Table 3).
TABLE 2Likelihood of subsequent stillbirth, preeclampsia, and small-for-gestational-age neonate
Data represent the likelihood of outcome in subjects with abnormal analyte ratio concentrations (above/below MoM cutoff) relative to patients with normal analyte ratio concentration multiples of the median cutoff;
Data represent the likelihood of outcome in subjects with abnormal analyte ratio concentrations (above/below MoM cutoff) relative to patients with normal analyte ratio concentration multiples of the median cutoff;
Odds ratio was adjusted for gestational age at venipuncture (continuous).
PlGF/sVEGFR-1 <0.12 MoM
71/1264 (5.6%)
4/5 (80%)
20.1
4.8–84.3
23.1
5.6–95.4
PlGF/sEng <0.2 MoM
137/1264 (10.8%)
3/5 (60%)
8.4
2.0–35.1
9.1
2.2–37.2
Medians were calculated among uncomplicated pregnancies (n = 886) by quantile regression (PlGF/sVEGFR-1: median = 1.8863 + (−0.0508 × gestational week); PlGF/sEng: median = 354.3280 + (−8.9791 × gestational week); cutoffs were selected based on inspection of receiver operating characteristic curves. The prediction of preeclampsia and small-for-gestational-age was adjusted for maternal age (continuous), combined parity and history of preeclampsia, prepregnancy body mass index (continuous), and tobacco use. PlGF/sVEGFR-1 MoM cutoff: <0.12 (or 5th -6th percentile of uncomplicated pregnancies) for stillbirth and <0.3 (or 17th percentile of uncomplicated pregnancies) for preeclampsia and small-for-gestational-age. PlGF/sEng MoM cutoff: <0.2 (or 11th percentile of uncomplicated pregnancies) for stillbirth and <0.3 (or 17th percentile of uncomplicated pregnancies) for preeclampsia and small-for-gestational-age.
Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.
a Data represent the likelihood of outcome in subjects with abnormal analyte ratio concentrations (above/below MoM cutoff) relative to patients with normal analyte ratio concentration multiples of the median cutoff;
b Odds ratio was adjusted for gestational age at venipuncture (continuous).
TABLE 3Predictive performance of maternal plasma concentrations of angiogenic and antiangiogenic factors for stillbirth and preeclampsia screening
Diagnostic performance metrics
Preeclampsia (n = 40)
Severe preeclampsia (n = 23)
Stillbirth (n = 5)
Estimate
95% CI
Estimate
95% CI
Estimate
95% CI
PlGF/sVEGFR-1
Sensitivity, %
58
40–73
74
52–90
80
28–100
Specificity, %
83
82–86
84
81–86
94
93–96
Positive predictive value, %
10
6–15
8
5–12
5
1–13
Negative predictive value, %
98
97–99
99
99–100
100
99–100
False positive rate, %
16
14–18
16
14–19
6
4–7
False negative rate, %
43
27–59
26
10–48
20
0.5–72
Positive likelihood ratio
3.6
2.6–4.8
4.5
3.4–5.9
14.2
8.7–23.3
Negative likelihood ratio
0.5
0.4–0.7
0.3
0.2–0.6
0.2
0.04–1.22
PlGF/sEng
Sensitivity, %
60
43–75
78
56–93
60
15–95
Specificity, %
84
82–86
84
82–86
89
87–91
Positive predictive value, %
11
7–16
8
5–13
2
0.4–6
Negative predictive value, %
98
97–99
99
99–100
99
99–100
False positive rate, %
16
14–18
16
14–18
11
9–13
False negative rate, %
40
25–57
22
7–44
40
5–85
Positive likelihood ratio
3.8
2.8–5.0
4.8
3.8–6.2
5.5
2.7–11.5
Negative likelihood ratio
0.5
0.3–0.7
0.3
0.1–0.6
0.4
0.2–1.3
Medians were calculated among uncomplicated pregnancies (n = 886) by quantile regression (PlGF/sVEGFR-1: median = 1.8863 + (−0.0508 × gestational week); PlGF/sEng: median = 354.3280 + (−8.9791 × gestational week); cutoffs were selected based on inspection of receiver operating characteristic curves. PlGF/sVEGFR-1 multiples of the median cutoff: <0.12 (or 5th -6th percentile of uncomplicated pregnancies) for stillbirth and <0.3 (or 17th percentile of uncomplicated pregnancies) for preeclampsia; PlGF/sEng multiples of the median cutoff: <0.2 (or 11th percentile of uncomplicated pregnancies) for stillbirth and <0.3 (or 17th percentile of uncomplicated pregnancies) for preeclampsia.
The addition of the PlGF/sEng or PlGF/sVEGFR-1 ratio to the clinical risk factors increased the AUC from 0.76 to 0.88 and 0.86, respectively, for the prediction of severe late preeclampsia (P = .03 and .06). With a fixed false-positive rate of 15%, both the PlGF/sEng ratio and PlGF/sVEGFR-1 ratios achieved a sensitivity of 74% in predicting severe PE. These biomarkers in the third trimester outperformed those obtained previously at 6-15 and 20-25 weeks of gestation and UADV that was assessed at 20-25 weeks of gestation for the prediction of severe late preeclampsia (each P ≤ .02; Figure 2). Further, the addition of the PlGF/sVEGFR-1 or the PlGF/sEng ratio measured in the third trimester to clinical risk factors (age, body mass index, combined parity and history of preeclampsia, and tobacco use) yielded significantly greater sensitivity (74%) at a fixed false-positive rate of 15%, compared with a model that used the same biomarker ratios measured in the second trimester, clinical risk factors, and abnormal UADV values that were obtained at 20-25 weeks of gestation (P = .008 and .03, respectively). The direction, magnitude, and significance of these associations also persisted during sensitivity analyses performed excluding patients with a history of preeclampsia (n = 37) based on their elevated a priori risk in the current pregnancy.
FIGURE 2Comparison of ROC curves for the identification of severe late preeclampsia
Areas under the ROC curves achieved by plasma concentrations of A, placental growth factor (PlGF)/soluble endoglin (sEng) ratio or B, PlGF/soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) ratio. Multiples of the median at 6-15, 20-25, and 30-34 weeks of gestation and uterine (Ut.) artery Doppler velocimetry at 20-25 weeks of gestation are shown.
Although patients with plasma PlGF/sVEGFR-1 or PlGF/sEng ratio concentrations of <0.3 MoM were more likely to experience SGA without preeclampsia (aOR, 2-3; Table 2), the addition of these biomarkers to demographic/perinatal data did not improve the AUC (0.64 vs 0.62; P = .2 and .6; respectively). Subgroup analysis that focused on patients with severe SGA (birthweight less than the third percentile; n = 23) indicated that the adjusted odds ratio of patients with PlGF/sVEGFR-1 or PlGF/sEng ratio <0.3 MoM to experience severe SGA ranged from 4.4–5.5 (Table 2). However, the addition of these biomarkers to clinical risk factors did not significantly improve the AUC (P > .05).
Patients with a PlGF/sVEGFR-1 ratio of <0.12 MoM were significantly more likely to have a stillbirth than patients with a MoM ratio at or above the threshold (aOR, 23.1; 95% CI, 5.6–95.4). This cutoff had a sensitivity of 80%, a specificity of 94%, and a likelihood ratio of a positive test of 14.2 for the identification of a subsequent stillbirth at >34 weeks of gestation (Table 3). Compared with a model that included only clinical data (maternal age, combined parity and history of preeclampsia, body mass index, and tobacco use), the addition of the PlGF/sVEGFR-1 ratio or the PlGF/sEng ratio to clinical data increased the AUC from 0.7 to 0.91 (P = .03 and .06, respectively; Figure 3). The association between an abnormal ratio of angiogenic/antiangiogenic factors and stillbirth at or near term was also observed in the subsequent case-control study performed in a different population (Table 4). A maternal plasma concentration of PlGF/sVEGFR-1 ratio ≤0.046 or PlGF/sEng ratio ≤11.7 pg/ng at 30-34 weeks had a sensitivity of 80% and a specificity of 93% for the identification of subsequent stillbirth (Figure 4).
FIGURE 3Comparison of ROC curves for the identification of stillbirth
Areas under the ROC curves achieved by plasma concentrations of placental growth factor (PIGF)/soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) ratio or PIGF/soluble endoglin (sEng) ratio at 30-34 weeks' gestation in combination with clinical data were compared with that achieved by clinical data alone.
Maternal plasma concentrations of placental growth factor (PlGF)/soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) or PlGF/soluble endoglin (sEng) at 30-34 weeks of gestation were used to identify patients at risk of stillbirth. Area under the ROC curves = 0.91 for both biomarkers.
Table 5 displays the obstetrical events at delivery, gestational age at venipuncture, and placental pathologic conditions of each patient with a stillbirth in the cohort and case-control studies. Among patients with a stillbirth in the cohort study, the interval from venipuncture to the diagnosis of stillbirth ranged from 2.2–6.1 weeks (median, 4.5 weeks). One patient was diagnosed with gestational diabetes mellitus, and another had an abruptio placentae. Three patients had histologic placental lesions consistent with maternal underperfusion, according to the criteria of the Society for Pediatric Pathology.
Chronic chorioamnionitis and hyalinized avascular villi (consistent with fetal thrombotic vasculopathy), were observed in the other 2 cases. None of the cases included in the cohort study had a fetal autopsy. Of 5 cases included in the case-control study, 2 women had a diagnosis of diabetes mellitus and the remaining 3 had a diagnosis of severe preeclampsia, chronic hypertension, and Marfan's syndrome, respectively. The interval from venipuncture to the diagnosis of stillbirth ranged from 2.4–5.4 weeks (median, 4 weeks). All 4 cases of stillbirth who had plasma concentrations of angiogenic/antiangiogenic factor ratios below the aforementioned cutoff in the case-control study also had lesions in the placenta suggestive of maternal underperfusion. Two stillbirths had a karyotype performed, and the results were 46 XY. Among 4 cases with available fetal autopsy results, 1 had lesions in the fetal brain consistent with acute hypoxic/ischemic damage in the grey matter.
TABLE 5Obstetrics events at delivery, gestational age at venipuncture, and placental lesions of patients with stillbirth
Case
Obstetrics events at delivery
PlGF/sVEGFR-1 ratio, MoM
Gestational age, wk
Birthweight in grams (percentile)
Placental lesions consistent with maternal underperfusion
Placental lesions
Fetal autopsy
At venous sampling
At delivery
Cohort study
1
Normal blood pressure
0.07
32 1/7
34 3/7
2200 (50%)
Yes
Diffuse chronic villitis; persistent muscularization of basal plate arteries
Not available
2
Gestational diabetes mellitus noncompliance with care
0.10
31 3/7
34 4/7
2280 (58%)
Yes
Increased syncytial knot
Not available
3
Blood pressure 140/90 mm Hg, urine protein dipstick negative, placental abruption
0.04
31
35 4/7
3000 (92%)
No
Chronic chorioamnionitis
Not available
4
Normal blood pressure, decreased fetal movement, thick meconium-stained amniotic fluid
0.08
33 3/7
39 3/7
3650 (74%)
Yes
Increased intervillous fibrin; prominent nucleated red blood cells, absence of physiologic change of the spiral arteries
This is the first prospective cohort study to evaluate the predictive performance of angiogenic/antiangiogenic factors in the third trimester for the identification of patients with late preeclampsia, severe late preeclampsia, SGA without preeclampsia, and stillbirth. The principal findings were: (1) A maternal plasma concentration of PlGF/sEng <0.3 MoM at 30-34 weeks' gestation was associated with late preeclampsia (aOR, 7) and severe late preeclampsia (aOR, 16). With a fixed false-positive rate of 15%, both the PlGF/sEng and PlGF/sVEGFR-1 ratios achieved a sensitivity of 74% for the identification of severe late preeclampsia; (2) The ratio of PlGF/sEng or PlGF/sVEGFR-1 in the third trimester outperformed ratios obtained at 6-15 and 20-25 weeks of gestation and abnormal UADV obtained at 20-25 weeks of gestation for the identification of severe late preeclampsia (comparisons of AUC; each P ≤ .02); (3) A maternal plasma concentration of PlGF/sVEGFR-1 ratio <0.12 MoM at 30-34 weeks of gestation was significantly associated with a subsequent stillbirth (aOR, 23). This cutoff had a sensitivity of 80%, a specificity of 94%, and a likelihood ratio of a positive result of 14 for the identification of patients destined to have a stillbirth; and (4) Although low maternal plasma concentrations of PlGF/sVEGFR-1 and PlGF/sEng ratios were associated with a significant increase in the likelihood of the development of SGA, these biomarkers did not improve the identification of SGA from the models using clinical factors alone.
Rationale for the examination of biomarkers in the third trimester
The following advantages underlie the use of biomarkers in the third trimester, in addition to the first or second trimesters: (1) Testing performed closer to the event of interest or diagnosis usually yields better results than tests performed earlier in gestation. Several studies of screening tests in the first or second trimester for conditions related to placental dysfunction (preeclampsia, SGA, or fetal death) using either biochemical markers
indicate that both are strongly associated with complications that develop earlier in pregnancy and therefore, are temporally close to the assessment of biomarkers.
Our findings that plasma concentrations of the ratio between angiogenic/antiangiogenic factors outperformed those that are obtained in the first 2 trimesters for the identification of patients with late preeclampsia strongly support this view; (2) The risk for a prospective stillbirth increases after 34 weeks of gestation
A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
and (3) The strategy of testing at the beginning of the third trimester to assess the risk of disease or pregnancy complications could be considered for patients who did not receive earlier prenatal care or undergo testing.
A disadvantage of performing a screening test in the third trimester is that this may be too late to implement therapeutic interventions that can reverse a pathophysiologic process responsible for disease. However, the precise mechanisms of late-onset preeclampsia are unknown, and there is no effective intervention even if at-risk patients are identified in early gestation. Although a recent metaanalysis suggests that the administration of aspirin at before 16 weeks of gestation may prevent preeclampsia,
Thus, a method to identify patients at risk for late preeclampsia is needed, given that it accounts for most severe maternal morbidity, including eclampsia,
Furthermore, this study, which examined the predictive performance of biomarkers in the third trimester in a low-risk population, is consistent with the recently proposed approach for the screening of adverse pregnancy outcomes that focuses on the prevention of pregnancy complications at term in low-risk, unselected populations.
However, although an appreciable magnitude of the association was observed between these biomarkers in the third trimester and late severe preeclampsia (which outperformed the screening implemented in earlier gestations), further studies are necessary to explain why some women with distinctly abnormal angiogenic/antiangiogenic profiles have uncomplicated pregnancies. Answers to this question would allow for even greater predictive performance than observed in this study.
Risk assessment for stillbirth: a neglected area of prenatal care
Our findings open new avenues for understanding the pathophysiologic process of the disease, risk assessment, and prevention of stillbirth, which is a neglected area of prenatal care.
In our cohort, there were no cases of intrapartum stillbirth. Among 8 stillbirths that were identified with these biomarkers from the cohort and case-control studies, 7 had placental lesions suggestive of maternal underperfusion.
Unexplained fetal death has a biological signature of maternal anti-fetal rejection: chronic chorioamnionitis and alloimmune anti-human leucocyte antigen antibodies.
Although the precise mechanisms responsible for stillbirth are unknown, it appears that the biomarkers investigated in this study may be able to identify a large fraction of stillbirths that result from placenta-related, rather than nonplacenta-related, causes (such as cord accident, fetal thrombosis, or fetomaternal hemorrhage). This interpretation is consistent with the findings from a recent study that demonstrated an association between stillbirth at or near term and UADV in the second trimester, which indicated that an increase in impedance to blood flow to the placenta is one of the major risk factors for stillbirth at term.
Moreover, because markers of placental dysfunction (such as high maternal serum alpha-fetoprotein or human chorionic gonadotropin), are associated with an increased risk of unexplained stillbirth and other pregnancy complications (such as preeclampsia or SGA),
Second-trimester prenatal screening for Down syndrome and the relationship of maternal serum biochemical markers to pregnancy complications with adverse outcome.
Second-trimester maternal serum marker screening: maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, estriol, and their various combinations as predictors of pregnancy outcome.
Association between second-trimester isolated high maternal serum maternal serum human chorionic gonadotropin levels and obstetric complications in singleton and twin pregnancies.
it is possible that a subset of unexplained stillbirths, preeclampsia, and SGA represent different clinical manifestations of a placental response from insults at different gestational ages. Evidence in support of this hypothesis is that rats subjected to reduced uteroplacental perfusion by the application of clips to the abdominal aorta at different gestational ages had a different magnitude of change in angiogenic/antiangiogenic imbalance, fetal growth restriction, and the severity of placental ischemia–induced systemic hypertension.
Our findings of an association between abnormal angiogenic/antiangiogenic factors and stillbirth, preeclampsia, and SGA without preeclampsia strengthen this hypothesis.
Limitations of the study
To facilitate comparison of the predictive performance of angiogenic/antiangiogenic factors in the third trimester with that of these biomarkers in the first and second trimester and UADV in the second trimester, a number of patients without plasma samples in the first 2 trimesters and those patients without UADV information were excluded from the cohort. However, the distribution of baseline characteristics (maternal age, body mass index, nulliparity, combined parity, and previous preeclampsia) did not differ significantly from patients included in the current study compared with the overall cohort, and there were no significant differences in the risk of stillbirth or SGA between the full and subcohort. However, by design, participants in the subcohort were more likely to deliver at >34 weeks of gestation. Patients in the subcohort were also less likely to experience late preeclampsia (3.2% vs 4.8%; P = .03), which indicates that our results for preeclampsia may be biased toward the null hypothesis.
Another limitation is that the determination of plasma concentrations of these biomarkers was performed in stored samples. However, serum PlGF and sVEGFR-1 concentrations are quite stable when stored at −80°C,
and the duration of sample storage did not differ significantly by pregnancy outcome. Therefore, this is unlikely to have introduced bias into our study.
A limited number of patients with stillbirth were included in this cohort of a low-risk population. This is a common problem of cohort studies with rare outcomes. We used statistical methods to address possible over-fitting of models and replicated our findings in a separate case-control study performed in a different population. Altogether, our results indicate that the ratio of maternal plasma concentrations of angiogenic/antiangiogenic factors in the third trimester have value in risk assessment for stillbirth, given the high likelihood ratio of a positive test (LR, 14.2), although further study is necessary both to validate these findings and to determine generalizability.
A low positive predictive value, consistent with that observed in this study, is often invoked as a limitation of a test. However, it is noteworthy that positive predictive values are dependent on the prevalence of the disease and will always be low when the condition under study is rare. This is the case for stillbirth, which had a prevalence of 0.4% in our study. Even if we had a test with 99% sensitivity and 99% specificity, the positive predictive value, given the prevalence of disease, would be 28%. Thus, most patients who are screened positive would not have the disease (false-positive). Yet, this scenario occurs daily in the practice of obstetrics (eg, most patients who undergo mid trimester amniocentesis or chorionic villous sampling do not have aneuploidy).
Conclusion
Risk assessment for severe late preeclampsia and stillbirth in the third trimester may be possible with the determination of maternal plasma concentrations of angiogenic and antiangiogenic factors at 30-34 weeks of gestation. Of interest, the StAmP trial (http://www.birmingham.ac.uk/research/activity/index.aspx), a randomized controlled trial to determine the effect of pravastatin on the changes of maternal angiogenic/antiangiogenic factor concentrations in patients with a diagnosis of early-onset preeclampsia, is ongoing in the United Kingdom. Statins have the potential to reverse the abnormalities in angiogenic/antiangiogenic factors
and may represent an intervention for patients identified with the approach herein. Other proposed therapeutic interventions to reverse an antiangiogenic state during pregnancy include the administration of VEGF 121
Accordingly, biomarkers investigated in this study in the third trimester may be useful as an additional tool for risk stratification in future interventional trials for the prevention of stillbirth and/or severe late preeclampsia at or near term. A specific clinical example is that patients at risk for stillbirth, after being identified by the markers proposed herein, can undergo intensive antepartum surveillance and deliver at or near term once the risks of prolonging pregnancy outweigh those of complications of prematurity.
Acknowledgment
The authors thank Professor Gordon C.S. Smith of Cambridge University, UK, for his advice and helpful discussions about the analyses performed in this study.
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A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
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Unexplained fetal death has a biological signature of maternal anti-fetal rejection: chronic chorioamnionitis and alloimmune anti-human leucocyte antigen antibodies.
Second-trimester prenatal screening for Down syndrome and the relationship of maternal serum biochemical markers to pregnancy complications with adverse outcome.
Second-trimester maternal serum marker screening: maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, estriol, and their various combinations as predictors of pregnancy outcome.
Association between second-trimester isolated high maternal serum maternal serum human chorionic gonadotropin levels and obstetric complications in singleton and twin pregnancies.
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