Journal Pre-proof

Placental growth factor predicts time to delivery in women with signs or symptoms of
early preterm preeclampsia: a prospective multicenter study.

John R. Barton, M.D., M.S., Doug A. Woelkers, M.D., Roger B. Newman, M.D., C.
Andrew Combs, M.D., PhD., Helen Y. How, M.D., Kim A. Boggess, M.D., James
N. Martin, Jr., M.D., Kenneth Kupfer, PhD., Baha M. Sibai, M.D., For the PETRA
(Preeclampsia Triage by Rapid Assay) Trial

PII: S0002-9378(19)31108-1
DOI: https://doi.org/10.1016/j.ajog.2019.09.003
Reference: YMOB 12874

To appear in: American Journal of Obstetrics and Gynecology

Received Date: 30 January 2019

Revised Date: 30 August 2019
Accepted Date: 3 September 2019

Please cite this article as: Barton JR, Woelkers DA, Newman RB, Combs CA, How HY, Boggess KA,
Martin Jr. JN, Kupfer K, Sibai BM, For the PETRA (Preeclampsia Triage by Rapid Assay) Trial, Placental
growth factor predicts time to delivery in women with signs or symptoms of early preterm preeclampsia:
a prospective multicenter study., American Journal of Obstetrics and Gynecology (2019), doi: https://
doi.org/10.1016/j.ajog.2019.09.003.

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 Placental growth factor predicts time to delivery in
women with signs or symptoms of early preterm
preeclampsia: a prospective multicenter study.

John R. Barton, M.D., M.S.1

Doug A. Woelkers, M.D. 2
Roger B. Newman, M.D. 3
C. Andrew Combs, M.D., PhD. 4
Helen Y. How, M.D. 5
Kim A. Boggess, M.D. 6
James N. Martin Jr., M.D. 7
Kenneth Kupfer, PhD.8
Baha M. Sibai, M.D. 9,
For the PETRA (Preeclampsia Triage by Rapid Assay) Trial

1. Baptist Health Lexington, Lexington, KY

2. University of California, San Diego, CA
3. Medical University of South Carolina, Charleston, SC
4. Obstetrix Medical Group, San Jose, CA
5. Norton Healthcare, Louisville, KY
6. University of North Carolina, Chapel Hill, NC
7. University of Mississippi, Jackson, MS
8. Alere San Diego, San Diego, CA
9. University of Texas-Houston, Houston, TX

Corresponding author: John R Barton M.D., M.S.

1740 Nicholasville Rd., Lexington, KY 40503
Phone: 859-260-6970, fax: 859-260-6649
Email: jbarton@bhsi.com

Word count: Abstract 454

Main text 2601

Presented at the 36th Annual Clinical Meeting of the Society for Maternal–Fetal
Medicine, Atlanta, GA, February 1-6, 2016.
 2

Disclosures This was an investigator-led study. The study was supported by

funds from Alere (San Diego). Alere had no role in the study design, patient
recruitment, data collection, analysis, interpretation of data or writing of the
manuscript. No author has been paid by Alere to write or submit this manuscript
for publication. As the corresponding author, Dr Barton had full access to all the
data in the study and had final responsibility to submit for publication. Dr
Woelkers and Dr Sibai have previously been paid as consultants for Alere. Dr
Kupfer was an employee of Alere (San Diego) at the time the study was
conducted. All other authors report no conflict of interest.

Condensation: In women < 35 weeks gestation, a low placental growth factor

level is highly predictive of preterm delivery irrespective of a diagnosis of

preeclampsia.

Short title: Low placental growth factor is predictive of preterm delivery

AJOG at a Glance: A. The temporal relationship between the measured

angiogenic factors and the time to delivery in women presenting with suspected

preeclampsia at <35weeks gestation remains to be clarified.

B. As compared to women with normal placental growth factor, women with a low

placental growth factor level (< 100 pg/mL) have a hazard ratio of 7.17 (5.08,

10.13) in Cox regression for time-to-delivery after adjusting for both gestational

age at enrollment and the final diagnosis of preeclampsia.

C. In women with suspected preeclampsia at < 35.0 weeks, a low placental

growth factor level was strongly correlated with preterm delivery independent of a

diagnosis of preeclampsia or gestational age at presentation. This suggests that

placental growth factor levels are superior to clinical markers in predicting

adverse pregnancy in women presenting with suspected preeclampsia.

Keywords: preeclampsia, placental growth factor, angiogenic factors, prediction,

hypertension in pregnancy
 3

Abstract

Background. There is a robust association between altered angiogenic factor

concentrations, including placental growth factor and clinically recognized

preeclampsia. Alterations in concentrations of angiogenic factors precede the

clinical onset of preeclampsia by several weeks. The temporal relationship

between the measured angiogenic factors and the time to delivery in women

presenting with suspected preeclampsia at <35weeks gestation, however

remains to be clarified.

Objectives. To examine the relationship between placental growth factor (PlGF)

and time-to-delivery in women <35 weeks’ gestation presenting with signs or

symptoms of preeclampsia, and to compare the performance of PlGF to other

clinical markers for prediction of time-to-delivery in preeclampsia.

Study Design. Women with signs or symptoms of preeclampsia between 20.0

and 35.0 weeks were enrolled in a prospective, observational study at 24

centers. Blood was collected at presentation for PlGF, and subjects were

evaluated and managed according to local protocols. Clinical outcomes were

obtained and all final diagnoses were adjudicated by an independent expert

panel according to 2013 ACOG Hypertension in Pregnancy criteria. PlGF was

measured retrospectively on the Alere, Inc. Triage platform. A normal PlGF was

defined as >100 pg/ml and the assay’s limit of detection is 12 pg/ml. 2x2 tables

were constructed for comparison of test outcomes including negative predictive

value (NPV); time-to-delivery was analyzed by survival curves and Cox

regression.
 4

Results. 753 subjects were enrolled; 538 (71%) had a final diagnosis of

preeclampsia. 542 (72%) delivered <37 weeks and 358 (47%) <34 weeks.

Among the 279 (37%) women with a normal PlGF at presentation, the NPV for

preeclampsia delivered within 14 days, or within 7 days was 90% and 93%,

respectively. As compared to women with normal PlGF, women with PlGF < 100

pg/mL have a hazard ratio of 7.17 (5.08, 10.13) in Cox regression for time-to-

delivery after adjusting for both gestational age at enrollment and the final

diagnosis of preeclampsia. The PlGF levels of normal (>100 pg/mL), low (12 to

100 pg/mL), and very low (<12 pg/mL), have well separated distributions of time-

to-delivery with median values of 45, 10, and 2 days, respectively. Subjects with

PlGF < 100 pg/mL have a perinatal death rate of 5.7% and an SGA rate of

51.7%, while subjects with PlGF > 100 pg/mL have a perinatal death rate of 0%

(no observations in this cohort) and an SGA rate of 16.8%.

Conclusions. In women with suspected preeclampsia at < 35.0 weeks, a low

PlGF was strongly correlated with preterm delivery independent of a diagnosis of

preeclampsia or gestational age at presentation, whereas a normal PlGF was

associated with pregnancy prolongation, even in patients who ultimately had a

final diagnosis of preeclampsia. This suggests that PlGF levels are superior to

clinical markers in predicting adverse pregnancy in women presenting with

suspected preeclampsia.
 5

Introduction

Preeclampsia is a heterogeneous syndrome of pregnancy recognized by

traditional clinical criteria of hypertension plus proteinuria or clinical symptoms

such as cerebral, hematological or gastrointestinal.1 Severe adverse outcomes

related to preeclampsia such as eclampsia, stroke, and perinatal mortality are

tragic, but relatively uncommon. In fact, the majority of perinatal morbidity and

medical costs due to preeclampsia are related to issues of prematurity incurred

from preterm delivery.2

The pathogenesis of preeclampsia is complex and heterogeneous. The

traditional diagnosis and staging of preeclampsia are difficult and despite recent

changes in diagnostic criteria, the current clinical definitions do not reliably

predict adverse pregnancy outcomes. Therefore, there is a great need for

adjunctive and objective quantitative biomarker tests to assist clinicians in

managing patients presenting with signs and symptoms suggestive of

preeclampsia.3

There is a robust association between altered angiogenic factor

concentrations, including placental growth factor (PlGF) and clinically recognized

preeclampsia.4 Because alterations in concentrations of angiogenic factors

precede the clinical onset of preeclampsia by several weeks, a predictive

potential of these factors has been reported.3 Indeed, Levine et al5 demonstrated

lower mean PlGF concentrations in subjects who later develop preeclampsia and

in subjects with established preeclampsia, compared to those who never

developed the disease.

 6

Using separate assays, two recent studies from European cohorts4,6 with

rates of confirmed disease within 1 and 2 weeks of screening of 18% and 55%

respectively, reported high sensitivity and high negative predictive values using

the ratio of sFlt:1:PlGF or PlGF alone for the prediction of preeclampsia.

Moreover, some studies reported that measurement of PlGF levels could be

clinically useful in predicting adverse pregnancy outcomes in women with

suspected preeclampsia4,7 or predict SGA in the absence of clinically recognized

preeclmpsia8 These data although encouraging, do not clarify the temporal

association between the measured angiogenic factors and the time to delivery in

women presenting with suspected preeclampsia at <35weeks gestation. The

objectives of this study are to examine the relationship between PlGF and time-

to-delivery (TTD) in women <35 weeks gestation presenting with signs or

symptoms of preeclampsia; and to compare the performance of PlGF to other

clinical markers for prediction of TTD.

Materials and Methods

The PETRA Trial (Preeclampsia Triage by Rapid Assay) was a

prospective, observational, IRB-approved assessment of women age 18-45

years presenting with any signs or symptoms of preeclampsia between 20 and

41 weeks at 24 North American centers. Principal investigators and their study

sites are provided in Appendix 1. Subjects were enrolled by gestational age

blocks and venous blood samples for this study were collected at enrollment

before a final diagnosis was made. The qualifying signs or symptoms of

 7

preeclampsia were intentionally broad, intending to mimic the diverse findings

encountered in clinical practice. These include either hypertension, proteinuria,

laboratory abnormalities, excessive maternal weight gain, fetal growth restriction

(estimated fetal weight less than the 10th percentile for gestational age), or

clinical symptoms (Table 1). All diagnoses were adjudicated by an independent

panel with the final diagnosis of preeclampsia according to the 2013 ACOG Task

Force on Hypertension in Pregnancy definitions.1

EDTA-anticoagulated plasma samples (Becton Dickinson K2 EDTA tube)

were labeled and transported to the laboratory where they were immediately

centrifuged at 1300 g for 10min at 2-8 C and the supernatants were kept frozen

at -80 C until assayed. Plasma was analyzed for PlGF retrospectively in batch

using the Triage PlGF Test in a central laboratory facility (Alere, San Diego,

California) using the Triage PlGF assay according to the manufacturer’s

instructions. The test uses fluorescently labeled monoclonal antibodies against

PlGF for PlGF quantification and contains chemistries for on-board positive and

negative controls systems to ensure that the quantitative PlGF result is valid.

Briefly, 230 ul of thawed plasma (room temperature) is pipetted into the sample

port of a new test cartridge. The cartridge is inserted into the meter, and the

results are displayed and printed in 15 to 20 minutes in pg/ml. Full details of

assay methods are available in Appendix 2. The dynamic range is 12 to 3000

pg/ml and the coefficient of variation is 13%.

The reference range of PlGF was previously studied in healthy pregnancy,

by Saffer et al which established the gestational age dependent 5th percentile of

 8

PlGF.9 In a clinical study of preeclampsia by Chappell et al.,4 in subjects enrolled

prior to 35 weeks of gestation, a single cutoff of 100 pg/mL was shown to be as

effective as a gestational age dependent cutoff (the 5th percentile); whereas a

cutoff value of 12 pg/ml represented the lower detection limit of the assay.4,9

Chappell et al.4 also showed that PlGF did not perform well for subjects enrolled

after 35 weeks of gestation, even when using the gestational age dependent

cutoff. Therefore, our study was conducted to validate a prospectively selected

single cutoff (100 pg/mL) in the cohort of women presenting prior to 35 weeks of

gestation.

The PlGF concentration was dichotomized at 100 pg/mL and the

dichotomous PlGF together with gestational age and established clinical markers

(blood pressures, proteinuria, etc.) to predict the time to delivery in Cox

proportional hazards regression. The most convincing way of combining the

standard of care clinical biomarkers was via the final adjudicated diagnosis of

PE. The final diagnosis represents a composite that includes the entire sequence

of blood pressure measurements, proteinuria measurements, and other clinical

information collected during enrollment and throughout the clinical course of the

subject. Adjusting for final diagnosis demonstrates that a single PlGF value

collected at enrollment is an independent predictor of time to delivery.

The minimum total sample size was calculated based on the width of the

95% confidence interval of the sensitivity and the specificity with goal of

achieving a width of 10% at a point estimate of 90%. This consideration resulted

in a minimum total sample size of 150 preeclamptic and 150 non-preeclamptic

 9

subjects. This minimum total sample size was exceeded to achieve a minimum of

at least 20 preeclamptic and 20 non-preeclamptic subjects in each of four

gestational age intervals spanning the enrollment range from 20 weeks to 35

weeks gestation, noting that the prevalence of preeclampsia was not controlled

by enrollment, resulting in a total of 753 subjects comprised of 538 preeclamptic

and 215 non-preeclamptic subjects. All laboratory staff were masked to clinical

outcomes. All participants had delivered and pregnancy outcomes recorded

before biomarker concentrations were analyzed and revealed. Patients were

managed and delivery timing and mode were determined per local protocols.

Nonparametric statistical comparisons were made by Chi Square or

Kruskal Wallis test. Cox Proportional Hazards assessment was performed with

MATLAB coxphfit for the prediction of TTD censored at 14 days using univariate

models incorporating either PlGF alone (positive < 100 mg/mL vs negative > 100

mg/mL) or final clinical diagnosis (preeclampsia versus not preeclampsia); or

adjusted models incorporating PlGF with gestational age at delivery, presence or

absence of proteinuria, or final diagnosis of preeclampsia. For analysis, perinatal

mortality was defined as stillbirth or neonatal death. Small for gestational age

(SGA) was defined as birth weight less than the 10th percentile according to

Alexander et al.10

Results

1,258 subjects were initially enrolled over the study period from November

2010 to January 2012. 24 subjects were ineligible or did not follow the protocol,
 10

and 11 were withdrawn or lost to follow up, leaving 1,223 subjects. Six enrollment

samples could not be evaluated on the Triage device, leaving 1,217 subjects in

the entire cohort. 753 women were enrolled at < 35 weeks, and these constitute

the subjects of the current analysis and are presented in the concert flowchart.

(Figure 1).

Table 1 describes signs and symptoms at time of enrollment. The majority

(79.8%) were enrolled with either new onset hypertension or worsening

preexisting hypertension.

Table 2 describes maternal characteristics at the time of study enrollment.

Most (58.2%) were obese; 680 pregnancies (90.3%) were singleton gestations.

BMI was included as a covariate in the Cox proportional hazards regression. The

hazard ratio for BMI is weak and the adjusted hazard ratio for PlGF is the same

regardless of the BMI. Therefore, the results are independent of BMI. The study

is underpowered to report results in the subset of non-singleton pregnancies.

Smoking has been reported to influence angiogenic factors. Baseline

characteristics of smoking included a history of smoking 184 (24.4%), quit

smoking before pregnancy 69 (9.2%), quit smoking after pregnancy 60 (8.0%)

and never quit smoking 55 (7.3%).

Table 3 summarizes the final adjudicated diagnoses according to the 2013

ACOG definitions.1 The overall prevalence of preeclampsia was 71.4%, however,

125 subjects (16.6%) did not ultimately meet diagnostic criteria for any

hypertensive disorder. The inclusion of women with pregnancies affected by fetal

growth restriction with no evidence of hypertensive disorder introduces

 11

heterogeneity, however in sub-analysis the odds ratio for PlGF predicting preterm

delivery in this subset of 125 subjects is 33.5 (95%CI 9.35 to 120), consistent

with the odds ratio of 26.0 (16.8, 40.3) reported for the overall cohort (Table 6).

The median gestational age at delivery was 34.1 weeks, with 72% delivering < 37

and 47% delivering < 34 weeks. The median gestational age at delivery varied by

the final diagnosis and was significantly earlier for the 538 subjects with a final

diagnosis of preeclampsia (33.1 weeks) versus the other 215 subjects (37.6

weeks). D. The frequency of preeclampsia within the enrollment gestational

week categories of < 24+0, 24+0 to 27+6, 28+0 to 31+6, and 32+0 to 34+6 was

50%, 66%, 74% and 74%, respectively.

Table 4 summarizes the TTD from enrollment according to final

adjudicated clinical diagnosis. Women with preeclampsia plus severe features

and those with superimposed preeclampsia were more likely to deliver within 2

days, 7 days, and within 14 days as compared to the other groups (two-tailed

Fisher exact p-value < 0.0001 for the contingency table comparing two groups of

subjects, preeclampsia plus severe features and superimposed preeclampsia,

versus all other, by two delivery intervals, 0-14 days, versus >14 days)..

Figure 2 compares TTD according to PlGF levels of normal (>100), low

(12-100), or very low (<12 pg/ml). Within the group of 280 subjects that had a

very low PLGF level (< 12 pg/mL), delivery within two days was 5.6 fold more

likely than delivery after 14 days following enrollment (174 subjects versus 31

subjects, P < 0.0001). Figure 3 is a survival curve summarizing the relationship

between PlGF concentration and time to delivery in the presence or absence of

 12

the final diagnosis of preeclampsia. The PlGF levels of normal, low, and very low

have well separated distributions of TTD with median values of 45, 10, and 2

days, respectively.

The strength of PlGF as a predictor of TTD can also be demonstrated by

calculating the hazard ratio in Cox regression. The test positive group (PlGF <

100 pg/mL) as compared to the test negative group (PlGF > 100) has a hazard

ratio of 9.37 (95% confidence interval of 6.69 to 13.12) in univariate Cox

regression. The hazard ratio is 9.21 (6.58, 12.91) when adjusted for the

gestational age at enrollment. The hazard ratio is 7.55 (5.35, 10.65) when

adjusted for both the gestational age at enrollment and the presence of

proteinuria at enrollment. The hazard ratio is 7.17 (5.08, 10.13) when adjusted for

both gestational age at enrollment and the final diagnosis of preeclampsia. The

final diagnosis is a relatively weak predictor compared to PlGF in the same

regression, where it has a hazard ratio of 2.43 (1.75, 3.36). These hazard ratios

demonstrate that PlGF is strong and independent predictor of TTD.

Table 5 illustrates the relationship between TTD, final diagnosis, PlGF

dichotomous category (Test+, or Test-), and two selected perinatal outcomes of

importance; perinatal mortality and SGA (birth weight <10th %tile) infant.

Regardless of the presence or absence of a final diagnosis of preeclampsia or

delivery within or after 2 weeks from enrollment, a PlGF value of <100 pg/ml is

associated with the higher risk of subsequent perinatal death (1.3 to 11.4%)

and/or delivery of a SGA infant (48.9 to 63.2%). To simplify, subjects with PlGF <

100 pg/mL have a perinatal death rate of 5.7% (95% confidence interval of 3.8 to
 13

8.2%) and an SGA rate of 51.7% (47.1 to 56.3%), while subjects with PlGF > 100

pg/mL have a perinatal death rate of 0% (0 to 1.3%) and an SGA rate of 16.8%

(12.6 to 21.8%).

Finally, the potential clinical utility of a single PlGF measurement above or

below 100 pg/ml in predicting preeclampsia and/or delivery within 7 or 14 days is

illustrated in Table 6. The sensitivity and specificity for a final diagnosis of

preeclampsia, within either 7 or 14 days, is only 76% and 68%, respectively. On

the other hand, for ruling out deliveries within 7 or 14 days of enrollment, with or

without preeclampsia, the sensitivities of a single PlGF measurement are 90% or

greater and the NPVs are high (approximately 90%). In addition, for prediction of

preterm delivery before 37 weeks, a PlGF < 100 pg/ml shows good specificity

and sensitivity (85% and 82%, respectively) and a high PPV of 93.5%. In the 280

patients with a very low PlGF (<12 pg/ml) as compared to the rest of the cohort,

the prediction of preterm delivery before 37 weeks is very strong, with a PPV of

98.9% and an odds ratio of 72.5 (95% CI 22.9, 229.4).

Comment

Principal Findings

This prospective multicenter study with blinded adjudication of

preeclampsia diagnosis by an independent expert panel shows that among

women presenting with signs or symptoms of preeclampsia at less than 35

weeks’ gestation, PlGF levels (dichotomized about 100 pg/mL) had a sensitivity

of 76% in predicting a final diagnosis of preeclampsia. Further, PlGF levels

 14

strongly and independently predict the time to delivery within 7 and 14 days

(sensitivity 91 to 94%) irrespective of a diagnosis of preeclampsia. In addition,

PlGF levels identified pregnancies at risk of perinatal mortality, preterm delivery,

and an SGA infant. Moreover, PlGF levels may be useful to identify women with

confirmed preeclampsia that are not at risk for preterm birth or other adverse

perinatal outcomes.

Results

Ischemic placental syndrome resulting from defective deep placentation is

associated with a wide spectrum of obstetric complications that can include one

or more of the following: preeclampsia, fetal growth restriction, preterm birth,

abruptio placentae, oligohydramnios or fetal death. Some authors refer to these

as the “Great Obstetrical Syndromes”.11 PlGF is produced in the placenta and is

considered a marker of placental function and potentially fetal growth restriction

and/or fetal death.12,13 Disease of the placental vascular bed resulting in

placental ischemic injury is associated with decreased PlGF production. Our

findings reveal that extremely low PlGF values can be a marker for the Great

Obstetrical Syndromes.

In a prospective multicenter study from the UK that had a similar design to

our study, Chappell et al4 examined the diagnostic accuracy of a low plasma

PlGF concentration (<5th centile for gestation, <100 pg/ml) in 287 women

presenting with suspected preeclampsia between 20 and <35.0 weeks’ gestation.

The overall prevalence of preeclampsia in their study was 48.9%. In addition,

they reported that a PlGF <5th centile had a high sensitivity (0.96; 95%
 15

confidence interval, 0.89–0.99) and a high negative predictive value (0.98; 0.93–

0.995) for developing preeclampsia within 14 days; however, the specificity was

lower (0.55; 0.48–0.61). The authors4 concluded that for women who present

before 35 weeks’ gestation with suspected preeclampsia, low PlGF level has

high sensitivity and high negative predictive value for preeclampsia developing

within 14 days and that low PlGF levels are superior to other currently used

biomarkers to predict preeclampsia. In a subsequent study, the same

investigators14 evaluated the role of PlGF in predicting delivery of a small-for-

gestational-age infant and other adverse perinatal outcome in women with

suspected preterm preeclampsia. The primary outcome was the delivery of an

SGA infant with a birthweight less than the 3rd percentile for gestational age. As

in our study, they found that low PlGF values could be a helpful adjunct for

identifying those at high risk for an SGA infant who could benefit from close

antenatal surveillance and timely delivery.14

The concentration of free PlGF is inversely related to the circulating

amount of its soluble receptor, FMS like tyrosine kinase receptor-1 (sFlt-1), which

is synthesized and secreted by the placenta.5 The ratio of sFlt-1 to PlGF has

been reported to accurately predict preeclampsia and time to delivery in women

with suspected preeclampsia.6 In a European cohort (n=550) between 24+0 to

36+6 weeks using an sFlt-1:PlGF ratio cutoff of 38, Zeisler, et al6 reported a

negative predictive value of 99.3% for preeclampsia and delivery within one week

of presentation, as compared to our NPV of 93.2%. Notably, the prevalence of

preeclampsia in this cohort was only 17.8%, much less than the rate of 71.4% in
 16

our cohort, demonstrating that PlGF retains a clinically useful sensitivity and

negative predictive value in a high prevalence and high risk early preterm

population.

Clinical Implications

This study was designed to collect specimens for the validation of the

PlGF assay. The statistical analysis plan for the validation was created following

Chappell et al4 which was the initial study to propose (1) a fixed 100 pg/mL cutoff

for women enrolled prior to 35 weeks gestation and (2) an endpoint of delivery

(or delivery for confirmed preeclampsia) within 14 days. Chappell and colleagues

proceeded to conduct an RCT for management of suspected preeclampsia

based on medical decision logic using PlGF at the 100 pg/mL.15 Although the

RCT reports excellent and important results, the medical decision logic (using

PlGF at the 100 pg/mL cutoff) is based on a single observational cohort, the

original Chappell 2013 study. Our study verifies the original observations that

were used as the basis for a successful RCT.

In contrast to the study of Chappell et al4, the rate of preeclampsia in our

cohort was 71.4%. This finding is likely the consequence of our stratified

enrollment from all gestational ages, rather than consecutively enrolled patients

as in their study. Nonetheless, our findings are in agreement with those of

Chappell et al4 regarding the sensitivity (92.5%) of low PlGF levels predicting

preeclampsia with delivery within 14 days, with a negative predictive value of

90.3%
 17

The overall incidence of SGA in our study participants was 38.9%,

whereas the reported incidence by Chappell et al4 was 47%. In addition, the

incidence of SGA was 43.4% in those with a final diagnosis of preeclampsia, but

was 17.8% in those without preeclampsia. It is important to note that the rate of

an SGA infant was substantially higher in those with low PlGF level irrespective

of the presence of preeclampsia. Furthermore, a low PlGF level was more likely

to be associated with preterm delivery and perinatal mortality. Indeed, all of the

perinatal losses in this study were associated with a low PlGF level.

Gestational age ≥ 35 weeks was excluded in the cohort, because the

cutoffs for effective use of PlGF after 35 weeks are unclear. This limitation was

shown in a previous study of preeclampia.4 The reference range of PlGF was

studied in healthy pregnancy where it was shown that healthy subjects have

rapidly dropping PlGF values as they approach term.9 These values are often

less than 100 pg/mL, making PlGF difficult to interpret for GA ≥ 35 weeks.

Furthermore, a test with applicable predictive value for 14 days would allow a

window of management up to 37 weeks gestation, when delivery for

preeclampsia is specified by ACOG recommendations.1

The time to delivery could be influenced by demographic variables and

clinical variables, including differences in the practice of medicine at the different

clinical sites where the study was performed. Statistical control of these variables

on the predictive performance of PlGF was considered. However, it was found

that the dominant variables that influence time to delivery were gestational age at
 18

enrollment and final diagnosis, which itself is a composite of all the clinical

variables collected during and after enrollment.

Research Implications

Alterations in concentrations of angiogenic factors have been shown to

precede the clinical onset of preeclampsia by several weeks. Levine et al5

demonstrated lower mean PlGF concentrations in subjects who later develop

preeclampsia and in subjects with established preeclampsia, compared to those

who never developed the disease. Our results would also suggest that low PlGF

levels might identify those at high risk for adverse pregnancy outcome, including

perinatal loss, irrespective of a final diagnosis of preeclampsia.

Since all previous studies including our current investigation were

observational in nature, we agree with the conclusions of Chappell et al4, Seely

et al16, and others that randomized trials are needed to determine the potential

clinical value of adding PlGF measurements or sFlt-1:PlGF ratio in women with

suspected preeclampsia to assist in triage decisions and management of such

women.

Strengths and Limitations

Strengths of this study include enrollment at 24 study sites and a large

participant cohort encompassing a wide demographic and ethnic profile across

North America. Plasma testing was performed in a central laboratory ensuring

 19

that results were obtained with rigorous quality control. All final diagnoses were

adjudicated by an independent expert panel with revisions to accommodate the

2013 ACOG definitions of hypertension in pregnancy.1

Study limitations include test results were not validated in a repeat sample

or by comparative testing at a second laboratory. Further, the inclusion of 24

study sites with potentially varying presentations of preeclampsia and indications

for delivery represents a potential study limitation. Some explanation is perhaps

required on why these data are delayed in their submission for publication.

Although study enrollment was conducted between November 2010 and January

2012, the specimens were banked and the PlGF analysis was not complete until

August 2016. The project was suspended for business reasons and in August

2018, the manufacturer allowed the investigators to move forward on the

publication.

Conclusions

In summary, in women presenting with suspected preeclampsia < 35

weeks, a low PlGF is strongly correlated with the need for preterm delivery

independent of a final diagnosis of preeclampsia or gestational age at

presentation, whereas a normal PlGF predicts pregnancy prolongation, even in

patients destined to acquire a final diagnosis of preeclampsia. Among women

delivering within 2 weeks of enrollment, 89% had a final diagnosis of

preeclampsia, of which, 92% had a low PlGF level with high rates of adverse

perinatal outcome. In contrast, only 8% of patients with preeclampsia who

 20

delivered within 2 weeks of enrollment had a normal PlGF but these subjects had

no perinatal mortality and a low rate of SGA. This suggests that in women

presenting with suspected preeclampsia, low PlGF levels identify those at high

risk for adverse pregnancy outcome irrespective of a final diagnosis of

preeclampsia.
 21

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Hypertension in pregnancy. Report of the American College of Obstetricians and

Gynecologists’ task force on hypertension in pregnancy. Obstet Gynecol

2013;122:1122-31.

2. Stevens W, Shih T, Incerti D, Ton TGN, Lee HC, Peneva D,,et al. Short-term

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 24

TABLE 1. Signs and symptoms at enrollment

Enrollment Assessment N (%)*

New onset hypertension 371 (49.3)

Worsening underlying hypertension 230 (30.5)

New onset proteinuria 390 (51.8)

Headaches 262 (34.8)

Epigastric or right upper quadrant abdominal pain 61 (8.1)

Estimated fetal weight <10th percentile 129 (17.1)

Nausea and vomiting 74 (9.8)

*Some women had more than one sign or symptom

 25

TABLE 2. Clinical characteristics at enrollment and delivery

Characteristics Median (IQR) or N (%)

Age (years) 29.9 (24.8, 34.3)

Obese (BMI ≥ 30 kg/m2) 438 (58.2)

(BMI ≥ 40 kg/m2) 164 (21.8)

Nulliparous 378 (50.2)

White 403 (53.5)

Black 209 (27.8)

Hispanic 101 (13.4)

Prior preeclampsia 159 (21.1)

Pre-gestational diabetes 47 (6.2)

Gestational diabetes 103 (13.7)

Anti-phospholipid antibody syndrome 5 (0.7)

Lupus 9 (1.2)

Polycystic ovarian syndrome 27 (3.6)

Singleton gestation 680 (90.3)

Spontaneous Labor without C-Section 52 (6.9)

C-Section* 516 (68.5)

Induced Labor, excluding C-Section 185 (24.6)

*Includes cases of C-Section initiated after spontaneous onset of labor.

 26

TABLE 3. Final adjudicated diagnoses

Final Diagnosis N (%)

Preeclampsia 538 (71.4)

Preeclampsia with severe features 401 (53.3)

Preeclampsia without severe features 32 (4.2)

Superimposed preeclampsia 105 (13.9)

Gestational hypertension only 43 (5.7)

Chronic hypertension 47 (6.2)

No hypertensive disorder 125 (16.6)

Small for gestational age (SGA)* 292 (38.8)

*Of the 292 satisfying the definition of SGA, 234 are classified as

preeclampsia and 58 are classified as non-preeclampsia.

 27

TABLE 4. Time to delivery according to final diagnosis (% of N with

column totals summing to 100)

Time to PE with Super- PE GHTN Chronic No

delivery severe imposed without HTN hypertensive

(day) features PE severe Disorder

features

(N=401) (N=105) (N=32) (N=43) (N=47) (N=125)

0-2 47.1 29.5 18.8 9.3 4.3 13.6

3-7 15.2 20.0 9.4 9.3 2.1 5.6

8-14 8.7 10.5 3.1 2.3 2.1 4.8

>14 28.9 40.0 68.8 79.1 91.5 76.0

PE = preeclampsia

GHTN = gestational hypertension

HTN = hypertension
 28

Table 5. Time to delivery and diagnosis by PlGF (dichotomized about 100

pg/ml) including perinatal outcomes in each category.

TTD n (%) Diagnosis n (%) PlGF Category n (%) Death n (%) SGA n (%)

Test-, 11 (25.6%) 0 (0%) 4 (36.4%)

Non-PE, 43
(10.7%)
Test+, 32 (74.4%) 3 (9.4%) 16 (50%)
≤ 14 Days,
401 (53.3%)
Test-, 27 (7.5%) 0 (0%) 5 (18.5%)

PE, 358 (89.3%)

Test+, 331 (92.5%) 19 (5.7%) 162 (48.9%)

Test-, 137 (79.7%) 0 (0%) 19 (13.9%)

Non-PE, 172
(48.9%)
Test+, 35 (20.3%) 4 (11.4%) 19 (54.3%)
Were >14
Days, 352
(46.7%)
Test-, 104 (57.8%) 0 (0%) 19 (18.3%)

PE, 180 (51.1%)

Test+, 76 (42.2%) 1 (1.3%) 48 (63.2%)

PlGF = placental growth factor, TTD = time to delivery

Test + defined as PlGF < 100 pg/ml, Test - defined as PlGF > 100 pg/ml

PE = preeclampsia, SGA = small for gestational age (< 10th % tile)

 29

Table 6. Predictions based on PlGF < 100 pg/ml with diagnostic

performance for each prediction
Prediction Sensitivity Specificity PPV NPV Diagnostic
OR (95% CI)

All preeclampsia 75.7 68.8 85.9 53.0 6.86 (4.84,9.73)

Preeclampsia 92.5 63.8 69.8 90.3 21.6 (13.9,33.6)

and delivery
within 14d
Preeclampsia 93.9 58.8 61.6 93.2 22.0 (13.3,31.6)
and delivery
within 7d
Any delivery 90.5 68.5 76.6 86.4 20.7 (13.9,31.0)
within 14d

Any delivery 92.5 62.2 67.5 90.7 20.2 (12.9,31.6)

within 7d

Preterm delivery 81.7 85.3 93.5 64.5 26.0 (16.8,40.3)

PPV = positive predictive value

NPV = negative predictive value

PlGF = placental growth factor

 30

Legends for figures

Figure 1. Flowchart of study participants.

PlGF = placental growth factor, Wks = weeks of gestation

Figure 2. Time to delivery by placental growth factor concentration.

PlGF levels: normal (>100 pg/ml), low (12-100 pg/ml), very low (<12 pg/ml)

Figure 3. Relationship between placental growth factor concentration and time

to delivery according to final diagnosis of preeclampsia.

PE = preeclampsia, GA = gestational age

PlGF levels: normal (>100 pg/ml), low (12-100 pg/ml), very low (<12 pg/ml)
 31

Appendix 1

PETRA investigators and institutions

Principal investigator Institution

R.H. Ball Maternal Fetal Services of Utah, Salt Lake City, UT
J.R. Barton Baptist Health Lexington, Lexington, KY
R. Beyerlein Clinical Trials of America, Inc., Eugene, OR
K.A. Boggess University of North Carolina, Chapel Hill, NC
C.A.Combs Obstetrix Medical Group, San Jose, CA
L. A. Friel University of Texas Health Sciences Center, Houston,
TX
C. Gyamfi-Bannerman Columbia University Medical Center, New York, NY
H.Y. How Norton Healthcare, Louisville, KY
S. Joy Carolinas HealthCare System, Charlotte, NC
A. Lee Corvallis Clinic, Corvallis, OR
B.J. Levine Phoenix OB/GYN, Moorestown, NJ
D.F. Lewis Jr Greater Cincinnati OB/GYN, Inc., Cincinnati, OH
J.N. Martin Jr University of Mississippi, Jackson, MS
A.L. Ness Saint Peter's University Hospital, New Brunswick, NJ
L. Pereira Oregon Health & Science University, Portland, OR
R.R. Reagan Women’s Healthcare, San Diego, CA
C.J. Robinson Medical University of South Carolina, Charleston, SC
S. Sadanandan Regional Obstetrical Consultants, Chattanooga, TN
C. Saffer West Coast OB/GYN, San Diego, CA
S. Sunderji ProMedica Physician Group,Toledo, OH
F. Tillman Discovery Clinical Trials, Dallas, TX
P. von Dadelszen BC Women’s Hospital and Health Centre, Vancouver,
BC, Canada
A. Wagner Saginaw Valley Medical Research Group, LLC,
Saginaw, MI
D.A. Woelkers University of California, San Diego, CA
 32

Appendix 2

Placental growth factor assay methods

The Alere Triage® PlGF Test (San Diego, CA, USA) is a fluorescence
immunoassay used with the Triage® Meter for the quantitative determination of
PlGF in EDTA-anticoagulated plasma specimens. The test procedure involves
the addition of several drops (230ul) of plasma to the sample port of the single-
use disposable plastic cartridge. The test uses fluorescently-labeled monoclonal
antibodies against PlGF for PlGF quantification and contains chemistries for on-
board positive and negative controls systems to ensure that the quantitative PlGF
result is valid. Calibration information is supplied by the manufacturer in the form
of a lot-specific EPROM chip that is provided with each kit of devices. The
measurable range of the assay is 12–3000 pg/mL. The total precision
(coefficient of variation) on plasma controls for PlGF at concentrations of 85 and
1300 pg/ml is 12.8% and 13.2%, respectively. The concentration of PlGF is
displayed on the meter screen approximately 15-20 minutes from the addition of
specimen.
Study Enrollment
1258
Enrolled
24 ineligible or
off protocol
1234
11 withdrawal or
lost to f/u
1223
6 unevaluable PlGF
1217
464 delivered >350 wks
753*
<350 wks

40 185 234 294

<240 wks 240-286 w 290-316 w 320-346 wks

PE = 20 PE = 128 PE = 171 PE = 219

PE = Preeclampsia,*62 Twins, 11 triplets