Dietmar Schlembach

An automated method for the determination of the sFlt-1/PIGF ratio in the assessment of preeclampsia

OBJECTIVE The angiogenic and antiangiogenic factors soluble fms-like tyrosine kinase (sFlt)-1 and placental growth factor (PIGF) have been implicated in the mechanisms of disease responsible for preeclampsia (PE). Moreover, it has been proposed that the concentrations of these markers in maternal serum/plasma may have predictive value. This study evaluates a newly developed Elecsys (Roche, Penzberg, Germany) assay for sFlt-1 and PIGF and tests the value of the sFlt-1/PIGF ratio in the assessment of PE. STUDY DESIGN This multicenter case-control study included 351 patients: 71 patients with PE and 280 gestational age-matched control subjects from 5 European study centers. A total of 595 serum samples were measured for sFlt-1 and PIGF using an automated platform. RESULTS Maternal serum concentrations of sFlt-1 and PIGF significantly separated healthy women and women with PE. The sFlt-1/PIGF ratio had an area under the receiver operating characteristic curve of 0.95. The best performance was obtained in the identification of early-onset PE (area under the receiver operating characteristic curve of 0.97). CONCLUSION Measurement of sFlt-1 and PIGF and calculation of sFlt-1/PIGF ratio can be performed quickly and in a platform available in clinical laboratories. This is a substantial step forward in bringing the determination of these analytes to clinical practice in obstetrics. We propose that sFlt-1, PIGF, and sFlt-1/PIGF ratio may be of value in the prediction of PE and in the differential diagnosis of patients with atypical presentations of PE, and perhaps in the differential diagnosis of women with chronic hypertension suspected to develop superimposed PE.

The sFlt-1/PlGF ratio in different types of hypertensive pregnancy disorders and its prognostic potential in preeclamptic patients

OBJECTIVE The soluble fms-like tyrosine kinase (sFlt-1)/placental growth factor (PlGF) ratio is a reliable tool in the assessment of preeclampsia. We tested the hypothesis that the sFlt-1/PlGF ratio is able to identify women at risk for imminent delivery. We characterized the sFlt-1/PlGF ratio in different types of hypertensive pregnancy disorders. STUDY DESIGN We investigated 388 singleton pregnancies with normal pregnancy outcome, 164 with PE, 36 with gestational hypertension, and 42 with chronic hypertension. sFlt-1 and PlGF were measured in serum samples. RESULTS Patients with preeclampsia had a significantly increased sFlt-1/PlGF ratio as compared with controls and with patients with chronic and gestational hypertension in <34 weeks and ≥34 weeks (P < .001). Time to delivery was significantly reduced in women with preeclampsia in the highest quartile of the sFlt-1/PlGF ratio (P < .001). CONCLUSION The sFlt-1/PlGF ratio allows the identification of women at risk for imminent delivery and is a reliable tool to discriminate between different types of pregnancy-related hypertensive disorders.

Angiogenic growth factors in maternal and fetal serum in pregnancies complicated by intrauterine growth restriction

The present study was performed to compare serum concentrations of maternal and fetal angiogenic growth factors in IUGR (intrauterine growth restriction) and normal pregnancy at the time of delivery. VEGF (vascular endothelial growth factor), PlGF (placental growth factor), sFlt-1 (soluble fms-like tyrosine kinase 1), sKDR (soluble kinase domain receptor) and bFGF (basic fibroblast growth factor) were measured by ELISA in serum from a maternal peripheral vein, the umbilical vein and the umbilical arteries in 15 women with pregnancies complicated by IUGR and 16 controls (women with normal pregnancies). In IUGR, sFlt-1 was increased, and PlGF and sKDR were decreased, in both maternal serum and serum from the umbilical vein. Additionally, bFGF was increased in serum from the umbilical vein of women with pregnancies complicated by IUGR. No significant differences in growth factor concentrations between the groups were found in serum from the umbilical artery. In both groups, levels of VEGF were higher and levels of sFlt-1 were lower in serum from the umbilical vein and umbilical artery compared with maternal serum. PlGF levels were found to be lower in serum from the umbilical vein compared with maternal serum in both groups, whereas PlGF levels in serum from the umbilical artery were significantly lower only in the control group. These findings suggest an imbalance of angiogenic and anti-angiogenic factors in IUGR, with formation of an anti-angiogenic state in maternal and, to a lesser extent, umbilical vein blood. The placenta appears to play a central role in the release of sFlt-1 into maternal and umbilical blood. Umbilical artery blood was unaffected in IUGR, indicating that the fetus does not contribute to changes in angiogenic growth factor concentrations.

Perinatal morbidity and mortality in early‐onset fetal growth restriction: cohort outcomes of the trial of randomized umbilical and fetal flow in Europe (TRUFFLE)

Few data exist for counseling and perinatal management of women after an antenatal diagnosis of early‐onset fetal growth restriction. Yet, the consequences of preterm delivery and its attendant morbidity for both mother and baby are far reaching. The objective of this study was to describe perinatal morbidity and mortality following early‐onset fetal growth restriction based on time of antenatal diagnosis and delivery.

2 year neurodevelopmental and intermediate perinatal outcomes in infants with very preterm fetal growth restriction (TRUFFLE): A randomised trial

BACKGROUND No consensus exists for the best way to monitor and when to trigger delivery in mothers of babies with fetal growth restriction. We aimed to assess whether changes in the fetal ductus venosus Doppler waveform (DV) could be used as indications for delivery instead of cardiotocography short-term variation (STV). METHODS In this prospective, European multicentre, unblinded, randomised study, we included women with singleton fetuses at 26-32 weeks of gestation who had very preterm fetal growth restriction (ie, low abdominal circumference [<10th percentile] and a high umbilical artery Doppler pulsatility index [>95th percentile]). We randomly allocated women 1:1:1, with randomly sized blocks and stratified by participating centre and gestational age (<29 weeks vs ≥29 weeks), to three timing of delivery plans, which differed according to antenatal monitoring strategies: reduced cardiotocograph fetal heart rate STV (CTG STV), early DV changes (pulsatility index >95th percentile; DV p95), or late DV changes (A wave [the deflection within the venous waveform signifying atrial contraction] at or below baseline; DV no A). The primary outcome was survival without cerebral palsy or neurosensory impairment, or a Bayley III developmental score of less than 85, at 2 years of age. We assessed outcomes in surviving infants with known outcomes at 2 years. We did an intention to treat study for all participants for whom we had data. Safety outcomes were deaths in utero and neonatal deaths and were assessed in all randomly allocated women. This study is registered with ISRCTN, number 56204499. FINDINGS Between Jan 1, 2005 and Oct 1, 2010, 503 of 542 eligible women were randomly allocated to monitoring groups (166 to CTG STV, 167 to DV p95, and 170 to DV no A). The median gestational age at delivery was 30·7 weeks (IQR 29·1-32·1) and mean birthweight was 1019 g (SD 322). The proportion of infants surviving without neuroimpairment did not differ between the CTG STV (111 [77%] of 144 infants with known outcome), DV p95 (119 [84%] of 142), and DV no A (133 [85%] of 157) groups (ptrend=0·09). 12 fetuses (2%) died in utero and 27 (6%) neonatal deaths occurred. Of survivors, more infants where women were randomly assigned to delivery according to late ductus changes (133 [95%] of 140, 95%, 95% CI 90-98) were free of neuroimpairment when compared with those randomly assigned to CTG (111 [85%] of 131, 95% CI 78-90; p=0.005), but this was accompanied by a non-significant increase in perinatal and infant mortality. INTERPRETATION Although the difference in the proportion of infants surviving without neuroimpairment was non-significant at the primary endpoint, timing of delivery based on the study protocol using late changes in the DV waveform might produce an improvement in developmental outcomes at 2 years of age. FUNDING ZonMw, The Netherlands and Dr Hans Ludwig Geisenhofer Foundation, Germany.

New Gestational Phase–Specific Cutoff Values for the Use of the Soluble fms-Like Tyrosine Kinase-1/Placental Growth Factor Ratio as a Diagnostic Test for Preeclampsia

To establish gestational phase adapted cutoffs for the use of the soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio as a diagnostic tool for preeclampsia in the clinical setting, a multicenter case–control study including a total of 1149 patients was performed. We report normal values of sFlt-1, PlGF, and the sFlt-1/PlGF ratio based on the analysis of a total of 877 patients with uneventful pregnancy outcome. A total of 234 patients with preeclampsia and a matched cohort consisting of 468 patients with normal pregnancy outcome were compared, and sFlt-1 and PlGF were measured on an automated platform. Separate cutoffs for the sFlt-1/PlGF ratio were determined for the early (20+0–33+6 weeks) and the late gestational phase (34+0 weeks–delivery). For each of the 2 gestational phases, 2 independent cutoffs framing an equivocal zone were determined: the first cutoff with focus on high sensitivity, and the second focusing on high specificity. Between 20+0 and 33+6 weeks, the cutoffs at ⩽33 and ≥85 resulted in a sensitivity/specificity of 95%/94% and 88%/99.5%, respectively. An sFlt-1/PlGF ratio of ⩽33 had the lowest likelihood of a negative test (0.05; 95% confidence interval, 0.02–0.13), whereas values ≥85 had the highest likelihood of a positive test (176; 95% confidence interval, 24.88–1245). After 34+0 weeks, the cutoffs at ⩽33 and ≥110 yielded a sensitivity/specificity of 89.6%/73.1% and 58.2%/95.5%, respectively. The approach to use multiple cutoffs for the early and late gestational phase enhances the diagnostic accuracy of the sFlt-1/PlGF ratio as a diagnostic tool for preeclampsia.

Anhydramnios associated with administration of trastuzumab and paclitaxel for metastatic breast cancer during pregnancy

A 38-year-old women in her second pregnancy presented with symptomatic metastatic spinal-cord compression 7 years after undergoing lumpectomy and axillary dissection for stage I primary breast cancer. Immunohistochemistry analysis of the tumour had shown that it was oestrogen-receptor negative, progesterone-receptor positive, and overexpressed ERBB2. The patient had received six cycles of cyclophosphamide, methotrexate, and fl uorouracil followed by radiotherapy and then tamoxifen, which she had taken for 5 years. 86 months after primary diagnosis the patient developed paresthesia and hypoesthesia of the left arm and pain in the cervical vertebrae. MRI showed diff use metastatic infi ltration of the corpus of the second cervical vertebra (fi gure 1) with spinal-cord compression. Additional lesions in the fourth thoracic vertebra and the left femur were also seen with bone scintigraphy, but no other signs of metastatic disease were identifi ed by clinical examination, chest radiograph, or abdominal ultrasound. At this time the patient was 17 weeks pregnant with normal fetal development. After counselling the patient decided to continue pregnancy and was started on hydromorphone hydrochloride. Palliative radiotherapy of 46 Gy given in 23 fractions was administered to the cervical vertebra, which resulted in clinically improved neurological symptoms and pain. Cervical radiotherapy was undertaken with lead shielding of the uterus to protect the fetus. At 25+6 weeks’ gestation the patient received trastuzumab (8 mg/kg loading dose) combined with 175 mg/m of paclitaxel, followed by another cycle at 28+5 weeks with the dose of trastuzumab reduced to 6 mg/kg and the dose of paclitaxel kept the same. Close fetal surveillance was undertaken. Between 26 weeks’ gestation and 32 weeks’ gestation, during two cycles of trastuzumab and paclitaxel, fetal abdominal circumference stopped increasing and the volume of amniotic fl uid decreased to almost anhydramnios (fi gure 2). The mother was of normal constitutional size with normal weight gain of 11 kg during pregnancy and no other risk factors for restriction of intrauterine growth. Tests for premature rupture of the membranes were negative. At 31+6 weeks the volume of both fetal kidneys was decreased below the fi fth percentile. Additionally, the urinary bladder was barely visible, suggesting reduced renal function, and doppler sonography showed increased resistance indices of both the renal arteries (fi gure 3). Doppler sonography of the fetal umbilical and maternal uterine arteries was normal, which suggested healthy placental function. Serial ultrasound measurements of femur length, biparietal diameter, and head circumference were all within normal limits. As a result of the evidence of fetal renal failure and cessation of abdominal growth, fetal lung maturation was induced with corticosteroids after two cycles of trastuzumab and paclitaxel, and a caesarean section was done at 32+1 weeks’ gestation. The male newborn infant weighed 1460 g (tenth percentile), had a body length of 39 cm, and had a head circumference of 29.5 cm. The pH value of the umbilical artery was 7.31. The placenta weighed 290 g and placental histology was normal. The newborn infant showed signs of bacterial sepsis with hypotension, transient renal failure, respiratory failure necessitating mechanical ventilation, and positive laboratory fi ndings (C-reactive protein 30 mg/dL). With antibiotic treatment blood pressure normalised after 2 days and mechanical ventilation was ended on day 6. Diuresis was adequate with serum creatinine slightly increased (1.6 mg/dL) until day 14. Ultrasonography of the fetal kidneys showed transient hyperechodensities in the renal parenchyma that resolved by day 28. These transient hyperechodensities are often noted in newborn infants with transient renal failure as a result of decreased renal perfusion. Echocardiography and cranial ultrasound examinations were normal. The infant was discharged at age 6 weeks weighing 2335 g and in healthy condition. Development at 12 weeks was normal. Lancet Oncol 2007; 8: 79–81

Angiogenic growth factor levels in maternal and fetal blood : correlation with doppler ultrasound parameters in pregnancies complicated by pre-eclampsia and intrauterine growth restriction

To correlate levels of angiogenic growth factors with Doppler ultrasound parameters in pregnancies complicated by pre‐eclampsia and intrauterine growth restriction (IUGR).

Calibrated automated thrombin generation in normal uncomplicated pregnancy

Pregnancy is associated with substantial changes in the haemostatic system and a six-fold higher incidence of venous thromboembolism. Conventional global tests, such as prothrombin time and activated partial thromboplastin time, do not definitely detect this hypercoagulable condition. We investigated whether the changes in haemostatic system during pregnancy are reflected in the calibrated automated thrombography (CAT). Thrombin generation was measured in platelet-poor plasma (PPP) of 150 healthy pregnant women without any pregnancy associated diseases by means of CAT. In addition, prothrombin (FII), antithrombin (AT), protein S, protein C, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TAT), and prothrombin fragments 1+2 (F1+2) were measured. Endogenous thrombin potential (ETP) and peak of thrombin generation increased significantly with gestational weeks, while lag time and time to peak remained unchanged. A significant increase of PAI-1, TFPI, F1+2 and TAT as well as a significant decrease of free protein S, protein S antigen, and protein S activity was observed. Levels of AT and protein C remained stable during pregnancy. Division of population in trimester of pregnancy and analysis of differences between the trimesters showed rather similar results. Our study shows that endogenous thrombin potential does increase with duration of normal uncomplicated pregnancy. Whether parameters of continuous thrombin generation will correlate with thrombembolic disease remains to be shown.

Implementation of the sFlt-1/PlGF ratio for prediction and diagnosis of pre-eclampsia in singleton pregnancy: implications for clinical practice

Pre-eclampsia (PE) is a leading cause of maternal and fetal/neonatal morbidity and mortality worldwide. Clinical diagnosis and definition of PE is commonly based on the measurement of non-specific signs and symptoms, principally hypertension and proteinuria1–3. However, due to the recognition that measurement of proteinuria is prone to inaccuracies and the fact that PE complications often occur before proteinuria becomes significant, most recent guidelines also support the diagnosis of PE on the basis of hypertension and signs of maternal organ dysfunction other than proteinuria3–5. Furthermore, the clinical presentation and course of PE is variable, ranging from severe and rapidly progressing early-onset PE, necessitating preterm delivery, to late-onset PE at term. There may be associated intrauterine growth restriction (IUGR), further increasing neonatal morbidity and mortality. These features suggest that the classical standards for the diagnosis of PE are not sufficient to encompass the complexity of the syndrome. Undoubtedly, proper management of pregnant women at high risk for PE necessitates early and reliable detection and intensified monitoring, with referral to specialized perinatal care centers, to reduce substantially maternal, fetal and neonatal morbidity6,7. In the decade since Maynard et al.8 reported that excessive placental production of soluble fms-like tyrosine kinase receptor-1 (sFlt-1), an antagonist of vascular endothelial growth factor and placental growth factor (PlGF), contributes to the pathogenesis of PE, extensive research has been published demonstrating the usefulness of angiogenic markers in both diagnosis and the subsequent prediction and management of PE and placenta-related disorders. Various reports have demonstrated that disturbances in angiogenic and antiangiogenic factors are implicated in the pathogenesis of PE and have possible relevance in the diagnosis and prognosis of the disease. Increased serum levels of sFlt-1 and decreased levels of PlGF, thereby resulting in an increased sFlt-1/PlGF ratio, can be detected in the second half of pregnancy in women diagnosed to have not only PE but also IUGR or stillbirth, i.e. placenta-related disorders. These alterations are more pronounced in early-onset rather than late-onset disease and are associated with severity of the clinical disorder. Moreover, the disturbances in angiogenic factors are reported to be detectable prior to the onset of clinical symptoms (disease), thereby allowing discrimination of women with normal pregnancies from those at high risk for developing pregnancy complications, primarily PE9–30. Plasma concentrations of angiogenic/antiangiogenic factors are of prognostic value in obstetric triage: similar to the progressively worsening clinical course observed in women with early-onset PE, changes in the angiogenic profile leading to a more antiangiogenic state can be found. Current definitions of PE are poor in predicting PE-related adverse outcomes. A diagnosis of PE based on blood pressure and proteinuria has a positive predictive value of approximately 30% for predicting PE-related adverse outcomes31. Estimation of the sFlt-1/PlGF ratio allows identification of women at high risk for imminent delivery and adverse maternal and neonatal outcome23,30,32–35. Moreover, it has also been shown that the time-dependent slope of the sFlt-1/PlGF ratio between repeated measurements is predictive for pregnancy outcome and the risk of developing PE, and repeated measurements have been suggested36. However, the ‘optimal’ time interval for a follow-up test remains unclear. Finally, high values are closely related to the need to deliver immediately22,23,37. Additionally, in normal and complicated pregnancies, angiogenic factors are correlated with Doppler ultrasound parameters, mainly uterine artery (UtA) indices38–42. Combining the sFlt-1/PlGF ratio with UtA Doppler ultrasound, at the time of diagnosis of early-onset PE, has prognostic value mainly for perinatal complications, being limited for the prediction of maternal complications37,43. The additional measurement of the sFlt-1/PlGF ratio has been shown to improve the sensitivity and specificity of Doppler measurement in predicting PE44–48, supporting its implementation in screening algorithms. Whereas studies on the predictive efficacy of the sFlt-1/PlGF ratio in the first trimester have yielded contradictory results49, reports on the use of this marker as an aid in prediction from the mid trimester onwards have led to its suggested use as a screening tool, especially for identifying all women developing PE and requiring delivery within the subsequent 4 weeks50–52. This short review of the literature highlights that measurement of the sFlt-1/PlGF ratio has the potential to become an additional tool in the management of PE, particularly as automated tests that allow rapid and easy measurement of these markers are now widely available. Nevertheless, although these markers were incorporated recently into the German guidelines53, no formal recommendation regarding how to use sFlt-1, PlGF or the sFlt-1/PlGF ratio has been established in any official protocol. The purpose of this paper is to answer questions that are frequently asked around the use of the sFlt-1/PlGF ratio in the diagnosis and prediction of PE and regarding the implications for clinical practice, in particular, ‘When?’ and ‘In which women?’ should the sFlt-1/PlGF ratio be measured and, ‘What should be done with the results?’, and to provide guidance to educate physicians on the use of the sFlt-1/PlGF ratio in clinical practice. To achieve this, international experts in the use of angiogenic markers have strived to develop a consensus statement on the clinical use of the sFlt-1/PlGF ratio and the consequential management in pregnant women with suspected PE or at a high risk of developing PE.