Typically, foetal flow is measured using Doppler ultrasonography to predict intrauterine foetal growth restriction, but Molvarec et al assess the comparative effectiveness of a new method, testing for the placental growth factor.
Hypertensive disorders are one of the most common complications of pregnancy, with a prevalence of between six and 22 percent.1,2 These conditions are responsible for the majority of maternal and perinatal morbidity and mortality, including intrauterine growth restriction (IUGR). A recent study has shown that unrecognised IUGR is the single largest risk factor to pregnancies that end in stillbirth.3 Research focus is now on improving antenatal detection of foetuses at risk, and allow selective management, timely delivery and minimisation of serious outcomes.
Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family. It is produced mainly by the placenta, and has potent pro-angiogenic effects. In normal uncomplicated pregnancy, PlGF levels rise until approximately pregnancy week 32 and then fall until delivery.4 In pregnancies complicated by preeclampsia before the 37th week, with or without IUGR, PlGF levels are significantly lower.5
The role of determination of sFlt-1, PlGF and other angiogenic factor levels in maternal peripheral blood in the prediction and diagnosis of preeclampsia has been extensively studied in recent years.6-20
We recently added to the limited information available about the levels of these factors in other forms of hypertension in pregnancy,21-24 and found that free PlGF measured before 35 weeks of pregnancy may predict preterm delivery in all forms of hypertensive disorders of pregnancy.25
There is still little information to date on their ability to predict foetal outcomes, such as intrauterine growth restriction, through their assessment of placental function.26
In this study, we used a new method, the Alere Triage PlGF test, for measuring free PlGF levels in the peripheral blood of hypertensive pregnant women before the 35th gestational week of pregnancy.
We examined its prognostic efficiency regarding adverse foetal outcomes in all forms of hypertensive disorders of pregnancy, compared with currently recommended assessment of foetal wellbeing.
In this observational study, 89 women with hypertensive disorders of pregnancy (19 women with preeclampsia, 12 with haemolysis, elevated liver enzymes and low platelet count (HELLP) syndrome, 17 with superimposed preeclampsia (SIPE), 24 with chronic hypertension, and 17 with gestational hypertension) were enrolled.
The study subjects were selected from the previously reported groups of hypertensive pregnant women25 based on availability of Doppler ultrasound results.
All subjects were Caucasian and resided in the same geographic area. Blood samples were taken in the First Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary between May 2008 and October 2010.
The blood draw occurred between the 22nd and 34th completed gestational week at the time of the first routine clinical blood test and repeated only if women were reclassified.
An interview was carried out with every subject after the diagnosis and again 12 weeks after the delivery, and if necessary, reclassification was done independently of the PlGF level.
Hypertension was diagnosed if systolic blood pressure (BP) was ≥ 140mmHg, or diastolic BP ≥ 90mmHg, on two occasions, six hours apart. According to the definitions of the ACOG and NHBPEP we classified the subjects’ diagnoses into four groups: chronic hypertension, gestational hypertension, preeclampsia, and superimposed preeclampsia.
We separately categorised any women with hypertension, proteinuria and the syndrome of haemolysis, elevated liver enzymes and low platelet count as HELLP.
Chronic hypertension was diagnosed if high blood pressure developed prior to pregnancy or before the 20th week of gestation, or if hypertension persisted for more than 12 weeks postpartum. Gestational hypertension was applied to women who developed new-onset hypertension after the 20th week of gestation, in the absence of proteinuria, confirmed after delivery.
Patients who later progressed to preeclampsia were excluded from this group, and following repeated blood draw, they were included in the preeclamptic group. Preeclampsia was defined as hypertension and proteinuria (≥ 300mg / 24 hours, or ≥ + by urine dipstick) with an onset after the 20th gestational week.
Severe preeclampsia was diagnosed if one of the following occurred: systolic BP ≥ 160mmHg, diastolic BP ≥ 110mmHg, proteinuria ≥ 5000mg per 24 hours or ≥ +++ by urine dipstick, partial HELLP syndrome, signs of renal insufficiency, pulmonary oedema or threatening eclampsia.
The diagnosis of HELLP syndrome was made based on characteristic laboratory findings (platelet count < 150G/l, SGOT and SGPT > 70U/l, LDH > 600U/l). It was made sure that all of the subjects in this group also met the criteria for severe preeclampsia.
Subjects with chronic hypertension who developed proteinuria after 20 weeks of gestation were categorised as superimposed preeclampsia. Subjects with preeclampsia, superimposed preeclampsia or HELLP syndrome were further grouped into early-onset (disease onset before 34th completed gestational week). Women with multiple gestations were not enrolled in this study.
A newborn was considered small for gestational age (SGA) if the birth weight was below the 10th percentile for gestational age and gender, according to a Hungarian birth weight percentile table.
All neonates with SGA had an asymmetric size (normal length, but low weight for gestational age at delivery), indicating that they had intrauterine growth restriction and were not constitutionally small. In addition, none of them had foetal structural abnormalities or genetic diseases.
For the determination of the abnormalities of foetal circulation we evaluated the last Doppler ultrasound results prior to delivery. Blood flow was examined in the umbilical and in the foetal middle cerebral arteries, as well as in the descending aorta of the foetus.
Abnormal foetal flow was diagnosed if diastolic block or reverse flow in the umbilical artery or descending aorta were present, or if there were signs of the centralisation of the foetal circulation (increased resistance in the umbilical artery or descending aorta with decreased resistance in the middle cerebral artery).
Our protocol for delivery dictates birth by caesarean section, as is clinically required, for women at any gestational age with HELLP syndrome, severe uncontrollable hypertension, severe proteinuria, with signs of renal insufficiency, pulmonary oedema, threatening eclampsia (persistent headache and visual disturbance), low platelet count (< 100,000/μl), elevated liver enzymes (SGOT > 70U/l) with epigastric pain, or evidence of foetal compromise (abnormal foetal flow or pathological CTG), severe IUGR or severe oligohydramnios. In the absence of any of these factors, we continue conservative management until the 37th completed gestational week.
After each blood draw, the EDTA-anticoagulated plasma samples were immediately centrifuged at 3000g for 10 minutes at 4°C and the supernatants were kept frozen at −80°C until assayed. Plasma was analysed for free PlGF using the Alere Triage PlGF assay according to the manufacturer’s instructions.
Using fluorescent-labelled monoclonal antibodies against PlGF for PlGF quantification, this immunoassay is run with a single-use disposable plastic assay test cartridge in conjunction with the Triage Meter.
Briefly, 250 microliter of thawed plasma (room temperature) is pipetted into the sample port of a new test cartridge. The cartridge is inserted into the meter and results are displayed in approximately 15 minutes in pg/ml. The cartridge contains chemistries for on-board positive and negative control systems. Control systems at the level of the cartridge and meter 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 contained within each kit of devices. The measurable range of the assay is between 12 and 3000pg/ml. Concentrations below 12pg/ml are value assigned based on the calibration curve, but this value is displayed to the user as a qualitative result ‘<12pg/ml’.
Women were tested up to 34 completed weeks, as recommended in the manufacturer’s product insert. The intra/inter-assay coefficients of variation at mean concentrations of 85.2 and 1300pg/ml were 12.1/12.8 percent and 11.7/13.2 percent, respectively.
Although PlGF concentrations fluctuate during pregnancy,4,27 original cut-off levels based on the 5th percentile of a PlGF level in a normal healthy pregnant population do not differ significantly from an absolute threshold of 100pg/ml in women presenting with signs and symptoms of preeclampsia before the 35th week of pregnancy.
Therefore levels were classified as normal (PlGF ≥ 100pg/ml), low (12pg/ml < PlGF < 100pg/ml) or very low (PlGF ≤ 12pg/ml).
Descriptive statistics were used to present the clinical characteristics. Outcomes were broken down into three PlGF groups and abnormal/normal foetal flow. All statistical analyses were conducted using MATLAB version 8.0. P-values were calculated using a two-tailed Fisher Exact test of each 2×2 contingency table (in this case a single PlGF cut-off of 100pg/ml was used).
The demographics and clinical characteristics of the 89 study participants are presented in Table 1.
By outcome, 61/89 women had a preterm birth and 22 neonates had IUGR. Table 2 shows the results of the foetal flow and PlGF tests by outcome (preterm birth and IUGR) and by diagnoses.
All 20 women with abnormal foetal flow had a PlGF < 100pg/ml so that the overall concordance between foetal flow and PlGF was high (p-value = 0.0023, two tailed Fisher Exact test of the contingency table dividing the 89 women by PlGF and foetal flow).
Considering the PlGF test results for the 28 women who had both a term birth and an infant of normal birth weight, one out of one woman with preeclampsia, two out of three women with SIPE, nine out of 11 women with GHT and five out of 13 women with CHT had a normal PlGF result. All of these women had a normal foetal flow.
Table 3 shows PlGF test results in women with normal and abnormal foetal flow. Of those who delivered preterm, 20/20 women with abnormal foetal flow and 36/41 women with normal foetal flow had low or very low PlGF.
Five women who had a preterm delivery had both a normal foetal flow and a normal PlGF test; of these five, two women had an early delivery because of premature rupture of membranes. All five women had infants with a normal birth weight.
Of the IUGR neonates, 22/22 had low or very low maternal PlGF and 10/22 had abnormal foetal flow. In the subset of 69 women who had normal foetal flow, a positive PlGF test result was significantly associated with adverse foetal outcomes (p-value < 0.0001 for preterm delivery and p-value = 0.0069 for IUGR).
Twenty-four of our study participants had pathological CTG, 21 of whom had low or very low PlGF. Oligohydramnios occurred in 27 cases, 20 of them had low or very low PlGF. Of those who delivered preterm, 19/20 women with pathological CTG and 37/41 women with normal CTG had low or very low PlGF.
In the preterm delivery group, 19/21 women with oligohydramnios and 37/40 women with normal amniotic fluid volume had low or very low PlGF. In the subset of 41 women with preterm delivery and normal foetal flow, 10/11 women with pathological CTG and 11/13 women with oligohydramnios had low or very low PlGF. Of the 22 IUGR neonates, 10 had pathological CTG and 10 had oligohydramnios.
We earlier evaluated the diagnostic accuracy of this test in the study groups and healthy pregnant control subjects,25 and found that using a gestational age-dependent threshold of five percent of a normal population, a positive PlGF test predicted delivery before 37 weeks in over 90 percent of hypertensive women.
In this study we compared the diagnostic value of PlGF measured before 35 weeks using two absolute thresholds with the last Doppler ultrasonography before delivery in identifying preterm birth and IUGR neonates.
We found that PlGF concentration below 100pg/ml identified all women with hypertensive disorders of pregnancy who required urgent delivery following an abnormal foetal flow result, and predicted all IUGR neonates, and 56/61 women who had a preterm delivery.
Nearly 60 percent of women with normal foetal flow results had a preterm delivery. Our protocol for delivery dictated birth by caesarean section, as clinically required, for women at any gestational age with evidence of foetal compromise including abnormal foetal flow, pathological CTG and severe oligohydramnios.
The Triage PlGF test identified the majority of women with normal foetal flow who needed to be delivered preterm due to pathological CTG or oligohydramnios. Considering both test results together, no woman with a normal foetal flow and normal PlGF had an IUGR infant, and only five had a preterm delivery, two of whom were due to premature rupture of membranes.
In a small study Benton et al found that Triage PlGF differentiated placental IUGR from constitutionally small foetuses with a sensitivity of 100 percent and a specificity of 86 percent.28
Consistently, all of our SGA neonates were asymmetrically small with no foetal structural abnormalities or genetic diseases suggesting placental IUGR and had low or very low PlGF.
In 2007 Schlembach et al correlated levels of angiogenic growth factors, including PlGF, with Doppler ultrasound parameters in women with preeclampsia and intrauterine growth restriction (suspected by ultrasound measurements and confirmed by birth weight).29
Maternal levels of PlGF were inversely correlated with PI values in both the umbilical and uterine arteries. PlGF levels in the umbilical vein were below the detection limit in nearly all samples of IUGR foetuses and lower than in those with preeclampsia (p < 0.001). In our study, we observed low or very low PlGF even in hypertensive women with normal foetal flow and an IUGR neonate.
Taylor et al reported in 2003 that maternal circulating PlGF levels in preeclampsia are lower if accompanied by IUGR.11 Furthermore they showed that in normotensive subjects with IUGR, PlGF concentrations are also decreased compared to controls. Our knowledge about IUGR in chronic and gestational hypertension in this regard is deficient.
We found a positive PlGF test in all women with an abnormal foetal flow, all women with an IUGR neonate, the majority of women with pathological CTG or oligohydramnios, as well as in a high proportion of women carrying normal sized foetuses, many of whom had a preterm delivery. Thus, the sensitivity of the PlGF test for foetal risk (IUGR, abnormal foetal flow, pathological CTG, oligohydramnios, preterm delivery) in the hypertension group with and without proteinuria was excellent.
The potential clinical impact of these findings is that PlGF may provide useful information before 35th gestational week to identify foetuses requiring urgent delivery, and those at risk of later adverse outcomes not identified by foetal flow Doppler ultrasonography.
Similarly, for all women with hypertensive disorders of pregnancy, a combination of a normal foetal flow and normal PlGF test may identify women at lower risk of adverse outcomes.
However, the weakness of our study is its retrospective observational design following normal clinical practice and the low case number. Additionally, we did not measure circulating levels of anti-angiogenic factors (sFlt-1, soluble endoglin).
In this retrospective observational study of a specific target population we are unable to calculate the specificity of either test with respect to preterm delivery or IUGR infant, or to calculate negative or positive predictive values. There is a need for prospective studies to prove the safety and efficiency of the test in the clinical management of hypertension in pregnancy.
The Triage test is a quick, reliable method for measuring circulating levels of free PlGF. The test may provide useful information before the 35th gestational week to identify foetuses requiring urgent delivery, and those at risk of later adverse outcomes not identified by foetal flow Doppler ultrasonography.
BMC Pregnancy and Childbirth 2013, 13:161 | doi:10.1186/1471-2393-13-161
© 2013 Molvarec et al.; licensee BioMed Central Ltd.
Attila Molvarec, First Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary.
Nóra Gullai, Balázs Stenczer, Gergely Fügedi, Bálint Nagy and János Rigó Jr.
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