sFlt-1/PlGF for prediction of early-onset pre-eclampsia: STEPS (Study of early pre-eclampsia in Spain)

Ultrasound Obstet Gynecol 2017; : 373–38250 Published online in Wiley Online Library (wileyonlinelibrary.com). 10.1002/uog.17373. This is an open access article under the termsDOI: of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. sFlt-1/PlGF for prediction of early-onset pre-eclampsia: STEPS (Study of Early Pre-eclampsia in Spain) A. PERALES1, J. L. DELGADO2, M. DE LA CALLE3, J. A. GARC´ IA-HERN ´ ANDEZ4, A. I. ESCUDERO5, J. M. CAMPILLOS6, M. D. SARABIA2, B. LA´ IZ1, M. DUQUE3, M. NAVARRO4, P. CALMARZA6, M. HUND7and F. V. ´ ALVAREZ5, on behalf of the STEPS investigators# 1Hospital Universitario y Polit´ecnico La Fe, Valencia, Spain; 2Universidad de Murcia and IMIB-Arrixaca, Murcia, Spain; 3Hospital Universitario La Paz, Madrid, Spain; 4Hospital Universitario Materno Infantil de Canarias, Gran Canaria, Spain; 5Hospital Universitario Central de Asturias, Oviedo, Spain; 6Hospital Universitario Miguel Servet, Zaragoza, Spain; 7Roche Diagnostics International Ltd, Rotkreuz, Switzerland KEYWORDS: biomarker; early-onset prediction; hypertension; PlGF; pre-eclampsia; sFlt-1; sFlt-1/PlGF ratio ABSTRACT Objective A high ratio of soluble fms-like tyrosine kinase-1 (sFlt-1) to placental growth factor (PlGF) has been linked to pre-eclampsia (PE). We evaluated the sFlt-1/PlGF ratio as a predictive marker for early-onset PE in women at risk of PE. Methods This prospective, Spanish, multicenter study included pregnant women with a risk factor for PE, including intrauterine growth restriction, PE, eclampsia or hemolysis, elevated liver enzymes and low platelet count syndrome in previous pregnancy, pregestational diabetes or abnormal uterine artery Doppler. The primary objective was to show that the sFlt-1/PlGF ratio at 20, 24 and 28 weeks’ gestation was predictive of early-onset PE ( 0 weeks). Serum sFlt-1 and PlGF were measured<34 + at 20, 24 and 28 weeks. Multivariate logistic regression was used to develop a predictive model. Results A total of 819 women were enrolled, of which 729 were suitable for analysis. Of these, 78 (10.7%) women developed PE (24 early onset and 54 late onset). Median sFlt-1/PlGF ratio at 20, 24 and 28 weeks was 6.3 (interquartile range (IQR), 4.1–9.3), 4.0 (IQR, 2.6–6.3) and 3.3 (IQR, 2.0–5.9), respectively, for women who did not develop PE (controls); 14.5 (IQR, 5.5–43.7), 18.4 (IQR, 8.2–57.9) and 51.9 (IQR, 11.5–145.6) for women with early-onset PE; and 6.7 (IQR, 4.6–9.9), 4.7 (IQR, 2.8–7.2) and 6.0 (IQR, 3.8–10.5) for women with late-onset PE. Compared with early-onset PE, the sFlt-1/PlGF ratio was significantly lower in controls (P<0.001 at each timepoint) and in women with chronic hypertension ( 0.001 at each timepoint), gestationalP< Correspondence to: Dr A. Perales, Hospital Universitario y Polit´ecnico La Fe, Avda F. Abril Martorell 106 Torre F, 3aPlanta, Valencia 46026, Spain (e-mail: [email protected]) The copyright line for this article was changed on 14 February 2018 after original online publication. #STEPS investigators are listed at the end of the article. Accepted: 11 November 2016 hypertension ( 0.001 at each timepoint) and late-onsetP< PE (P<0.001 at each timepoint). A prediction model for early-onset PE was developed, which included the sFlt-1/PlGF ratio plus mean arterial pressure, being parous and previous PE, with areas under the receiver–operating characteristics curves of 0.86 (95% CI, 0.77–0.95), 0.91 (95% CI, 0.85–0.97) and 0.93 (95% CI, 0.86–0.99) at 20, 24 and 28 weeks, respectively, and was superior to models using the sFlt-1/PlGF ratio alone or uterine artery mean pulsatility index. Conclusions The sFlt-1/PlGF ratio can improve prediction of early-onset PE for women at risk of this condition. ©2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. INTRODUCTION Pre-eclampsia (PE) affects 2–5% of pregnancies1–5 and can result in intrauterine growth restriction (IUGR), renal or hepatic impairment, HELLP syndrome (hemolysis, elevated liver enzyme levels and low platelet count), eclampsia, and maternal and fetal mortality5–7. Early and late manifestations of PE differ in time of onset of symptoms, relative frequency, placental morphology, genetic risk and risk of adverse outcomes8–13. Early-onset PE is associated with a higher incidence of adverse perinatal outcomes, including oligohydramnios, Apgar score 7, stillbirth and early neonatal death, compared< with late-onset PE14,15. As early intervention is important to improve maternal and fetal outcomes16 and the classical ©2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd ORIGINAL PAPER on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. Printed by [Obstetrics and Gynecology - 095.022.056.057 - /doi/epdf/10.1002/uog.17373] at [15/03/2021]. 374 Perales et al. clinical markers of PE (hypertension and proteinuria) are poorly predictive of those who will develop the condition, markers of angiogenesis have been examined as aids to PE prediction. A key feature of PE is placental insufficiency. Dysregulation of proand antiangiogenic factors is thought to be causally linked to the condition7,17,18; before and during PE, maternal serum concentrations of antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) are increased and levels of proangiogenic placental growth factor (PlGF) are decreased19,20. A high sFlt-1/PlGF ratio has been linked with PE and demonstrated before clinical onset of the condition, and differences in sFlt-1 and PlGF have been observed between earlyand late-onset PE21–29. The Elecsys®immunoassay sFlt-1/PlGF ratio is CE-IVD (Conformit´e Europ´eenne–In Vitro Diagnostics) approved as a diagnostic aid for PE with gestational age-specific cut-off values, and as an aid in short-term prediction of PE in women with suspected PE26,30,31. The Prediction of Short-Term Outcome in Pregnant Women with Suspected PE Study (PROGNOSIS) developed a cut-off-based PE prediction model. Optimum sFlt-1/PlGF ratio cut-off levels of 38 and≤>38 were identified to rule out and rule in, respectively, PE, in women with singleton pregnancy at 24 +0 to 36 +6 weeks’ gestation32 . However, the predictive value of the sFlt-1/PlGF ratio has not been examined specifically for early-onset PE. This study, the Study of Early Pre-eclampsia in Spain (STEPS), aimed to evaluate the sFlt-1/PlGF ratio at 20, 24 and 28 weeks as a predictive marker for early-onset PE in women at risk of PE. METHODS Study design and participants STEPS was a prospective, double-blind, multicenter (10 study sites in Spain) study, performed between October 2010 and March 2013, and enrolled pregnant women at risk of PE. To be considered at risk of PE, women had to meet one of the following inclusion criteria: PE, eclampsia, HELLP syndrome or IUGR in a previous pregnancy; pre-existing chronic hypertension without proteinuria; gestational hypertension (new-onset hypertension in pregnancy); pre-existing renal disease (kidney transplantation or creatinine clearance <60 mL/min); pre-existing diabetes mellitus Type I (insulin dependent); mean uterine artery Doppler pulsatility index (UtA-PI) >1.45 (at 19–20 weeks); thrombophilia (antiphospholipid syndrome, protein C deficiency, protein S deficiency, antithrombin deficiency, factor V Leiden mutation); multiple pregnancy; age ≥40 years and conceived with assisted reproductive technologies (ART). Women with two or more of the following risk factors were also included: nulliparity; body mass index ≥35 kg/m2; diastolic blood pressure >80 mmHg at study inclusion; age 40 years; and family history≥ (mother or sister) of PE, eclampsia or HELLP syndrome. Women were excluded if they were both hypertensive and had proteinuria or if major fetal malformations/chromosome disorders were observed. Women provided informed, signed consent. The protocol was approved by applicable national/regional independent ethics committees and institutional review boards (Table S1). The study adhered to the Guidelines for Good Clinical Practice. The primary objective of the study was to demonstrate that the sFlt-1/PlGF ratio was a predictive marker for early-onset PE. Secondary objectives included evaluation of sFlt-1/PlGF ratio as a predictor of late-onset PE and the use of the sFlt-1/PlGF ratio for differentiation of hypertension from PE. Diagnostic criteria For consistency, investigators used predefined diagnostic criteria (Table 1) based on the Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy33. PE was defined as newly occurring hypertension (systolic blood pressure 140 mmHg and/or diastolic blood pres-≥ sure ≥90 mmHg) with newly occurring proteinuria after 20 weeks. To be considered early onset, PE had to occur before 34 +0 weeks. Data collection and visits At gestational weeks 19–20 (Visit 1), 23–24 (Visit 2) and 27–28 (Visit 3), participants underwent a blood test to determine the sFlt-1/PlGF ratio, Doppler examination of the uterine arteries and assessment of blood pressure (measured by validated automated devices), proteinuria, PE status, hemoglobin, platelets and uric acid levels. Postpartum, additional data were collected, including blood pressure, type of delivery, Apgar score, weight of placenta, neonatal outcomes (perinatal/fetal death, delivery 34 weeks, IUGR, placental abruption, respiratory< distress syndrome, necrotizing enterocolitis, intraventricular hemorrhage) and maternal outcomes (maternal death, pulmonary edema, acute renal failure, cerebral hemorrhage, cerebral thrombosis, disseminated intravascular coagulation). Unplanned visits could be carried out in the event of complications. Serum samples (≥2 mL) were collected according to a standard operating procedure and were analyzed at the individual study sites. Results were checked for consistency between study sites by central analysis at Hospital Universitario Central de Asturias. Maternal serum levels of sFlt-1 and PlGF were determined using the fully automated Elecsys sFlt-1 and Elecsys PlGF assays on the cobas®e electrochemiluminescence immunoassay platform (Roche Diagnostics GmbH, Mannheim, Germany) and the sFlt-1/PlGF ratio was calculated27,30,31. The sFlt-1/PlGF ratio results were concealed from both patients and carers to ensure that they did not affect the clinical monitoring of patients. Adverse events were recorded, although the study was non-interventional. ©2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2017; : 373–382.50 on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. Printed by [Obstetrics and Gynecology - 095.022.056.057 - /doi/epdf/10.1002/uog.17373] at [15/03/2021]. sFlt-1/PlGF STEPS 375 Table 1 Diagnostic criteria in Study of Early Pre-eclampsia in Spain (STEPS) Diagnosis Criteria Hypertension Systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg (on two occasions at least 6 h apart) Chronic hypertension Hypertension (systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg) diagnosed before pregnancy or in first half of pregnancy (<20weeks) and continued for >12 weeks after delivery Proteinuria For determination of urinary protein using test strips, a value of 1 was not considered reliable for diagnosis+ of PE. Data were reconfirmed with protein test on 24-h urine ( 0.3g protein/24 h); in an emergency, if it≥ was not possible to determine protein in 24-h urine, protein determination was carried out on isolated urine sample (≥30 mg protein/dL or protein/creatinine ratio≥ 30 mg protein/mmol creatinine) Gestational hypertension New-onset hypertension (systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg) after 20 weeks of pregnancy, which resolved by 12 weeks postpartum PE New-onset hypertension (systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg) and new-onset proteinuria after 20 weeks of pregnancy Severe PE PE plus one or more of the following: systolic blood pressure≥160 mmHg and/or diastolic blood pressure ≥110 mmHg (on two occasions at least 6 h apart); proteinuria (>5 g protein/24 h or test strip ≥ +3 in two urine samples collected at random at least 4 h apart); impairment of renal function (serum creatinine ≥1.2 mg/dL unless known to be elevated previously or oliguria<500 mL/24 h); pulmonary edema; impairment of hepatic function (elevated liver enzymes, epigastric pain or right upper quadrant pain caused by distension of Glisson’s capsule); neurological symptoms (cerebral or visual disturbances, severe headache); hematological disturbances (thrombocytopenia, hemolysis); IUGR Eclampsia New-onset tonic–clonic convulsions in women with PE, not attributable to any other cause Earlyand late-onset PE Early onset: PE developing<34 34+ ≥0 weeks; late onset: PE developing +0 weeks HELLP syndrome Increased ASAT ( 70 IU/L); decreased platelet count ( L); increased LDH (> < 100 000/μ>600 IU/L) IUGR Estimated fetal weight or abdominal circumference<10th percentile (adjusted for gender/race in accordance with tables normally used by study center). Presence of pathological process that inhibits expression of normal intrinsic growth potential. Pathological process must be demonstrated at least once after 22 weeks, according to either oligohydramnios (amniotic fluid index <10th percentile) or pathological flow in umbilical artery (pulsatility index>95th percentile) SGA neonate Estimated fetal weight or abdominal circumference<10th percentile (adjusted for gender/race in accordance with tables normally used by study center); no pathological process Preterm birth Delivery before end of 37 weeks (e.g. gestational age of 36 +6 weeks would be recorded as 36 completed weeks of pregnancy and baby would be defined as preterm) ASAT, aspartate aminotransferase; HELLP, hemolysis, elevated liver enzymes and low platelet count; IUGR, intrauterine growth restriction; LDH, lactate dehydrogenase; PE, pre-eclampsia; SGA, small-for-gestational-age. Statistical analysis To obtain 100 cases of PE, it was calculated that 800 pregnant women would need to be included in the study, based on a presumed prevalence of PE of 12% (including both singleton and multiple pregnancies). The sFlt-1/PlGF ratio was log-transformed to correct for right skewness prior to any calculation. Differences in means between independent groups were assessed using analysis of variance (ANOVA) or Student’s -test in the case of homogeneity oft variances, and using generalized least squares in the case of heteroscedasticity. Appropriateness of the methods was assessed by evaluation of the plots of residuals. To develop a predictive model of PE, multivariate logistic regression was used considering maternal characteristics, medical history and biomarkers as potential predictors. The variables that were finally included in the early-PE prediction model were selected according to the results of a logistic regression model with L1 penalization (‘lasso’ technique)34. The coefficients derived from the multivariate analysis were used as weights in a nomogram to predict early PE. Performances of the models were evaluated by receiver–operating characteristics (ROC) curves and areas under the curve (AUC) with 95% CIs. All statistical analyses were performed using R (version 3.1.2) and R-packages rms (version 4.2-1) and ROCR (version 1.0-5). RESULTS Study participants Overall, 729 women were eligible for analysis, including 447 with singleton pregnancy and 282 with multiple pregnancy (twin pregnancy, 276; triplet pregnancy,n= n=6). A total of 78 (10.7%) women developed PE (singleton pregnancy, 42; multiple pregnancy,n=n=36), of which 24 were early-onset PE (singleton pregnancy, n n=14; multiple pregnancy, =10) and 54 were late-onset PE (singleton pregnancy, 28; multiple pregnancy,n= n=26) (Figure 1). The number of participants per study site is reported in Table S2. Women who developed early-onset PE had higher systolic and diastolic blood pressures, mean arterial blood pressure (MAP) and lower gestational age at delivery compared with the control group (women who did not develop PE/hypertension during the entire pregnancy) (Table 2). sFlt-1, PlGF and sFlt-1/PlGF ratio measurements In the control group, median sFlt-1/PlGF ratio remained low ( 7) between 20 and 28 weeks’ gestation (Table 3).< In women who developed early-onset PE, median sFlt-1/PlGF ratio was already higher (14.5) at 20weeks’ gestation and increased further to 18.4 at 24 weeks and ©2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2017; : 373–382.50 on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. Printed by [Obstetrics and Gynecology - 095.022.056.057 - /doi/epdf/10.1002/uog.17373] at [15/03/2021]. 376 Perales et al. Participants excluded* ( 90)n= (S, 75; M, 15)n=n= Participants who did not develop PE ( 651)n= (S, 405; M, 246)n=n= Participants who developed PE ( 78)n= (S, 42; M, 36)n=n= Participants with late-onset PE ( 54)n= (S, n= 28; M, 26)n= Participants with early-onset PE ( 24)n= (S, n= 14; M, 10)n= Participants with IUGR ( 28)n= (S, n= 15; M, 13)n= Participants with gestational hypertension ( 35)n= (S, n= 22; M, 13)n= Participants with chronic hypertension ( 33)n= (S, n= 32; M, n= 1) Participants without hypertension ( 555)n= (S, 336;n= M, n= 219) Participants enrolled ( 819)n= (S, 522; M, 297)n=n= Participants eligible for analysis ( 729)n= (S, 447; M, 282)n=n= Figure 1 Flowchart of participants in Study of Early Pre-eclampsia in Spain (STEPS). *Reasons for exclusion: inclusion criteria not met (n=4); signed consent given but did not start study (n=28); miscarriage (n=7); termination of pregnancy due to fetal malformations ( 13); placental abruption at 26 weeks (n=8); lost to follow-up (n=n=1); completed follow-up until 28 weeks but data could not be retrieved because of delivery in another setting (n=29). IUGR, intrauterine growth restriction; M, multiple pregnancy; PE, pre-eclampsia; S, singleton pregnancy. Table 2 Baseline characteristics of women who developed earlyor late-onset pre-eclampsia (PE) and those who did not develop PE (controls) Characteristic Controls (n=651) Early-onset PE (n=24) Late-onset PE (n=54) Age (years) 34.6 ±5.3 35.6 ±3.9 34.7 ±0.7 Body mass index (kg/m2) 26.7 6.0 28.5 6.4 27.9 7.3± ± ± Systolic blood pressure (mmHg) 119.1 ±13.7 127.9 ±13.5* 125.0 16.0*± Diastolic blood pressure (mmHg) 73.7 ±11.3 77.5 8.5* 77.7± ± 12.0* Mean arterial pressure (mmHg) 88.9 ±11.1 94.3 ±8.1* 93.4 ±12.2* Multiple pregnancy 246 (37.8) 10 (41.7) 26 (48.1) Gestational age at delivery (weeks) 37.5 ±2.7 31.8 3.5* 36.6 1.4± ± Birth weight of first infant (g) 2911 ±721 ( 646) 1745 830 ( 584 ( 54)n= ± n=22)* 2759 ±n= Birth weight of second infant (g) 2303 ±552 ( 243) 1807 378 ( 364 ( 26)n= ± n=9)† 2285 ±n= Birth weight of third infant (g) 1221 ±689 ( 4) 1445 304 ( 2) — ( 0)n= ± n=n= Nulliparous 272 (41.8) 15 (62.5) 31 (57.4) Previous PE 101 (15.5) 9 (37.5)* 14 (25.9) Family history of PE 23 (3.5) 1 (4.2) 5 (9.3) Previous IUGR 55 (8.4) 3 (12.5) 5 (9.3) Chronic hypertension 81 (12.4) 6 (25.0) 12 (22.2) Gestational hypertension 4 (0.6) 0 (0) 3 (5.6) Nephropathy 6 (0.9) 0 (0) 1 (1.9) Diabetes mellitus Type 1 42 (6.5) 1 (4.2) 1 (1.9) Thrombophilia 50 (7.7) 1 (4.2) 3 (5.6) Conceived by assisted reproduction 93 (14.3) 5 (20.8) 11 (20.4) Smoker at enrollment 80 (12.3) 1 (4.2) 3 (5.6) Abnormal UtA Doppler 8 (1.2) 1 (4.2) 2 (3.7) Data are given as mean 0.05, after adjustment by±SD or n P(%). PE groups compared with controls using Dunnett’s test: * <0.001; †P< Bonferroni correction. IUGR, intrauterine growth restriction; UtA, uterine artery. ©2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2017; : 373–382.50 on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. Printed by [Obstetrics and Gynecology - 095.022.056.057 - /doi/epdf/10.1002/uog.17373] at [15/03/2021]. sFlt-1/PlGF STEPS 377 Table 3 Measurements of soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF) and sFlt-1/PlGF ratio in maternal serum at 20, 24 and 28weeks in women who developed earlyor late-onset pre-eclampsia (PE) and in those who did not develop PE (controls) Biomarker Controls Early-onset PE Late-onset PE 20 weeks n612 21 52 PlGF (pg/mL) 264.5 (172.0–403.6) 193.1 (68.0–262.4) 267.8 (151.5–414.0) sFlt-1 (pg/mL) 1623.0 (1081.0–2531.0) 1972.0 (1331.0–3473.0) 1967.0 (1120.5–2903.8) sFlt-1/PlGF ratio 6.3 (4.1–9.3) 14.5 (5.5–43.7) 6.7 (4.6–9.9) 24 weeks n580 20 52 PlGF (pg/mL) 424.5 (277.0–615.6) 168.9 (62.1–329.7) 415.0 (259.7–595.7) sFlt-1 (pg/mL) 1725.0 (1123.5–2674.3) 3127.5 (1961.8–4202.5) 1882.5 (1134.5–3115.8) sFlt-1/PlGF ratio 4.0 (2.6–6.3) 18.4 (8.2–57.9) 4.7 (2.8–7.2) 28 weeks n557 16 49 PlGF (pg/mL) 540.0 (339.0–821.5) 176.5 (67.2–278.6) 335.0 (263.0–485.9) sFlt-1 (pg/mL) 1826.0 (1231.0–2766.0) 6370.0 (2385.3–8788.3) 2499.0 (1522.0–3681.0) sFlt-1/PlGF ratio 3.3 (2.0–5.9) 51.9 (11.5–145.6) 6.0 (3.8–10.5) Data are given as median (interquartile range) unless stated otherwise. 51.9 at 28weeks. There was little change in the median sFlt-1/PlGF ratio between 20 and 28weeks in women who developed late-onset PE, remaining low throughout at <7. Mean sFlt-1 levels and PlGF levels were significantly different between singleton and multiple pregnancies at 20, 24 and 28 weeks (P=0.001). However, the mean sFlt-1/PlGF ratio was only significantly different at 28weeks’ gestation ( 0.001) (Table S3) and, atP= all timepoints, the difference between median values in singleton and multiple pregnancies was small. sFlt-1/PlGF ratio: prediction of PE Compared with control participants, the sFlt-1/PlGF ratio was consistently significantly higher in women with early-onset PE ( 0.001 at all timepoints) (Figure 2).P< Women with early-onset PE also had significantly higher sFlt-1/PlGF ratios at 20, 24 and 28weeks relative to women with chronic or gestational hypertension and women with late-onset PE (Figure 3). Differences between early-onset PE and control/hypertension/late-onset PE became more pronounced as the pregnancy progressed. Women with late-onset PE were not easily differentiated from control participants by the sFlt-1/PlGF ratio at 20 and 24weeks (difference was non-significant at 20 (P P=0.15) and 24 ( =0.21) weeks, Figure 2). At 28 weeks, there was a statistically significant difference in the sFlt-1/PlGF ratio between women with late-onset PE and control participants (P<0.001), although the numerical difference in the median ratio was small (2.7) (Table 3). Development of a prediction model for early-onset PE Prediction models for early-onset PE were developed, which included variations of the following factors: sFlt-1/PlGF ratio, PlGF, UtA-PI, MAP, being parous, previous PE and use of ART. The AUC was optimal for a model including the sFlt-1/PlGF ratio, MAP, being parous 20 3 5 7 10 20 sFlt-1/PlGF ratio (log scale) 50 75 24 Gestational age (weeks) 28 † Figure 2 Soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio at 20, 24 and 28weeks in control group of women who did not develop pre-eclampsia (PE; ) and in those who developed early-onset ) or late-onset ) PE. Comparison with controls: *P<0.001; † 0.15; ‡ 0.21.P=P= and previous PE (hereafter referred to as the ‘early-onset PE prediction model’) (Figure S1) compared with models that used the sFlt-1/PlGF ratio alone or UtA-PI alone (Table 4). The accuracy of the prediction model was not substantially improved by including ART or UtA-PI and ART in the model. At 20 and 24 weeks, including these two parameters in the model increased the AUC from 0.86 (95% CI, 0.77–0.95) to 0.87 (95% CI, 0.79–0.96), and from 0.91 (95% CI, 0.85–0.97) to 0.92 (95% CI, 0.85–0.97), respectively. However, at 28weeks, including UtA-PI and ART in the model reduced the AUC from 0.93 (95% CI, 0.86–0.99) to 0.91 (95% CI, 0.82–0.99) (Figure 4). A nomogram for prediction risk is presented in Figures S2 and S3. The detection rate for early-onset PE using different prediction models is reported in Table 5. We also compared the performance of a standard prediction model (maternal history, MAP and UtA-PI) ©2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2017; : 373–382.50 on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. Printed by [Obstetrics and Gynecology - 095.022.056.057 - /doi/epdf/10.1002/uog.17373] at [15/03/2021]. 378 Perales et al. Controls 0.5 1 5 10 100 † * * * * * * † † † 50 sFlt-1/PlGF ratio (log scale) 500 1000 (a) GH Late PECH Early PE Controls GH Late PECH Early PE Controls GH Late PECH Early PE 0.5 1 5 10 100 † * * * † † † 50 sFlt-1/PlGF ratio (log scale) 500 1000 (b) 0.5 1 5 10 100 † * * * † † † 50 sFlt-1/PlGF ratio (log scale) 500 1000 (c) Figure 3 Box-and-whisker plots of soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio in control women who did not develop pre-eclampsia (PE) and in those who developed chronic hypertension (CH), gestational hypertension (GH), late-onset PE or early-onset PE at: (a) 20 weeks, (b) 24 weeks and (c) 28 weeks. †P<0.001 in comparison with early-onset PE. Boxes with internal lines represent median and interquartile range, whiskers are 1.5 interquartile range and stars are outliers.× with the same model but including the sFlt-1/PlGF immunoassay ratio to estimate early-onset PE risk at 20, 24 and 28weeks’ gestation. The addition of the sFlt-1/PlGF immunoassay ratio substantially increased the detection rate at all gestational ages studied (assuming a false-positive rate of both 5% and 10%) (Table 6). sFlt-1 and PlGF as single biomarkers: development of PE Women with early-onset PE had lower PlGF (P<0.001 at 20, 24 and 28 weeks) and higher sFlt-1 (P=0.018, P P<0.001 and <0.001 at 20, 24 and 28 weeks, respectively) compared with those who did not develop early-onset PE (women who developed late-onset PE and those who did not develop any PE combined) (Figure S4). A comparison of prediction models that included the sFlt-1/PlGF ratio with models that used sFlt-1 or PlGF alone was performed by evaluating their respective AUCs and Akaike information criterion (AIC), which measures goodness of fit35. The AUC for prediction models that included the sFlt-1/PlGF ratio (0.86–0.87, 0.91–0.92 and 0.91–0.93 at 20, 24 and 28 weeks’ gestation, respectively) was greater than that of models that used single biomarkers (0.81–0.83, 0.88–0.90 and 0.88–0.91 for sFlt-1 and 0.79–0.83, 0.85–0.89 and 0.86–0.89 for PlGF at 20, 24 and 28 weeks’ gestation, respectively) (Table S4). Data consistency No inconsistencies were found between site and central testing (data not shown). DISCUSSION Substantial evidence supports the use of the sFlt-1/PlGF ratio in PE diagnosis and prediction20,21,23,27,36–40. However, differences between earlyand late-onset PE suggest different etiologies; thus, different ‘rules’ for the sFlt-1/PlGF ratio could be applied. In a study of 257 women with suspected PE, the optimal sFlt-1/PlGF ratio cut-off to diagnose PE <34 weeks’ and ≥34 weeks’ gestation was 23 (92.0% sensitivity, 81.1% specificity) and 45 (83.7% sensitivity, 72.6% specificity), respectively41. In PROGNOSIS, a sFlt-1/PlGF ratio cut-off ≤38 ruled out PE within 1 week in women with suspected PE and singleton pregnancy at 24 +0 to 36 +6 weeks’ gestation32 . PROGNOSIS had a higher prevalence of PE compared with our study (19% 11%, respectively),vs possibly due to the fact that PROGNOSIS enrolled women with suspicion of PE, while we enrolled women with a moderate or high risk of developing PE. The prevalence of PE in our study falls between the estimated ranges for women with moderate or high risk for PE (5.29–6.19% and 16.09–19.49%, respectively)42. In STEPS, the sFlt-1/PlGF ratio was significantly different between women who did not develop PE and those who did. The combination of the sFlt-1/PlGF ratio with other clinical measures produced a predictive model with considerably increased specificity and sensitivity compared with using UtA-PI or sFlt-1/PlGF ratio alone. We also evaluated how the models used to estimate early-onset PE risk would perform when using the single biomarkers, instead of the sFlt-1/PlGF ratio. Based on both AUC and AIC, models with the sFlt-1/PlGF ratio demonstrated consistently the highest predictive performance. Using our early-onset PE prediction model (sFlt-1/PlGF ratio, MAP, being parous, previous PE), early-onset PE could be predicted from 20weeks onward, with an AUC of 0.86 and 60% sensitivity for a false-positive rate of 10%. A previous model developed without serum biomarkers, which used a history of diabetes, hypertension and MAP, reported an AUC of 0.83 with 55% sensitivity for a false-positive rate of 10% (these were not high-risk women)43. ©2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2017; : 373–382.50 on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. Printed by [Obstetrics and Gynecology - 095.022.056.057 - /doi/epdf/10.1002/uog.17373] at [15/03/2021]. sFlt-1/PlGF STEPS 379 Table 4 Prediction of early-onset pre-eclampsia (PE) at 20, 24 and 28 weeks using different individual parameters and early-onset PE prediction model Prediction parameter AUC (95% CI) 20 weeks Early-onset PE prediction model 0.86 (0.77–0.95) MAP 0.67 (0.55–0.79) UtA-PI 0.50 (0.35–0.66) PlGF 0.70 (0.58–0.82) sFlt-1 0.61 (0.49–0.74) sFlt-1/PlGF ratio 0.77 (0.65–0.89) 24 weeks Early-onset PE prediction model 0.91 (0.85–0.97) MAP 0.72 (0.62–0.83) UtA-PI 0.55 (0.39–0.72) PlGF 0.81 (0.72–0.90) sFlt-1 0.71 (0.58–0.84) sFlt-1/PlGF ratio 0.86 (0.76–0.96) 28 weeks Early-onset PE prediction model 0.93 (0.86–0.99) MAP 0.77 (0.66–0.89) UtA-PI 0.63 (0.45–0.80) PlGF 0.86 (0.78–0.94) sFlt-1 0.81 (0.67–0.95) sFlt-1/PlGF ratio 0.89 (0.79–0.98) Early-onset PE prediction model includes soluble fms-like tyrosine kinase 1(sFlt-1)/placental growth factor (PlGF) ratio, mean arterial pressure (MAP), being parous and previous PE. AUC, area under the receiver–operating characteristics curve; UtA-PI, uterine artery pulsatility index. Other studies have included sFlt-1 and PlGF in their models. An observational study of women at high risk of PE developed a prediction model for early-onset PE using sFlt-1 at 28 +0 to 31 +6 weeks’ gestation, which had an AUC of 0.85 (67% sensitivity, 96% specificity)44. A model including gestational age, UtA-PI and sFlt-1/PlGF ratio showed an association with perinatal complications with an AUC of 0.89 (64% sensitivity, 95% specificity)45. Another model combined PlGF with maternal characteristics, obstetric history and UtA-PI to predict early-onset PE in the first trimester with an AUC of 0.9446 . Although an abnormal UtA-PI was associated with the development of PE in our study, it was not included in our model since it did not substantially improve PE prediction. From a practical perspective, the UtA can be difficult to locate in the first trimester and the International Society of Ultrasound in Obstetrics and Gynecology does not include UtA Doppler as part of the routine first-trimester fetal ultrasound examination47. Other studies have also not included UtA-PI in their models48,49 . A recent study demonstrated that a prospective screening model at 19–24 weeks’ gestation, involving maternal factors, UtA-PI, MAP and PlGF, was superior to screening by maternal factors alone. The performance of the model was inversely related to the gestational age at which delivery became necessary; detection rates (false-positive rate of 10%) for PE 32 weeks, between< 32 0 and 36 37 weeks were 99%, 85%+ + ≥6 weeks, and and 46%, respectively. However, this study evaluated 1.0 0.8 0.6 0.4 0.2 0 True-positive rate 0 0.2 0.4 False-positive rate 0.6 0.8 1.0 1.0 0.8 0.6 0.4 0.2 0 True-positive rate 0 0.2 0.4 False-positive rate 0.6 0.8 1.0 (b) (a) 1.0 0.8 0.6 0.4 0.2 0 True-positive rate 0 0.2 0.4 False-positive rate 0.6 0.8 1.0 (c) Figure 4 Receiver–operating characteristcs curves for prediction of early-onset pre-eclampsia (PE) using different models at 20 weeks (a), 24 weeks (b) and 28 weeks (c). Tables S4 and 5 present numerical values for areas under curves. Early-onset PE prediction model ) includes soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio, mean arterial pressure (MAP), being parous and previous PE. ART, assisted reproductive technologies; UtA-PI, uterine artery pulsatility index. , sFlt-1/PlGF ratio, MAP, being parous, previous PE, ART. , sFlt-1/PlGF ratio, MAP, being parous, previous PE, UtA-PI, ART. , MAP, being parous, previous PE, ART, PlGF. , MAP, being parous, previous PE, UtA-PI, ART, PlGF. PlGF and sFlt-1 separately; it did not assess the sFlt-1/PlGF ratio50 . Of note, the study defined early-onset PE as requiring delivery before 32 weeks’ gestation, rather than before 34weeks. A related study showed that a two-stage screening model, in which UtA-PI and PlGF measurements were reserved for at-risk individuals, achieved similar detection rates for preterm PE ( 37 weeks’ gestation),< compared with screening the whole population by maternal factors, MAP, UtA-PI and PlGF51. Various guidelines recommend PE screening based on maternal history52–54. However, the addition of MAP, ©2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2017; : 373–382.50 on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. Printed by [Obstetrics and Gynecology - 095.022.056.057 - /doi/epdf/10.1002/uog.17373] at [15/03/2021]. 380 Perales et al. Table 5 Prediction rates of early-onset pre-eclampsia (PE) using different models at 20, 24 and 28weeks Detection rate (%) Prediction model FPR =5% FPR =10% 20 weeks Early-onset PE prediction model 45 60 sFlt-1/PlGF ratio, MAP, being parous, previous PE, UtA-PI and ART 50 60 sFlt-1/PlGF ratio, MAP, being parous, previous PE and ART 55 60 MAP, being parous, previous PE, UtA-PI, ART and PlGF 35 55 MAP, being parous, previous PE, ART and PlGF 45 45 24 weeks Early-onset PE prediction model 60 70 sFlt-1/PlGF ratio, MAP, being parous, previous PE, UtA-PI and ART 72 78 sFlt-1/PlGF ratio, MAP, being parous, previous PE and ART 60 70 MAP, being parous, previous PE, UtA-PI, ART and PlGF 56 67 MAP, being parous, previous PE, ART and PlGF 56 67 28 weeks Early-onset PE prediction model 81 81 sFlt-1/PlGF ratio, MAP, being parous, previous PE, UtA-PI and ART 73 80 sFlt-1/PlGF ratio, MAP, being parous, previous PE and ART 81 81 MAP, being parous, previous PE, UtA-PI, ART and PlGF 53 53 MAP, being parous, previous PE, ART and PlGF 53 53 Early-onset PE prediction model includes soluble fms-like tyrosine kinase 1(sFlt-1)/placental growth factor (PlGF) ratio, mean arterial pressure (MAP), being parous and previous PE. Areas under receiver–operating chracteristics curves for each model are provided in Table S4. ART, assisted reproductive technologies; FPR, false-positive rate; UtA-PI, uterine artery pulsatility index. Table 6 Performance of standard prediction model and same model plus soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio to estimate risk of early-onset pre-eclampsia at 20, 24 and 28weeks Detection rate (%) Prediction model AUC (95% CI) FPR=5% FPR =10% 20 weeks Standard prediction model (maternal history, MAP, UtA-PI) 0.81 (0.71–0.89) 17 48 Standard prediction model plus sFlt-1/PlGF ratio 0.91 (0.85–0.97) 60 70 24 weeks Standard prediction model (maternal history, MAP, UtA-PI) 0.87 (0.79–0.94) 40 60 Standard prediction model plus sFlt-1/PlGF ratio 0.95 (0.90–0.99) 72 83 28 weeks Standard prediction model (maternal history, MAP, UtA-PI) 0.89 (0.83–0.95) 42 58 Standard prediction model plus sFlt-1/PlGF ratio 0.95 (0.90–1.00) 80 80 AUC, area under receiver–operating characteristics curve; FPR, false-positive rate; MAP, mean arterial pressure; UtA-PI, uterine artery pulsatility index. UtA-PI and angiogenic serum markers to the assessment of maternal history has been shown to increase the PE detection rate between 12 and 36 weeks’ gestation55–58. In STEPS, the addition of the sFlt-1/PlGF ratio increased the detection rate at all gestational ages studied, supporting the inclusion of the sFlt-1/PlGF ratio in the risk estimation of early-onset PE. The prospective, longitudinal design and large cohort in our study provided a robust dataset and the angiogenic marker results were hidden from the investigators to avoid bias in the diagnosis of outcomes. However, despite the large sample size, there were relatively small numbers of women in the early-onset PE group and the results of this study, which included women at risk of developing PE, cannot be applied to a low-risk population, i.e. in screening for PE. The data were validated using the Elecsys immunoassay sFlt-1/PlGF ratio and the predictive value may differ when other assays are used. The developed prediction model for early-onset PE has to be validated in an independent prospective study cohort in a comparable target population, and interventional studies are required to confirm the clinical utility of the results. STEPS provides further evidence that the addition of the sFlt-1/PlGF ratio to clinical protocols for women at risk of PE improves prediction of early-onset PE in the second trimester. This complements the findings of PROGNOSIS, which showed that, in women with signs and/or symptoms of PE and a sFlt-1/PlGF ratio above 38, the positive predictive value for PE within the following 4 weeks was 36.7%32 . In STEPS, women who developed early-onset PE had a median sFlt-1/PlGF ratio at 28weeks’ gestation of 51.9, indicating that these women had an increased risk of developing PE in the following 4 weeks before 34 0 weeks’ gestation.+ Better prediction of PE could facilitate targeting of monitoring and therapeutic procedures towards at-risk women and allow better utilization of healthcare resources. ©2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2017; : 373–382.50 on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. Printed by [Obstetrics and Gynecology - 095.022.056.057 - /doi/epdf/10.1002/uog.17373] at [15/03/2021]. sFlt-1/PlGF STEPS 381 ACKNOWLEDGMENTS We thank all women who participated in the STEPS study and the recruitment officers, nurses, midwives and midwifery staff who supported the study. We thank the STEPS investigators and MaJos´e Ramirez and Nuria Piella (Roche Diagnostics, Spain). Support for third-party writing assistance for this manuscript was provided by Emma McConnell, PhD (Gardiner-Caldwell Communications), and was funded by Roche Diagnostics. DISCLOSURES The STEPS study was sponsored by Roche Diagnostics Spain who were involved in study design, interpretation of the data and writing of the manuscript. ELECSYS and cobas are trademarks of Roche. A.P. is a consultant for Roche, GE Healthcare, Ferring, Italfarmaco, EFFIK, Merck and Gynea. J.L.D. is a consultant for Roche and Italfarmaco. M.H. is employed by Roche Diagnostics and has shares in F. Hoffmann-La Roche. STEPS INVESTIGATORS Azahar Romero and Francisco Cabrera, Hospital Materno Infantil de Canarias, Gran Canaria, Spain; Mar´ ıa V ´azquez, Francisco Moreno and ´ Oscar Vaquerizo, Hospital Universitario Central de Asturias, Oviedo, Spain; Myriam Miguel, Catalina de Paco, Miriam Pertegal and Alicia Arteaga, Hospital Virgen de la Arrixaca, Murcia, Spain; M. Jes´us Franco and Blanca Envid, Hospital Miguel Servet, Zaragoza, Spain; Elena Martin, Jos´e Luis Bartha and Antonio Bu ˜ no, Hospital de la Paz, Madrid, Spain; Gema P´erez, Silvia Roig, Rosa G´omez and David Herv ´as, Hospital Universitario y Polit ´ecnico La Fe, Valencia, Spain; ´ Angel Aguaron de la Cruz and Nieves L´ opez, Hospital Gregorio Mara ˜n ´on, Madrid, Spain; Victoria Melero and Mercedes Calero, Hospital Puerta del Mar, Cadiz, Spain; Marta de Ram´on and Antonio Paya, .Hospital del Mar, Barcelona, Spain REFERENCES 1. WHO. 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Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd Ultrasound Obstet Gynecol 2017; : 373–382.50 on behalf of the International Society of Ultrasound in Obstetrics and Gynecology. Printed by [Obstetrics and Gynecology - 095.022.056.057 - /doi/epdf/10.1002/uog.17373] at [15/03/2021].