Placental growth factor testing at 19-23 weeks of gestation as a guide to subsequent care in pregnancy: A prospective observational study.

OBJECTIVE: To determine whether serum placental growth factor (PlGF) at 19-23 weeks of gestation can improve the identification of risk for adverse outcomes. DESIGN: Prospective observational cohort study. SETTING: Two English maternity units. POPULATION: Unselected singleton pregnancies attending routine ultrasound at 19-23 weeks of gestation. METHODS: Outcomes ascertained by health record review. Diagnostic test properties evaluated clinical risk factors for pre-eclampsia (according to National Institute of Care Excellence) or fetal growth restriction (according to Royal College of Obstetricians and Gynaecologists), low PlGF at 19-23 weeks of gestation (<5th percentile) or both. MAIN OUTCOME MEASURES: Pre-eclampsia, gestational hypertension, stillbirth, birthweight below third percentile or neonatal intensive care unit (NICU) admission for ≥48 h. RESULTS: In 30 013 pregnancies, risk factors were present in 9941 (33.1%), low PlGF was present in 1501 (5.0%) and both ('two-stage' screening) were present in 547 (1.8%) pregnancies. Risk factors detected 41.7%-54.7% of adverse outcomes, and could not meaningfully revise the risk (all positive likelihood ratios, +LR, <5.0; all negative likelihood ratios, -LR, ≥0.2). Low PlGF detected 8.5%-17.4% of adverse outcomes, but meaningfully increased risks (other than NICU admission) associated with delivery <37 weeks of gestation (+LR = 5.03-15.55); all -LRs were ≥0.2. 'Two-stage' screening detected 4.2%-8.9% of adverse outcomes, with meaningful +LRs (6.28-18.61) at <37 weeks of gestation, except for NICU admission of ≥48 h, which had an +LR of 7.56 at <34 weeks of gestation; all -LRs were ≥0.2. No screening strategy meaningfully increased or decreased the detection of adverse outcome risk at term. CONCLUSIONS: Clinical risk factor screening has a high screen-positive rate and a poor detection of adverse outcomes. False positives cannot be reduced by PlGF testing at 19-23 weeks of gestation; therefore, this cannot be recommended as a useful strategy on its own.

The additive role of angiogenic markers for women with confirmed preeclampsia.

BACKGROUND: Hypertension complicates 2% to 8% of all pregnancies and is a leading cause of maternal and perinatal morbidity and mortality globally. Given the prognostic role that angiogenic markers play in evaluation of patients with "suspected preeclampsia," the International Society for the Study of Hypertension in Pregnancy incorporated angiogenic imbalance into the 2021 definition of preeclampsia. As women with "suspected preeclampsia" are a heterogeneous group, with some already meeting the diagnostic criteria for preeclampsia, we evaluated whether the soluble fms-like tyrosine kinase-1/placental growth factor ratio adds prognostic value among these women. OBJECTIVE: This study aimed to assess the additive value of soluble fms-like tyrosine kinase-1/placental growth factor ratio when the diagnostic criteria for preeclampsia have already been met. STUDY DESIGN: This was a secondary analysis of a prospective cohort study of patients presenting to obstetrical triage with suspected preeclampsia at ≥20+0 weeks' gestation from July 2009 to June 2012 in Boston, United States. Clinicians were masked to soluble fms-like tyrosine kinase-1/placental growth factor ratio results. Clinical records were reviewed for maternal and neonatal care and outcomes. The value of the soluble fms-like tyrosine kinase-1/placental growth factor ratio (≤38, >38, or >85) was assessed for identifying women at low or high risk of evolving into preeclampsia with severe features within 2 weeks of the triage visit, with preeclampsia with severe features being defined by the American College of Obstetricians and Gynecologists (2013 definition). Based on information in obstetrical triage, preeclampsia among triage patients was defined either by: (1) The International Society for the Study of Hypertension in Pregnancy "restrictive" criteria (ie, new-onset hypertension and proteinuria at ≥20 weeks), or (2) The International Society for the Study of Hypertension in Pregnancy "broad" maternal criteria (ie, new-onset hypertension with proteinuria or one/more relevant maternal end-organ complications). RESULTS: Of 1043 patients included, 459 presented at 20+0 to 34+6 weeks and 584 at ≥35+0 weeks. In triage, 25.8% of women with "suspected preeclampsia" already met the preeclampsia criteria based on the International Society for the Study of Hypertension in Pregnancy broad criteria and 22.0% based on the restrictive criteria. In separate multivariable analyses adjusted for gestational age, a soluble fms-like tyrosine kinase-1/placental growth factor ratio >38 was independently associated with preeclampsia with severe features within 2 weeks even after adjusting for preeclampsia diagnosis in obstetrical triage, whether that preeclampsia were defined restrictively (odds ratio, 15.62; 95% confidence interval, 8.91-27.40) or broadly (odds ratio, 14.56; 95% confidence interval, 8.30-25.56). A soluble fms-like tyrosine kinase-1/placental growth factor ratio ≤38 was good at ruling out development of preeclampsia with severe features within 2 weeks among all patients and among those meeting the restrictive or broad definitions of preeclampsia (negative likelihood ratios, ≤0.16), driven by performance of the ratio before 35 weeks (ie, negative likelihood ratio ≤0.12). A soluble fms-like tyrosine kinase-1/placental growth factor ratio >85 was good at ruling-in preeclampsia with severe features within 2 weeks among women with suspected preeclampsia, either before (positive likelihood ratio, 8.20) or after 35 weeks (positive likelihood ratio, 6.00) and fair at ruling-in preeclampsia with severe features within 2 weeks when preeclampsia had already been confirmed in patients at <35 weeks (restrictively positive likelihood ratio, 3.48, or broadly positive likelihood ratio, 3.40). CONCLUSION: Our findings support the prognostic value of the soluble fms-like tyrosine kinase-1/placental growth factor ratio among patients with confirmed preeclampsia, particularly to identify those both likely and unlikely to progress toward the development of severe features in the next 2 weeks and those who may be most appropriate for expectant and potentially outpatient care. Our findings support the incorporation of angiogenic imbalance into the definition of preeclampsia, particularly at 20-34+0 weeks.

Effect of race on the measurement of angiogenic factors for prediction and diagnosis of pre-eclampsia.

OBJECTIVE: To examine the effect of self-declared race on serum placental growth factor (PlGF) and sFlt-1/PlGF ratio and the impact on pre-eclampsia (PE) prediction. DESIGN: Prospective observational study. SETTING: Two UK maternity hospitals. POPULATION: 29 035 women with singleton pregnancies attending a routine 35+0 to 36+6 weeks' gestation hospital visit, including 654 (2.3%) who subsequently developed PE. METHODS: The predictive performance of PlGF and sFlt-1/PlGF for PE in minority racial groups (versus white) was examined. MAIN OUTCOME MEASURE: Delivery with PE. RESULTS: Compared with white women, mean PlGF was higher and sFlt-1/PlGF ratio lower in black, South Asian, East Asian and mixed race women. In white women at a PlGF concentration cut-off corresponding to a screen-positive rate (SPR) of 10%, detection rates (DRs) were 49.1% for PE at any time and 72.3% for PE within 2 weeks after screening. In black women, at the same PlGF concentration cut-off for white women, the SPR was 5.5%, and DRs 33.6% and 55.0%, respectively; the number of PE cases was too small to evaluate screening performance in other racial groups. Using a fixed cut-off in sFlt-1/PlGF ratio to identify women at risk of developing PE, similarly diagnostically disadvantaged black women. Bias was overcome by adjusting metabolite concentrations for maternal characteristics and use of the competing risks model to estimate patient-specific risks. CONCLUSION: Screening for PE with fixed cut-offs in PlGF or sFlt-1/PlGF diagnostically disadvantages black women. It is essential that measured levels of PlGF be adjusted for race as well as other maternal characteristics.

Genomic imbalances in the placenta are associated with poor fetal growth.

BACKGROUND: Fetal growth restriction (FGR) is associated with increased risks for complications before, during, and after birth, in addition to risk of disease through to adulthood. Although placental insufficiency, failure to supply the fetus with adequate nutrients, underlies most cases of FGR, its causes are diverse and not fully understood. One of the few diagnosable causes of placental insufficiency in ongoing pregnancies is the presence of large chromosomal imbalances such as trisomy confined to the placenta; however, the impact of smaller copy number variants (CNVs) has not yet been adequately addressed. In this study, we confirm the importance of placental aneuploidy, and assess the potential contribution of CNVs to fetal growth. METHODS: We used molecular-cytogenetic approaches to identify aneuploidy in placentas from 101 infants born small-for-gestational age (SGA), typically used as a surrogate for FGR, and from 173 non-SGA controls from uncomplicated pregnancies. We confirmed aneuploidies and assessed mosaicism by microsatellite genotyping. We then profiled CNVs using high-resolution microarrays in a subset of 53 SGA and 61 control euploid placentas, and compared the load, impact, gene enrichment and clinical relevance of CNVs between groups. Candidate CNVs were confirmed using quantitative PCR. RESULTS: Aneuploidy was over tenfold more frequent in SGA-associated placentas compared to controls (11.9% vs. 1.1%; p = 0.0002, OR = 11.4, 95% CI 2.5-107.4), was confined to the placenta, and typically involved autosomes, whereas only sex chromosome abnormalities were observed in controls. We found no significant difference in CNV load or number of placental-expressed or imprinted genes in CNVs between SGA and controls, however, a rare and likely clinically-relevant germline CNV was identified in 5.7% of SGA cases. These CNVs involved candidate genes INHBB, HSD11B2, CTCF, and CSMD3. CONCLUSIONS: We conclude that placental genomic imbalances at the cytogenetic and submicroscopic level may underlie up to ~ 18% of SGA cases in our population. This work contributes to the understanding of the underlying causes of placental insufficiency and FGR, which is important for counselling and prediction of long term outcomes for affected cases.

Impact of new definitions of preeclampsia at term on identification of adverse maternal and perinatal outcomes.

BACKGROUND: Any definition of preeclampsia should identify women and babies at greatest risk of adverse outcomes. OBJECTIVE: This study aimed to investigate the ability of the American College of Obstetricians and Gynecologists and International Society for the Study of Hypertension in Pregnancy definitions of preeclampsia at term gestational age (≥37 0/7 weeks) to identify adverse maternal and perinatal outcomes. STUDY DESIGN: In this prospective cohort study at 2 maternity hospitals in England, women attending a routine hospital visit at 35 0/7 to 36 6/7 weeks' gestation underwent assessment that included history; ultrasonographic estimated fetal weight; Doppler measurements of the pulsatility index in the uterine, umbilical, and fetal middle cerebral arteries; and serum placental growth factor-to-soluble fms-like tyrosine kinase-1 ratio. Obstetrical records were examined for all women with chronic hypertension and those who developed new-onset hypertension, with preeclampsia (de novo or superimposed on chronic hypertension) defined in 5 ways: traditional, based on new-onset proteinuria; American College of Obstetricians and Gynecologists 2013 definition; International Society for the Study of Hypertension in Pregnancy maternal factors definition; International Society for the Study of Hypertension in Pregnancy maternal factors plus fetal death or fetal growth restriction definition, defined according to the 35 0/7 to 36 6/7 weeks' gestation scan as either estimated fetal weight <3rd percentile or estimated fetal weight at the 3rd to 10th percentile with any of uterine artery pulsatility index >95th percentile, umbilical artery pulsatility index >95th percentile, or middle cerebral artery pulsatility index <5th percentile; and International Society for the Study of Hypertension in Pregnancy maternal-fetal factors plus angiogenic imbalance definition, defined as placental growth factor <5th percentile or soluble fms-like tyrosine kinase-1-to-serum placental growth factor >95th percentile. Detection rates for outcomes of interest (ie, severe maternal hypertension, major maternal morbidity, perinatal mortality or major neonatal morbidity, neonatal unit admission ≥48 hours, and birthweight <10th percentile) were compared using the chi-square test, and P<.05 was considered significant. RESULTS: Among 15,248 singleton pregnancies, the identification of women with preeclampsia varied by definition: traditional, 15 of 281 (1.8%; 248); American College of Obstetricians and Gynecologists, 15 of 326 (2.1%; 248); International Society for the Study of Hypertension in Pregnancy maternal factors, 15 of 400 (2.6%; 248); International Society for the Study of Hypertension in Pregnancy maternal-fetal factors, 15 of 434 (2.8%; 248); and International Society for the Study of Hypertension in Pregnancy maternal-fetal factors plus angiogenic imbalance, 15 of 500 (3.3%; 248). Compared with the traditional definition of preeclampsia, the International Society for the Study of Hypertension in Pregnancy maternal-fetal factors plus angiogenic imbalance best identified the adverse outcomes: severe hypertension (40.6% [traditional] vs 66.9% [International Society for the Study of Hypertension in Pregnancy maternal-fetal factors plus angiogenic imbalance, P<.0001], 59.2% [International Society for the Study of Hypertension in Pregnancy maternal-fetal factors, P=.004], 56.2% [International Society for the Study of Hypertension in Pregnancy maternal factors, P=.013], 46.1% [American College of Obstetricians and Gynecologists, P=.449]); P<.0001); composite maternal severe adverse event (72.2% [traditional] vs 100% for all others; P=.046); composite of perinatal mortality and morbidity (46.9% [traditional] vs 71.1% [International Society for the Study of Hypertension in Pregnancy maternal-fetal factors plus angiogenic imbalance, P=.002], 62.2% [International Society for the Study of Hypertension in Pregnancy maternal-fetal factors, P=.06], 59.8% [International Society for the Study of Hypertension in Pregnancy maternal factors, P=.117], 49.4% [American College of Obstetricians and Gynecologists, P=.875]); neonatal unit admission for ≥48 hours (51.4% [traditional] vs 73.4% [International Society for the Study of Hypertension in Pregnancy maternal-fetal factors plus angiogenic imbalance, P=.001], 64.5% [International Society for the Study of Hypertension in Pregnancy maternal-fetal factors, P=.070], 60.7% [International Society for the Study of Hypertension in Pregnancy maternal factors, P=.213], 53.3% [American College of Obstetricians and Gynecologists, P=.890]); birthweight <10th percentile (40.5% [traditional] vs 78.7% [International Society for the Study of Hypertension in Pregnancy maternal-fetal factors plus angiogenic imbalance, P<.0001], 70.1% [International Society for the Study of Hypertension in Pregnancy maternal-fetal, P<.0001], 51.3% [International Society for the Study of Hypertension in Pregnancy maternal factors, P=.064], 46.3% [American College of Obstetricians and Gynecologists, P=.349]). CONCLUSION: Our findings present an evidence base for the broad definition of preeclampsia. Our data suggest that compared with a traditional definition, a broad definition of preeclampsia can better identify women and babies at risk of adverse outcomes. Compared with the American College of Obstetricians and Gynecologists definition, the more inclusive International Society for the Study of Hypertension in Pregnancy definition of maternal end-organ dysfunction seems to be more sensitive. The addition of uteroplacental dysfunction to the broad definition optimizes the identification of women and babies at risk, particularly when angiogenic factors are included.

Validation and development of models using clinical, biochemical and ultrasound markers for predicting pre-eclampsia: an individual participant data meta-analysis.

BACKGROUND: Pre-eclampsia is a leading cause of maternal and perinatal mortality and morbidity. Early identification of women at risk is needed to plan management. OBJECTIVES: To assess the performance of existing pre-eclampsia prediction models and to develop and validate models for pre-eclampsia using individual participant data meta-analysis. We also estimated the prognostic value of individual markers. DESIGN: This was an individual participant data meta-analysis of cohort studies. SETTING: Source data from secondary and tertiary care. PREDICTORS: We identified predictors from systematic reviews, and prioritised for importance in an international survey. PRIMARY OUTCOMES: Early-onset (delivery at < 34 weeks' gestation), late-onset (delivery at ≥ 34 weeks' gestation) and any-onset pre-eclampsia. ANALYSIS: We externally validated existing prediction models in UK cohorts and reported their performance in terms of discrimination and calibration. We developed and validated 12 new models based on clinical characteristics, clinical characteristics and biochemical markers, and clinical characteristics and ultrasound markers in the first and second trimesters. We summarised the data set-specific performance of each model using a random-effects meta-analysis. Discrimination was considered promising for C-statistics of ≥ 0.7, and calibration was considered good if the slope was near 1 and calibration-in-the-large was near 0. Heterogeneity was quantified using I2 and τ2. A decision curve analysis was undertaken to determine the clinical utility (net benefit) of the models. We reported the unadjusted prognostic value of individual predictors for pre-eclampsia as odds ratios with 95% confidence and prediction intervals. RESULTS: The International Prediction of Pregnancy Complications network comprised 78 studies (3,570,993 singleton pregnancies) identified from systematic reviews of tests to predict pre-eclampsia. Twenty-four of the 131 published prediction models could be validated in 11 UK cohorts. Summary C-statistics were between 0.6 and 0.7 for most models, and calibration was generally poor owing to large between-study heterogeneity, suggesting model overfitting. The clinical utility of the models varied between showing net harm to showing minimal or no net benefit. The average discrimination for IPPIC models ranged between 0.68 and 0.83. This was highest for the second-trimester clinical characteristics and biochemical markers model to predict early-onset pre-eclampsia, and lowest for the first-trimester clinical characteristics models to predict any pre-eclampsia. Calibration performance was heterogeneous across studies. Net benefit was observed for International Prediction of Pregnancy Complications first and second-trimester clinical characteristics and clinical characteristics and biochemical markers models predicting any pre-eclampsia, when validated in singleton nulliparous women managed in the UK NHS. History of hypertension, parity, smoking, mode of conception, placental growth factor and uterine artery pulsatility index had the strongest unadjusted associations with pre-eclampsia. LIMITATIONS: Variations in study population characteristics, type of predictors reported, too few events in some validation cohorts and the type of measurements contributed to heterogeneity in performance of the International Prediction of Pregnancy Complications models. Some published models were not validated because model predictors were unavailable in the individual participant data. CONCLUSION: For models that could be validated, predictive performance was generally poor across data sets. Although the International Prediction of Pregnancy Complications models show good predictive performance on average, and in the singleton nulliparous population, heterogeneity in calibration performance is likely across settings. FUTURE WORK: Recalibration of model parameters within populations may improve calibration performance. Additional strong predictors need to be identified to improve model performance and consistency. Validation, including examination of calibration heterogeneity, is required for the models we could not validate. STUDY REGISTRATION: This study is registered as PROSPERO CRD42015029349. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 72. See the NIHR Journals Library website for further project information.

WHAT IS THE PROBLEM?: Pre-eclampsia, a condition in pregnancy that results in raised blood pressure and protein in the urine, is a major cause of complications for the mother and baby. WHAT IS NEEDED?: A way of accurately identifying women at high risk of pre-eclampsia to allow clinicians to start preventative interventions such as administering aspirin or frequently monitoring women during pregnancy. WHERE ARE THE RESEARCH GAPS?: Although over 100 tools (models) have been reported worldwide to predict pre-eclampsia, to date their performance in women managed in the UK NHS is unknown. WHAT DID WE PLAN TO DO?: We planned to comprehensively identify all published models that predict the risk of pre-eclampsia occurring at any time during pregnancy and to assess if this prediction is accurate in the UK population. If the existing models did not perform satisfactorily, we aimed to develop new prediction models. WHAT DID WE FIND?: We formed the International Prediction of Pregnancy Complications network, which provided data from a large number of studies (78 studies, 25 countries, 125 researchers, 3,570,993 singleton pregnancies). We were able to assess the performance of 24 out of the 131 models published to predict pre-eclampsia in 11 UK data sets. The models did not accurately predict the risk of pre-eclampsia across all UK data sets, and their performance varied within individual data sets. We developed new prediction models that showed promising performance on average across all data sets, but their ability to correctly identify women who develop pre-eclampsia varied between populations. The models were more clinically useful when used in the care of first-time mothers pregnant with one child, compared to a strategy of treating them all as if they were at high-risk of pre-eclampsia. WHAT DOES THIS MEAN?: Before using the International Prediction of Pregnancy Complications models in various populations, they need to be adjusted for characteristics of the particular population and the setting of application.

Predisposition to superimposed preeclampsia in women with chronic hypertension: endothelial, renal, cardiac, and placental factors in a prospective longitudinal cohort.

OBJECTIVE: To assess the contribution of maternal and placental factors to the development of superimposed preeclampsia in women with chronic hypertension. METHODS: Endothelial and renal function markers were serially assessed in 90 pregnant women with chronic hypertension and controls. RESULTS: Syndecan-1 concentrations were lower at 26-27+6 weeks in women with chronic hypertension who subsequently developed superimposed preeclampsia compared with those who did not. Decreased PlGF and raised urine albumin:creatinine ratio were also associated with development of superimposed preeclampsia. CONCLUSION: Decreased syndecan-1 and PlGF concentrations implicate endothelial glycocalyx disturbance and reduced placental angiogenic capacity, respectively, in the pathophysiology of superimposed preeclampsia.

NPFF increases fusogenic proteins syncytin 1 and syncytin 2 via GCM1 in first trimester primary human cytotrophoblast cells.

Neuropeptide FF (NPFF) is well-known for its roles in the central nervous system. Despite studies demonstrating that NPFF receptor 2 (NPFFR2) mRNA is highest in placenta, nothing is known about NPFF-NPFFR2 functions in placental development. Here, we investigated the effects of NPFF-NPFFR2 on expression of syncytial [human chorionic gonadotropin (hCG) ß] and fusogenic [syncytin 1, syncytin 2, and glial cells missing 1 (GCM1)] genes in first trimester primary human cytotrophoblast cells. By analyzing two publicly available microarray data sets, we found that NPFF is consistently expressed throughout gestation whereas NPFFR2 increases in first trimester and is elevated in placenta samples from women with preeclampsia. Immunohistochemistry showed that NPFFR2, syncytin 1/2, and GCM1 each displayed unique patterns of expression among different trophoblast populations in first trimester placenta. Treatment of primary human cytotrophoblast cells with NPFF increased the mRNA and protein levels of hCG ß, syncytin 1, syncytin 2, and GCM1; and knockdown of NPFFR2 abolished these effects. Interestingly, GCM1 mediated NPFF-induced upregulation of syncytin 1 and syncytin 2, but not hCG ß, in primary human cytotrophoblasts. Our results demonstrate that NPFF acts via NPFFR2 to enhance production of hCG ß and promote GCM1-dependent expression of syncytin 1 and 2 in human cytotrophoblasts.

Risk factors for postpartum sepsis: a nested case-control study.

BACKGROUND: The Majority (99%) of maternal deaths occur in low and middle-income countries. The three most important causes of maternal deaths in these regions are postpartum hemorrhage, pre-eclampsia and puerperal sepsis. There are several diagnostic criteria used to identify sepsis and one of the commonly used criteria is systematic inflammatory response syndrome (SIRS). However, these criteria require laboratory investigations that may not be feasible in resource-constrained settings. Therefore, this study aimed to develop a model based on risk factors and clinical signs and symptoms that can identify sepsis early among postpartum women. METHODS: A case-control study was nested in an ongoing cohort of 4000 postpartum women who delivered or were admitted to the study hospital. According to standard criteria of SIRS, 100 women with sepsis (cases) and 498 women without sepsis (controls) were recruited from January to July 2017. Information related to the socio-demographic status, antenatal care and use of tobacco were obtained via interview while pregnancy and delivery related information, comorbid and clinical sign and symptoms were retrieved from the ongoing cohort. Multivariable logistic regression was performed and discriminative performance of the model was assessed using area under the curve (AUC) of the receiver operating characteristic (ROC). RESULTS: Multivariable analysis revealed that 1-4 antenatal visits (95% CI 0.01-0.62). , 3 or more vaginal examinations (95% CI 1.21-3.65), home delivery (95% CI 1.72-50.02), preterm delivery, diabetes in pregnancy (95% CI 1.93-20.23), lower abdominal pain (95% CI 1.15-3.42)) vaginal discharge (95% CI 2.97-20.21), SpO2 < 93% (95% CI 4.80-37.10) and blood glucose were significantly associated with sepsis. AUC was 0.84 (95% C.I 0.80-0.89) which indicated that risk factors and clinical sign and symptoms-based model has adequate ability to discriminate women with and without sepsis. CONCLUSION: This study developed a non-invasive tool that can identify postpartum women with sepsis as accurately as SIRS criteria with good discriminative ability. Once validated, this tool has the potential to be scaled up for community use by frontline health care workers.

PREPARE: protocol for a stepped wedge trial to evaluate whether a risk stratification model can reduce preterm deliveries among women with suspected or confirmed preterm pre-eclampsia.

BACKGROUND: Preeclampsia (PE) is a major cause of short and long-term morbidity for affected infants, including consequences of fetal growth restriction and iatrogenic prematurity. In Brazil, this is a special problem as PE accounts for 18% of preterm births (PTB). In the PREPARE (Prematurity REduction by Pre-eclampsia cARE) study, we will test a novel system of integrated care based on risk stratification and knowledge transfer, to safely reduce PTB. METHODS: This is a stepped wedge cluster randomised trial that will include women with suspected or confirmed PE between 20 + 0 and 36 + 6 gestational weeks. All pregnant women presenting with these findings at seven tertiary centres in geographically dispersed sites, throughout Brazil, will be considered eligible and evaluated in terms of risk stratification at admission. At randomly allocated time points, sites will transition to risk stratification performed according to sFlt-1/PlGF (Roche Diagnostics) measurement and fullPIERS score with both results will be revealed to care providers. The healthcare providers of women stratified as low risk for adverse outcomes (sFlt-1/PlGF ≤38 AND fullPIERS< 10% risk) will receive the recommendation to defer delivery. sFlt-1/PlGF will be repeated once and fullPIERS score twice a week. Rates of prematurity due to preeclampsia before and after the intervention will be compared. Additionally, providers will receive an active program of knowledge transfer about WHO recommendations for preeclampsia, including recommendations regarding antenatal corticosteroids for foetal benefits, antihypertensive therapy and magnesium sulphate for seizure prophylaxis. This study will have 90% power to detect a reduction in PTB associated with PE from a population estimate of 1.5 to 1.0%, representing a 33% risk reduction, and 80% power to detect a reduction from 2.0 to 1.5% (25% risk reduction). The necessary number of patients recruited to achieve these results is 750. Adverse events, serious adverse events, both anticipated and unanticipated will be recorded. DISCUSSION: The PREPARE intervention expects to reduce PTB and improve care of women with PE without significant adverse side effects. If successful, this novel pathway of care is designed for rapid translation to healthcare throughout Brazil and may be transferrable to other low and middle income countries. TRIAL REGISTRATION: ClinicalTrials.gov : NCT03073317.

The International Federation of Gynecology and Obstetrics (FIGO) initiative on pre-eclampsia: A pragmatic guide for first-trimester screening and prevention.

Pre­eclampsia (PE) is a multisystem disorder that typically affects 2%­5% of pregnant women and is one of the leading causes of maternal and perinatal morbidity and mortality, especially when the condition is of early onset. Globally, 76 000 women and 500 000 babies die each year from this disorder. Furthermore, women in low­resource countries are at a higher risk of developing PE compared with those in high­resource countries. Although a complete understanding of the pathogenesis of PE remains unclear, the current theory suggests a two­stage process. The first stage is caused by shallow invasion of the trophoblast, resulting in inadequate remodeling of the spiral arteries. This is presumed to lead to the second stage, which involves the maternal response to endothelial dysfunction and imbalance between angiogenic and antiangiogenic factors, resulting in the clinical features of the disorder. Accurate prediction and uniform prevention continue to elude us. The quest to effectively predict PE in the first trimester of pregnancy is fueled by the desire to identify women who are at high risk of developing PE, so that necessary measures can be initiated early enough to improve placentation and thus prevent or at least reduce the frequency of its occurrence. Furthermore, identification of an "at risk" group will allow tailored prenatal surveillance to anticipate and recognize the onset of the clinical syndrome and manage it promptly. PE has been previously defined as the onset of hypertension accompanied by significant proteinuria after 20 weeks of gestation. Recently, the definition of PE has been broadened. Now the internationally agreed definition of PE is the one proposed by the International Society for the Study of Hypertension in Pregnancy (ISSHP). According to the ISSHP, PE is defined as systolic blood pressure at ≥140 mm Hg and/or diastolic blood pressure at ≥90 mm Hg on at least two occasions measured 4 hours apart in previously normotensive women and is accompanied by one or more of the following new­onset conditions at or after 20 weeks of gestation: 1.Proteinuria (i.e. ≥30 mg/mol protein:creatinine ratio; ≥300 mg/24 hour; or ≥2 + dipstick); 2.Evidence of other maternal organ dysfunction, including: acute kidney injury (creatinine ≥90 µmol/L; 1 mg/dL); liver involvement (elevated transaminases, e.g. alanine aminotransferase or aspartate aminotransferase >40 IU/L) with or without right upper quadrant or epigastric abdominal pain; neurological complications (e.g. eclampsia, altered mental status, blindness, stroke, clonus, severe headaches, and persistent visual scotomata); or hematological complications (thrombocytopenia­platelet count <150 000/µL, disseminated intravascular coagulation, hemolysis); or 3.Uteroplacental dysfunction (such as fetal growth restriction, abnormal umbilical artery Doppler waveform analysis, or stillbirth). It is well established that a number of maternal risk factors are associated with the development of PE: advanced maternal age; nulliparity; previous history of PE; short and long interpregnancy interval; use of assisted reproductive technologies; family history of PE; obesity; Afro­Caribbean and South Asian racial origin; co­morbid medical conditions including hyperglycemia in pregnancy; pre­existing chronic hypertension; renal disease; and autoimmune diseases, such as systemic lupus erythematosus and antiphospholipid syndrome. These risk factors have been described by various professional organizations for the identification of women at risk of PE; however, this approach to screening is inadequate for effective prediction of PE. PE can be subclassified into: 1.Early­onset PE (with delivery at <34+0 weeks of gestation); 2.Preterm PE (with delivery at <37+0 weeks of gestation); 3.Late­onset PE (with delivery at ≥34+0 weeks of gestation); 4.Term PE (with delivery at ≥37+0 weeks of gestation). These subclassifications are not mutually exclusive. Early­onset PE is associated with a much higher risk of short­ and long­term maternal and perinatal morbidity and mortality. Obstetricians managing women with preterm PE are faced with the challenge of balancing the need to achieve fetal maturation in utero with the risks to the mother and fetus of continuing the pregnancy longer. These risks include progression to eclampsia, development of placental abruption and HELLP (hemolysis, elevated liver enzyme, low platelet) syndrome. On the other hand, preterm delivery is associated with higher infant mortality rates and increased morbidity resulting from small for gestational age (SGA), thrombocytopenia, bronchopulmonary dysplasia, cerebral palsy, and an increased risk of various chronic diseases in adult life, particularly type 2 diabetes, cardiovascular disease, and obesity. Women who have experienced PE may also face additional health problems in later life, as the condition is associated with an increased risk of death from future cardiovascular disease, hypertension, stroke, renal impairment, metabolic syndrome, and diabetes. The life expectancy of women who developed preterm PE is reduced on average by 10 years. There is also significant impact on the infants in the long term, such as increased risks of insulin resistance, diabetes mellitus, coronary artery disease, and hypertension in infants born to pre­eclamptic women. The International Federation of Gynecology and Obstetrics (FIGO) brought together international experts to discuss and evaluate current knowledge on PE and develop a document to frame the issues and suggest key actions to address the health burden posed by PE. FIGO's objectives, as outlined in this document, are: (1) To raise awareness of the links between PE and poor maternal and perinatal outcomes, as well as to the future health risks to mother and offspring, and demand a clearly defined global health agenda to tackle this issue; and (2) To create a consensus document that provides guidance for the first­trimester screening and prevention of preterm PE, and to disseminate and encourage its use. Based on high­quality evidence, the document outlines current global standards for the first­trimester screening and prevention of preterm PE, which is in line with FIGO good clinical practice advice on first trimester screening and prevention of pre­eclampsia in singleton pregnancy.1 It provides both the best and the most pragmatic recommendations according to the level of acceptability, feasibility, and ease of implementation that have the potential to produce the most significant impact in different resource settings. Suggestions are provided for a variety of different regional and resource settings based on their financial, human, and infrastructure resources, as well as for research priorities to bridge the current knowledge and evidence gap. To deal with the issue of PE, FIGO recommends the following: Public health focus: There should be greater international attention given to PE and to the links between maternal health and noncommunicable diseases (NCDs) on the Sustainable Developmental Goals agenda. Public health measures to increase awareness, access, affordability, and acceptance of preconception counselling, and prenatal and postnatal services for women of reproductive age should be prioritized. Greater efforts are required to raise awareness of the benefits of early prenatal visits targeted at reproductive­aged women, particularly in low­resource countries. Universal screening: All pregnant women should be screened for preterm PE during early pregnancy by the first­trimester combined test with maternal risk factors and biomarkers as a one­step procedure. The risk calculator is available free of charge at https://fetalmedicine.org/research/assess/preeclampsia. FIGO encourages all countries and its member associations to adopt and promote strategies to ensure this. The best combined test is one that includes maternal risk factors, measurements of mean arterial pressure (MAP), serum placental growth factor (PLGF), and uterine artery pulsatility index (UTPI). Where it is not possible to measure PLGF and/or UTPI, the baseline screening test should be a combination of maternal risk factors with MAP, and not maternal risk factors alone. If maternal serum pregnancy­associated plasma protein A (PAPP­A) is measured for routine first­trimester screening for fetal aneuploidies, the result can be included for PE risk assessment. Variations to the full combined test would lead to a reduction in the performance screening. A woman is considered high risk when the risk is 1 in 100 or more based on the first­trimester combined test with maternal risk factors, MAP, PLGF, and UTPI. Contingent screening: Where resources are limited, routine screening for preterm PE by maternal factors and MAP in all pregnancies and reserving measurements of PLGF and UTPI for a subgroup of the population (selected on the basis of the risk derived from screening by maternal factors and MAP) can be considered. Prophylactic measures: Following first­trimester screening for preterm PE, women identified at high risk should receive aspirin prophylaxis commencing at 11­14+6 weeks of gestation at a dose of ~150 mg to be taken every night until 36 weeks of gestation, when delivery occurs, or when PE is diagnosed. Low­dose aspirin should not be prescribed to all pregnant women. In women with low calcium intake (<800 mg/d), either calcium replacement (≤1 g elemental calcium/d) or calcium supplementation (1.5­2 g elemental calcium/d) may reduce the burden of both early­ and late­onset PE.

Development and internal validation of the multivariable CIPHER (Collaborative Integrated Pregnancy High-dependency Estimate of Risk) clinical risk prediction model.

BACKGROUND: Intensive care unit (ICU) outcome prediction models, such as Acute Physiology And Chronic Health Evaluation (APACHE), were designed in general critical care populations and their use in obstetric populations is contentious. The aim of the CIPHER (Collaborative Integrated Pregnancy High-dependency Estimate of Risk) study was to develop and internally validate a multivariable prognostic model calibrated specifically for pregnant or recently delivered women admitted for critical care. METHODS: A retrospective observational cohort was created for this study from 13 tertiary facilities across five high-income and six low- or middle-income countries. Women admitted to an ICU for more than 24 h during pregnancy or less than 6 weeks post-partum from 2000 to 2012 were included in the cohort. A composite primary outcome was defined as maternal death or need for organ support for more than 7 days or acute life-saving intervention. Model development involved selection of candidate predictor variables based on prior evidence of effect, availability across study sites, and use of LASSO (Least Absolute Shrinkage and Selection Operator) model building after multiple imputation using chained equations to address missing data for variable selection. The final model was estimated using multivariable logistic regression. Internal validation was completed using bootstrapping to correct for optimism in model performance measures of discrimination and calibration. RESULTS: Overall, 127 out of 769 (16.5%) women experienced an adverse outcome. Predictors included in the final CIPHER model were maternal age, surgery in the preceding 24 h, systolic blood pressure, Glasgow Coma Scale score, serum sodium, serum potassium, activated partial thromboplastin time, arterial blood gas (ABG) pH, serum creatinine, and serum bilirubin. After internal validation, the model maintained excellent discrimination (area under the curve of the receiver operating characteristic (AUROC) 0.82, 95% confidence interval (CI) 0.81 to 0.84) and good calibration (slope of 0.92, 95% CI 0.91 to 0.92 and intercept of -0.11, 95% CI -0.13 to -0.08). CONCLUSIONS: The CIPHER model has the potential to be a pragmatic risk prediction tool. CIPHER can identify critically ill pregnant women at highest risk for adverse outcomes, inform counseling of patients about risk, and facilitate bench-marking of outcomes between centers by adjusting for baseline risk.

Timing of delivery in a high-risk obstetric population: a clinical prediction model.

BACKGROUND: The efficacy of antenatal corticosteroid treatment for women with threatened preterm birth depends on timely administration within 7 days before delivery. We modelled the probability of delivery within 7 days of admission to hospital among women presenting with threatened preterm birth, using routinely collected clinical characteristics. METHODS: Data from the Canadian Perinatal Network (CPN) were used, 2005-11, including women admitted to hospital with preterm labour, preterm pre-labour rupture of membranes, short cervix without contractions, or dilated cervix or prolapsed membranes without contractions at preterm gestation. Women with fetal anomaly, intrauterine fetal demise, twin-to-twin transfusion syndrome, and quadruplets were excluded. Logistic regression was undertaken to create a predictive model that was assessed for its calibration capacity, stratification ability, and classification accuracy (ROC curve). RESULTS: We included 3012 women admitted at 24-28 weeks gestation, or readmitted at up to 34 weeks gestation, to 16 tertiary-care CPN hospitals. Of these, 1473 (48.9%) delivered within 7 days of admission. Significant predictors of early delivery included maternal age, parity, gestational age at admission, smoking, preterm labour, prolapsed membranes, preterm pre-labour rupture of membranes, and antepartum haemorrhage. The area under the ROC curve was 0.724 (95% CI 0.706-0.742). CONCLUSION: We propose a useful tool to improve prediction of delivery within 7 days after admission among women with threatened preterm birth. This information is important for optimal corticosteroid treatment.

The Usefulness of the APACHE II Score in Obstetric Critical Care: A Structured Review.

OBJECTIVE: To assess the performance of the Acute Physiology and Chronic Health Evaluation II (APACHE II) mortality prediction model in pregnant and recently pregnant women receiving critical care in low-, middle-, and high-income countries during the study period (1985-2015), using a structured literature review. DATA SOURCES: Ovid MEDLINE, Embase, Web of Science, and Evidence-Based Medicine Reviews, searched for articles published between 1985 and 2015. STUDY SELECTION: Twenty-five studies (24 publications), of which two were prospective, were included in the analyses. Ten studies were from high-income countries (HICs), and 15 were from low- and middle-income countries (LMICs). Median study duration and size were six years and 124 women, respectively. DATA SYNTHESIS: ICU admission complicates 0.48% of deliveries, and pregnant and recently pregnant women account for 1.49% of ICU admissions. One quarter were admitted while pregnant, three quarters of these for an obstetric indication and for a median of three days. The median APACHE II score was 10.9, with a median APACHE II-predicted mortality of 16.6%. Observed mortality was 4.6%, and the median standardized mortality ratio was 0.36 (interquartile range 0.23 to 0.73). The standardized mortality ratio was < 0.9 in 24 of 25 studies. Women in HICs were more frequently admitted with a medical comorbidity but were less likely to die than were women in LMICs. CONCLUSION: The APACHE II score consistently overestimates mortality risks for pregnant and recently pregnant women receiving critical care, whether they reside in HICs or LMICs. There is a need for a pregnancy-specific outcome prediction model for these women.

Preventing deaths due to the hypertensive disorders of pregnancy.

In this chapter, taking a life cycle and both civil society and medically oriented approach, we will discuss the contribution of the hypertensive disorders of pregnancy (HDPs) to maternal, perinatal and newborn mortality and morbidity. Here we review various interventions and approaches to preventing deaths due to HDPs and discuss effectiveness, resource needs and long-term sustainability of the different approaches. Societal approaches, addressing sustainable development goals (SDGs) 2.2 (malnutrition), 3.7 (access to sexual and reproductive care), 3.8 (universal health coverage) and 3c (health workforce strengthening), are required to achieve SDGs 3.1 (maternal survival), 3.2 (perinatal survival) and 3.4 (reduced impact of non-communicable diseases (NCDs)). Medical solutions require greater clarity around the classification of the HDPs, increased frequency of effective antenatal visits, mandatory responses to the HDPs when encountered, prompt provision of life-saving interventions and sustained surveillance for NCD risk for women with a history of the HDPs.

The Placental Distal Villous Hypoplasia Pattern: Interobserver Agreement and Automated Fractal Dimension as an Objective Metric.

The distal villous hypoplasia (DVH) pattern is a placental correlate of fetal growth restriction. Because the pattern seems to involve less complexity than do appropriately developed placental villi, we postulated that it may be associated with lower fractal dimension-a mathematical measure of complexity. Our study objectives were to evaluate interobserver agreement related to the DVH pattern among expert pathologists and to determine whether pathologist classification of DVH correlates with fractal dimension. A study set of 30 images of placental parenchyma at ×4 magnification was created by a single pathologist from a digital slide archive. The images were graded for the DVH pattern according to pre-specified definitions and included 10 images graded as "no DVH" (grade  =  0), 10 with mild to moderate DVH (grade  =  1), and 10 with severe DVH (grade  =  2). The images were randomly sorted and shown to a panel of 4 international experts who similarly graded the images for DVH. Weighted kappas were calculated. For each image, fractal dimension was calculated by the Box Counting method. The correlation coefficient between (1) the averaged DVH scores obtained by the 5 pathologists and (2) fractal dimension was calculated. The mean weighted kappa score among the observers was 0.59 (range: 0.42-0.70). The correlation coefficient between fractal dimension and the averaged DVH score was -0.915 (P < 0.001). Expert pathologists achieve fair to substantial agreement in grading DVH, indicating consensus on the definition of DVH. Distal villous hypoplasia correlates extremely well with fractal dimension and represents an objective measure for DVH.

Genome-wide DNA methylation identifies trophoblast invasion-related genes: Claudin-4 and Fucosyltransferase IV control mobility via altering matrix metalloproteinase activity.

Previously we showed that extravillous cytotrophoblast (EVT) outgrowth and migration on a collagen gel explant model were affected by exposure to decidual natural killer cells (dNK). This study investigates the molecular causes behind this phenomenon. Genome wide DNA methylation of exposed and unexposed EVT was assessed using the Illumina Infinium HumanMethylation450 BeadChip array (450 K array). We identified 444 differentially methylated CpG loci in dNK-treated EVT compared with medium control (P < 0.05). The genes associated with these loci had critical biological roles in cellular development, cellular growth and proliferation, cell signaling, cellular assembly and organization by Ingenuity Pathway Analysis (IPA). Furthermore, 23 mobility-related genes were identified by IPA from dNK-treated EVT. Among these genes, CLDN4 (encoding claudin-4) and FUT4 (encoding fucosyltransferase IV) were chosen for follow-up studies because of their biological relevance from research on tumor cells. The results showed that the mRNA and protein expressions of both CLDN4 and FUT4 in dNK-treated EVT were significantly reduced compared with control (P < 0.01 for both CLDN4 and FUT4 mRNA expression; P < 0.001 for CLDN4 and P < 0.01 for FUT4 protein expression), and were inversely correlated with DNA methylation. Knocking down CLDN4 and FUT4 by small interfering RNA reduced trophoblast invasion, possibly through the altered matrix metalloproteinase (MMP)-2 and/or MMP-9 expression and activity. Taken together, dNK alter EVT mobility at least partially in association with an alteration of DNA methylation profile. Hypermethylation of CLDN4 and FUT4 reduces protein expression. CLDN4 and FUT4 are representative genes that participate in modulating trophoblast mobility.

STRIDER: Sildenafil Therapy In Dismal prognosis Early-onset intrauterine growth Restriction--a protocol for a systematic review with individual participant data and aggregate data meta-analysis and trial sequential analysis.

BACKGROUND: In pregnancies complicated by early-onset extreme fetal growth restriction, there is a high risk of preterm birth and an overall dismal fetal prognosis. Sildenafil has been suggested to improve this prognosis. The first aim of this review is to assess whether sildenafil benefits or harms these babies. The second aim is to analyse if these effects are modified in a clinically meaningful way by factors related to the women or the trial protocol. METHODS/DESIGN: The STRIDER (Sildenafil Therapy In Dismal prognosis Early-onset intrauterine growth Restriction) Individual Participant Data (IPD) Study Group will conduct a prospective IPD and aggregate data systematic review with meta-analysis and trial sequential analysis. The STRIDER IPD Study Group started trial planning and funding applications in 2012. Three trials will be launched in 2014, recruiting for three years. Further trials are planned to commence in 2015.The primary outcome for babies is being alive at term gestation without evidence of serious adverse neonatal outcome. The latter is defined as severe central nervous system injury (severe intraventricular haemorrhage (grade 3 and 4) or cystic periventricular leukomalacia, demonstrated by ultrasound and/or magnetic resonance imaging) or other severe morbidity (bronchopulmonary dysplasia, retinopathy of prematurity requiring treatment, or necrotising enterocolitis requiring surgery). The secondary outcomes are improved fetal growth velocity assessed by ultrasound abdominal circumference measurements, gestational age and birth weight (centile) at delivery, and age-adequate performance on the two-year Bayley scales of infant and toddler development-III (composite cognitive score and composite motor score). Subgroup and sensitivity analyses in the IPD meta-analysis include assessment of the influence of several patient characteristics: an abnormal or normal serum level of placental growth factor, absent/reversed umbilical arterial end diastolic flow at commencement of treatment, and other patient characteristics available at baseline such as gestational age and estimated fetal weight. The secondary outcomes for mothers include co-incidence and severity of the maternal syndrome of pre-eclampsia, mortality, and other serious adverse events. DISCUSSION: Trials are expected to start in 2013-2014 and end in 2016-2017. Data analyses of individual trials are expected to finish in 2019. Given the pre-planned and agreed IPD protocol, these results should be available in 2020.

CMV infection, TLR-2 and -4 expression, and cytokine profiles in early-onset preeclampsia with HELLP syndrome.

PROBLEM: Cytomegalovirus (CMV) infection was previously reported in pregnancy complications. However, its seroprevalence and associated Toll-like receptor (TLR) expression in early-onset preeclampsia (EOPE) with hemolysis, elevated liver enzyme and low platelets syndrome (HELLPs) are unexplored. METHOD OF STUDY: A case­control study was performed to examine maternal CMV antibodies, neutrophil Toll-like receptor (TLR)-2 and -4 expression as well as the cytokine profile in EOPE with HELLPs (EOPE-HELLPs) (n = 10), late-onset preeclampsia (LOPE) (n = 20), normal pregnancy (n = 60), and non-pregnancy (n = 20) controls. RESULTS: EOPE-HELLPs had significantly increased CMV IgG sero-positivity, upregulated TLR-2/-4 mRNA expression, increased serum IL-6 and TNF-α, and reduced IL-10 compared with matched normal and non-pregnancy controls. No significant difference was observed between LOPE and normal pregnancy controls. CONCLUSION: We observed a significant association between CMV IgG sero-positivity and innate immune response in EOPE-HELLPs. Our data suggest that CMV infection may be a risk factor for this disorder.

Widespread DNA hypomethylation at gene enhancer regions in placentas associated with early-onset pre-eclampsia.

Pre-eclampsia is a serious complication of pregnancy that can affect both maternal and fetal outcomes. Early-onset pre-eclampsia (EOPET) is a severe form of pre-eclampsia that is associated with altered physiological characteristics and gene expression in the placenta. DNA methylation is a relatively stable epigenetic modification to DNA that can reflect gene expression, and can provide insight into the mechanisms underlying such expression changes. This case-control study focused on DNA methylation and gene expression of whole chorionic villi samples from 20 EOPET placentas and 20 gestational age-matched controls from pre-term births. DNA methylation was also assessed in placentas affected by late-onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR). The Illumina HumanMethylation450 BeadChip was used to assess DNA methylation at >480 000 cytosine-guanine dinucleotide (CpG) sites. The Illumina HT-12v4 Expression BeadChip was used to assess gene expression of >45 000 transcripts in a subset of cases and controls. DNA methylation analysis by pyrosequencing was used to follow-up the initial findings in four genes with a larger cohort of cases and controls, including nIUGR and LOPET placentas. Bioinformatic analysis was used to identify overrepresentation of gene ontology categories and transcription factor binding motifs. We identified 38 840 CpG sites with significant (false discovery rate <0.01) DNA methylation alterations in EOPET, of which 282 had >12.5% methylation difference compared with the controls. Significant sites were enriched at the enhancers and low CpG density regions of the associated genes and the majority (74.5%) of these sites were hypomethylated in EOPET. EOPET, but not associated clinical features, such as intrauterine growth restriction (IUGR), presented a distinct DNA methylation profile. CpG sites from four genes relevant to pre-eclampsia (INHBA, BHLHE40, SLC2A1 and ADAM12) showed different extent of changes in LOPET and nIUGR. Genome-wide expression in a subset of samples showed that some of the gene expression changes were negatively correlated with DNA methylation changes, particularly for genes that are responsible for angiogenesis (such as EPAS1 and FLT1). Results could be confounded by altered cell populations in abnormal placentas. Larger sample sizes are needed to fully address the possibility of sub-profiles of methylation within the EOPET cohort. Based on DNA methylation profiling, we conclude that there are widespread DNA methylation alterations in EOPET that may be associated with changes in placental function. This property may provide a useful tool for early screening of such placentas. This study identifies DNA methylation changes at many loci previously reported to have altered gene expression in EOPET placentas, as well as in novel biologically relevant genes we confirmed to be differentially expressed. These results may be useful for DNA- methylation-based non-invasive prenatal diagnosis of at-risk pregnancies.