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Circulating maternal placental growth factor responses to low-molecular-weight heparin in pregnant patients at risk of placental dysfunction

Published:August 27, 2021DOI:https://doi.org/10.1016/j.ajog.2021.08.027

      Background

      Patients at high risk of severe preeclampsia and fetal growth restriction have low circulating levels of placental growth factor and features of maternal vascular malperfusion placental pathology at delivery. Multimodal screening and commencement of aspirin prophylaxis at 11 to 13 weeks’ gestation markedly reduces the risk of preterm delivery with preeclampsia. However, the additional role of low-molecular-weight heparin and mechanisms of action remain uncertain. Because low-molecular-weight heparin augments the production and release of placental growth factor in vitro by both placental villi and vascular endothelium, it may be effective to suppress the risk of severe preeclampsia in a niche group of high-risk patients with low circulating placental growth factor in the early second trimester.

      Objective

      This study aimed to define a gestational age-specific reference range for placental growth factor and to test the hypothesis that prophylactic low-molecular-weight heparin administered in the early second trimester may restore deficient circulating placental growth factor levels and thereby prolong pregnancy.

      Study Design

      Centile curves for circulating placental growth factor levels from 12 to 36 weeks’ gestation were derived using quantile regression of combined data from a published cohort of 4207 unselected nulliparous patients in Cambridge, United Kingdom, at 4 sampling time points (12, 20, 28, and 36 weeks’ gestation) and the White majority (n=531) of a healthy nulliparous cohort in Toronto, Canada, at 16 weeks’ gestation using the same test platform. Within a specialty high-risk clinic in Toronto, a niche group of 7 patients with a circulating placental growth factor at the <10th centile in the early second trimester received daily prophylactic low-molecular-weight heparin (enoxaparin; 40 mg subcutaneously) and were followed up until delivery (group 1). Their baseline characteristics, delivery details, and placental pathologies were compared with 5 similar patients who did not receive low-molecular-weight heparin during the observation period (group 2) and further with 21 patients who delivered with severe preeclampsia (group 3) in the same institution.

      Results

      A gestational age-specific reference range for placental growth factor levels at weekly intervals between 12 and 36 weeks was established for White women with singleton pregnancies. Within group 1, 5 of 7 patients demonstrated a sustained increase in circulating placental growth factor levels, whereas placental growth factor levels did not increase in group 2 or group 3 patients who did not receive low-molecular-weight heparin. Group 1 patients receiving low-molecular-weight heparin therapy exhibited a later gestation at delivery, relative to groups 2 and 3 (36 weeks [33–37] vs 23 weeks [22–26] and 28 weeks [27–31], respectively), and consequently had higher birthweights (1.93 kg [1.1–2.7] vs 0.32 kg [0.19–0.39] and 0.73 kg [0.52–1.03], respectively). The incidence of stillbirth was lowest in group 1 (14% [1 of 7]), relative to groups 2 and 3 (80% [4 of 5] and 29% [6 of 21], respectively). Maternal vascular malperfusion was the most common placental pathology found in association with abnormal uterine artery Doppler.

      Conclusion

      In patients at high risk of a serious adverse pregnancy outcome owing to placental disease, the addition of low-molecular-weight heparin to aspirin prophylaxis in the early second trimester may restore deficient circulating placental growth factor to mediate an improved perinatal outcome. These data support the implementation of a multicenter pilot randomized control trial where patients are recruited primarily based on the assessment of placental function in the early second trimester.

      Key words

      Click Supplemental Materials under article title in Contents at ajog.org

      Introduction

      Placental growth factor (PlGF) is a proangiogenic protein produced by both the maternal vascular endothelium and the trophoblast layer covering the placental villi.
      • McLaughlin K.
      • Nadeem L.
      • Wat J.
      • Baczyk D.
      • Lye S.J.
      • Kingdom J.C.
      Low molecular weight heparin promotes transcription and release of placental growth factor from endothelial cells.
      ,
      • Clark D.E.
      • Smith S.K.
      • Licence D.
      • Evans A.L.
      • Charnock-Jones D.S.
      Comparison of expression patterns for placenta growth factor, vascular endothelial growth factor (VEGF), VEGF-B and VEGF-C in the human placenta throughout gestation.
      Circulating PlGF levels rise steadily in maternal blood until the beginning of the third trimester, reflecting both the development of the uteroplacental circulation and placental growth.
      • Sovio U.
      • Gaccioli F.
      • Cook E.
      • Hund M.
      • Charnock-Jones D.S.
      • Smith G.C.
      Prediction of preeclampsia using the Soluble fms-Like tyrosine kinase 1 to placental growth factor Ratio: a Prospective Cohort Study of Unselected Nulliparous Women.
      ,
      • Agrawal S.
      • Shinar S.
      • Cerdeira A.S.
      • Redman C.
      • Vatish M.
      Predictive performance of PlGF (placental growth factor) for screening preeclampsia in asymptomatic women: a systematic review and meta-analysis.
      Between 20 and 36 weeks’ gestation, a single cutoff value of 100 pg/mL has high diagnostic test precision for patients with suspected preeclampsia and placenta-mediated fetal growth restriction.
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      • Duckworth S.
      • Seed P.T.
      • et al.
      Diagnostic accuracy of placental growth factor in women with suspected preeclampsia: a prospective multicenter study.
      ,
      • Benton S.J.
      • McCowan L.M.
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      • et al.
      Placental growth factor as a marker of fetal growth restriction caused by placental dysfunction.
      Consequently, real-time PlGF testing is an effective clinical tool for high-risk pregnancy management.
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      • Audette M.C.
      • et al.
      PlGF (Placental growth factor) testing in clinical practice: evidence From a Canadian Tertiary Maternity Referral Center.

      Why was this study conducted?

      A meta-analysis of randomized control trials suggests that a subgroup at highest risk of developing severe early-onset preeclampsia may benefit from low-molecular-weight heparin (LMWH) in addition to low-dose aspirin but the underlying mechanism of action of LMWH in this context is unknown. Women who develop severe early-onset preeclampsia have low maternal circulating placental growth factor (PlGF) levels at presentation and before disease onset. LMWH exerts several non-anticoagulant actions in vitro, including an ability to enhance PlGF release by placental villi and the maternal vascular endothelium.

      Key findings

      A reference range for maternal circulating PlGF was generated for unselected nulliparous White women at 12 to 36 weeks’ gestation. In a small number of patients at high risk of placenta-mediated adverse pregnancy outcomes with low circulating PlGF in the early second trimester, the administration of LMWH induced sustained elevations in PlGF.

      What does this add to what is known?

      Future trials evaluating the potential benefits of LMWH for the prevention of placenta-mediated complications should consider recruitment of women in the early second trimester with low circulating PlGF, so as to focus on those at highest risk.
      Before 20 weeks’ gestation, PlGF measurements may be useful to screen pregnancies for substantial placental dysfunction causing early-onset preeclampsia, for example, in combination with clinical risk factors and mean uterine artery Doppler at 16 weeks.
      • Espinoza J.
      • Romero R.
      • Nien J.K.
      • et al.
      Identification of patients at risk for early onset and/or severe preeclampsia with the use of uterine artery Doppler velocimetry and placental growth factor.
      ,
      • Myers J.E.
      • Kenny L.C.
      • McCowan L.M.
      • et al.
      Angiogenic factors combined with clinical risk factors to predict preterm pre-eclampsia in nulliparous women: a predictive test accuracy study.
      Multimodal screening at an earlier gestation of 11 to 13 weeks’ gestation, incorporating PlGF and pregnancy-associated plasma protein-A [PAPP-A] with mean uterine artery Doppler, blood pressure, and maternal characteristics, is an effective tool to prevent preterm delivery owing to severe preeclampsia with the institution of low-dose aspirin prophylaxis.
      • Rolnik D.L.
      • Wright D.
      • Poon L.C.
      • et al.
      Aspirin versus Placebo in pregnancies at high risk for preterm preeclampsia.
      • Wright A.
      • Wright D.
      • Syngelaki A.
      • Georgantis A.
      • Nicolaides K.H.
      Two-stage screening for preterm preeclampsia at 11-13 weeks’ gestation.
      • Guy G.P.
      • Leslie K.
      • Diaz Gomez D.
      • et al.
      Implementation of routine first trimester combined screening for pre-eclampsia: a clinical effectiveness study.
      Despite this important advancement, aspirin is not universally effective and may be of no benefit in specific subgroups, such as patients with chronic hypertension.
      • Poon L.C.
      • Wright D.
      • Rolnik D.L.
      • et al.
      Aspirin for Evidence-Based preeclampsia Prevention trial: effect of aspirin in prevention of preterm preeclampsia in subgroups of women according to their characteristics and medical and obstetrical history.
      Comprehensive assessment of more complex high-risk individuals on aspirin prophylaxis at 16 weeks may identify those destined to fail treatment and deliver preterm with placenta-mediated complications.
      The dominant placental pathology underlying severe early-onset preeclampsia is termed maternal vascular malperfusion (MVM).
      • Kingdom J.C.
      • Audette M.C.
      • Hobson S.R.
      • Windrim R.C.
      • Morgen E.
      A placenta clinic approach to the diagnosis and management of fetal growth restriction.
      This complex disease is a constellation of gross and microscopic pathologic findings initiated by diseased spiral arteries, whereby the injured placenta secretes reduced amounts of PlGF followed by excessive amounts of the vascular endothelial growth factor (VEGF) antagonist soluble fms-like tyrosine Flt-1.
      • Espinoza J.
      • Romero R.
      • Nien J.K.
      • et al.
      Identification of patients at risk for early onset and/or severe preeclampsia with the use of uterine artery Doppler velocimetry and placental growth factor.
      ,
      • O’Brien M.
      • Baczyk D.
      • Kingdom J.C.
      Endothelial dysfunction in severe preeclampsia is mediated by soluble factors, rather than extracellular vesicles.
      ,
      • Zur R.L.
      • Kingdom J.C.
      • Parks W.T.
      • Hobson S.R.
      The placental basis of fetal growth restriction.
      Although aspirin is principally known to be an antiplatelet agent, the beneficial actions of aspirin in the context of preeclampsia prevention are largely unknown.
      Low-molecular-weight heparin (LMWH) is a complex macromolecule, frequently prescribed safely in pregnancy for its anticoagulant properties. However, LMWH is also capable of exerting a range of non-anticoagulant actions that are highly relevant to the maternal circulatory and placental pathogenesis of severe preeclampsia summarized in Figure 1. These actions include stimulation of PlGF production by explanted human placental villi and the vascular endothelium.
      • McLaughlin K.
      • Nadeem L.
      • Wat J.
      • Baczyk D.
      • Lye S.J.
      • Kingdom J.C.
      Low molecular weight heparin promotes transcription and release of placental growth factor from endothelial cells.
      ,
      • Wat J.M.
      • Hawrylyshyn K.
      • Baczyk D.
      • Greig I.R.
      • Kingdom J.C.
      Effects of glycol-split low molecular weight heparin on placental, endothelial, and anti-inflammatory pathways relevant to preeclampsia.
      ,
      • Drewlo S.
      • Levytska K.
      • Sobel M.
      • Baczyk D.
      • Lye S.J.
      • Kingdom J.C.
      Heparin promotes soluble VEGF receptor expression in human placental villi to impair endothelial VEGF signaling.
      Favorable acute vascular changes accompany augmented PlGF levels after a bolus administration of LMWH to pregnant patients at risk of severe placental dysfunction,
      • McLaughlin K.
      • Baczyk D.
      • Potts A.
      • Hladunewich M.
      • Parker J.D.
      • Kingdom J.C.
      Low molecular weight heparin improves endothelial function in pregnant women at high risk of preeclampsia.
      whereas pregnant patients chronically anticoagulated with higher-dose LMWH exhibit substantial elevations in circulating PlGF.
      • Yinon Y.
      • Ben Meir E.
      • Margolis L.
      • et al.
      Low molecular weight heparin therapy during pregnancy is associated with elevated circulatory levels of placental growth factor.
      Collectively, these data form a compelling argument in favor of exploring the potential value of LMWH to reduce disease severity in a subgroup of patients considered most at risk of early-onset preeclampsia, a conclusion that is supported by the most recent meta-analysis of randomized controlled trials.
      • McLaughlin K.
      • Scholten R.R.
      • Parker J.D.
      • Ferrazzi E.
      • Kingdom J.C.P.
      Low molecular weight heparin for the prevention of severe preeclampsia: where next?.
      ,
      • Cruz-Lemini M.
      • Vázquez J.C.
      • Ullmo J.
      • Llurba E.
      Low-molecular-weight heparin for prevention of preeclampsia and other placenta-mediated complications: a systematic review and meta-analysis.
      Figure thumbnail gr1
      Figure 1Systemic effects of LMWH in pregnancy
      • McLaughlin K.
      • Nadeem L.
      • Wat J.
      • Baczyk D.
      • Lye S.J.
      • Kingdom J.C.
      Low molecular weight heparin promotes transcription and release of placental growth factor from endothelial cells.
      ,
      • Wat J.M.
      • Hawrylyshyn K.
      • Baczyk D.
      • Greig I.R.
      • Kingdom J.C.
      Effects of glycol-split low molecular weight heparin on placental, endothelial, and anti-inflammatory pathways relevant to preeclampsia.
      • McLaughlin K.
      • Baczyk D.
      • Potts A.
      • Hladunewich M.
      • Parker J.D.
      • Kingdom J.C.
      Low molecular weight heparin improves endothelial function in pregnant women at high risk of preeclampsia.
      • Yinon Y.
      • Ben Meir E.
      • Margolis L.
      • et al.
      Low molecular weight heparin therapy during pregnancy is associated with elevated circulatory levels of placental growth factor.
      • Mello G.
      • Parretti E.
      • Fatini C.
      • et al.
      Low-molecular-weight heparin lowers the recurrence rate of preeclampsia and restores the physiological vascular changes in angiotensin-converting enzyme DD women.
      • Ellison J.
      • Thomson A.J.
      • Conkie J.A.
      • McCall F.
      • Walker D.
      • Greer A.
      Thromboprophylaxis following caesarean section—a comparison of the antithrombotic properties of three low molecular weight heparins--dalteparin, enoxaparin and tinzaparin.
      • Quenby S.
      • Mountfield S.
      • Cartwright J.E.
      • Whitley G.S.
      • Vince G.
      Effects of low-molecular-weight and unfractionated heparin on trophoblast function.
      • Manduteanu I.
      • Dragomir E.
      • Voinea M.
      • Capraru M.
      • Simionescu M.
      Enoxaparin reduces H2O2-induced activation of human endothelial cells by a mechanism involving cell adhesion molecules and nuclear transcription factors.
      • Torricelli M.
      • Reis F.M.
      • Florio P.
      • et al.
      Low-molecular-weight heparin improves the performance of uterine artery Doppler velocimetry to predict preeclampsia and small-for-gestational age infant in women with gestational hypertension.
      LMWH, low-molecular-weight heparin; PlGF, placental growth factor.
      McLaughlin et al. Low-molecular-weight heparin in pregnancies at risk of placental dysfunction. Am J Obstet Gynecol 2022.
      Based on these observations, our goal was to explore the potential of daily LMWH administration to restore deficient circulating levels of PlGF in clinically high-risk patients, beginning in the second trimester.

      Materials and Methods

      Placental growth factor reference range

      A reference range for PlGF was constructed by merging data from 2 studies of unselected nulliparous patients using the same assay method (Roche Diagnostics, Germany). Data at 4 sampling time points (12, 20, 28, and 36 weeks’ gestation) from a cohort of 4207 unselected nulliparous patients (92% White) in Cambridge, United Kingdom,
      • Gaccioli F.
      • Lager S.
      • Sovio U.
      • Charnock-Jones D.S.
      • Smith G.C.S.
      The pregnancy outcome prediction (POP) study: investigating the relationship between serial prenatal ultrasonography, biomarkers, placental phenotype and adverse pregnancy outcomes.
      were combined with data at the single time point of 16 weeks’ gestation from the White majority (531 of 773; 68.7%) within a healthy nulliparous cohort in Toronto, Canada.
      • Wright E.
      • Audette M.C.
      • Ye X.Y.
      • et al.
      Maternal vascular malperfusion and adverse perinatal outcomes in low-risk nulliparous women.
      The PlGF assay coefficient of variation was 2.7% to 4.1% in the Cambridge study
      • Sovio U.
      • Gaccioli F.
      • Cook E.
      • Hund M.
      • Charnock-Jones D.S.
      • Smith G.C.
      Prediction of preeclampsia using the Soluble fms-Like tyrosine kinase 1 to placental growth factor Ratio: a Prospective Cohort Study of Unselected Nulliparous Women.
      and 3.8% to 6.7% in the Toronto study.
      • Wright E.
      • Audette M.C.
      • Ye X.Y.
      • et al.
      Maternal vascular malperfusion and adverse perinatal outcomes in low-risk nulliparous women.

      Study population

      We conducted a single-center pilot observational study within a maternal-fetal medicine placenta clinic program of care focused on placental dysfunction disorders.
      • Kingdom J.C.
      • Audette M.C.
      • Hobson S.R.
      • Windrim R.C.
      • Morgen E.
      A placenta clinic approach to the diagnosis and management of fetal growth restriction.
      Eligible patients included those at the age of >18 years with a live singleton fetus and followed up in the period between July 2017 and March 2021. Patients were not eligible if they were already on LMWH therapy for a maternal indication or if previously diagnosed with a major thrombophilia disorder or antiphospholipid syndrome. Approval by the research ethics board of Mount Sinai Hospital was obtained (REB 20-0067-E). Patients at high risk of severe placental dysfunction in the current pregnancy, based on prepregnancy health and obstetrical history, received low-dose (162 mg nightly) aspirin prophylaxis for preeclampsia from 12 weeks’ gestation as part of standard clinical management.
      • Rolnik D.L.
      • Wright D.
      • Poon L.C.
      • et al.
      Aspirin versus Placebo in pregnancies at high risk for preterm preeclampsia.
      At the 16-week assessment, each patient completed an early placental health assessment comprising fetal biometry, amniotic fluid, and mean uterine artery Doppler ultrasound. Mean uterine artery pulsatility index (PI) values at the >95th centile were considered abnormal.
      • Gómez O.
      • Figueras F.
      • Fernández S.
      • et al.
      Reference ranges for uterine artery mean pulsatility index at 11-41 weeks of gestation.
      Based on previous cohort data, we included same-day PlGF testing, because this test became available for the real-time management of high-risk pregnant patients in 2017 using the Elecsys platform (Roche Diagnostics, Germany).
      • McLaughlin K.
      • Snelgrove J.W.
      • Audette M.C.
      • et al.
      PlGF (Placental growth factor) testing in clinical practice: evidence From a Canadian Tertiary Maternity Referral Center.
      ,
      • Espinoza J.
      • Romero R.
      • Nien J.K.
      • et al.
      Identification of patients at risk for early onset and/or severe preeclampsia with the use of uterine artery Doppler velocimetry and placental growth factor.
      PlGF values <10th centile at 16 weeks’ gestation were considered abnormal. Between July 2017 and March 2021, 12 pregnant patients were identified for study inclusion with PlGF results at the <10th centile between 16 and 20 weeks’ gestation.
      An additional cohort of 21 high-risk pregnant patients were identified between 20 and 24 weeks’ gestation with low circulating PlGF (<100 pg/mL) who were not on LMWH therapy and subsequently developed early-onset preeclampsia with delivery at <34 weeks’ gestation. All patients received standardized high-risk obstetrical care in Mount Sinai Hospital by a group practice of 3 maternal-fetal medicine physicians with appointments every 2 to 4 weeks and delivered at the same hospital.
      • Kingdom J.C.
      • Audette M.C.
      • Hobson S.R.
      • Windrim R.C.
      • Morgen E.
      A placenta clinic approach to the diagnosis and management of fetal growth restriction.
      After delivery, the placenta was sent for histopathology testing by a dedicated perinatal pathologist blinded to treatment during pregnancy. This observational study was not registered as a clinical trial, because LMWH therapy is approved for use in high-risk pregnant patients for the prevention of placental complications, including preeclampsia.
      • Magee L.A.
      • Pels A.
      • Helewa M.
      • Rey E.
      • von Dadelszen P.
      Canadian Hypertensive Disorders of Pregnancy Working Group. Diagnosis, evaluation, and management of the hypertensive disorders of pregnancy: executive summary.

      Study intervention

      Based on previous experimental data supporting the therapeutic potential of LMWH to restore circulating PlGF,
      • McLaughlin K.
      • Baczyk D.
      • Potts A.
      • Hladunewich M.
      • Parker J.D.
      • Kingdom J.C.
      Low molecular weight heparin improves endothelial function in pregnant women at high risk of preeclampsia.
      ,
      • Yinon Y.
      • Ben Meir E.
      • Margolis L.
      • et al.
      Low molecular weight heparin therapy during pregnancy is associated with elevated circulatory levels of placental growth factor.
      the option of adjunctive prophylactic LMWH (enoxaparin 40 mg/d subcutaneously) for the prevention of preeclampsia was reviewed with 12 patients who had low circulating PlGF before 20 weeks’ gestation. Of these, 7 received prophylactic LMWH therapy in addition to aspirin and 5 continued aspirin alone. Notably, 5 patients who initiated LMWH therapy had 2 PlGF tests between 16 and 20 weeks’ gestation at the <10th centile, whereas 2 had a single abnormal PlGF test. An additional 21 high-risk pregnant patients with low PlGF levels between 20 and 24 weeks’ gestation who did not initiate LMWH therapy and subsequently developed early-onset preeclampsia with delivery <34 weeks’ gestation were identified. All participants continued to receive maternal-fetal medicine obstetrical care at the placenta clinic, with appointments every 2 to 4 weeks, and delivered at Mount Sinai Hospital.

      Outcomes

      The primary outcome of this pilot observational study was change in circulating maternal PlGF levels in response to LMWH, measured every 2 to 4 weeks in the placenta clinic appointments. Maternal PlGF levels after 16 weeks’ gestation were overlaid on the newly derived reference range for comparative purposes. Secondary outcomes included change in mean uterine artery Doppler PI, gestational age at delivery, maternal and perinatal outcomes, and placental pathology diagnosis. Preeclampsia was defined as systolic blood pressure of ≥140 mm Hg or diastolic blood pressure of ≥90 mm Hg on 2 occasions at least 4 hours apart after 20+0 weeks’ gestation, with evidence of related organ injury: proteinuria (urine protein to creatinine ratio of ≥30 mg/mmol), thrombocytopenia (platelets of <100×109/L), renal compromise (serum creatinine of ≥1.1 mg/dL), or impaired liver function (aspartate aminotransferase of ≥70 U/L or alanine aminotransferase of ≥70 U/L).
      Hypertension in pregnancy. Report of the American College of Obstetricians and Gynecologists’ Task Force on Hypertension in Pregnancy.
      Placental pathology diagnoses were established according to the Amsterdam Criteria of Standardized Placental Classification.
      • Khong T.Y.
      • Mooney E.E.
      • Ariel I.
      • et al.
      Sampling and definitions of placental lesions: Amsterdam placental workshop group consensus statement.

      Statistical analysis

      The reference range of PlGF levels across gestation in healthy pregnant patients generated by the Cambridge, United Kingdom, cohort and the White majority of the Toronto, Canada, cohort was described by quantile regression curves using the LMS method described by Cole and Green
      • Cole T.J.
      • Green P.J.
      Smoothing reference centile curves: the LMS method and penalized likelihood.
      and implemented in the gamlss package
      • Rigby R.A.
      • Stasinopoulos D.M.
      Generalized additive models for location, scale and shape.
      within the R statistical computing system (version 4.0.2, R Foundation for Statistical Computing, Vienna, Austria). The flexibility of the quantile curves was controlled by 5 degrees of freedom, to reflect the 5 broad groupings of gestational ages across the Cambridge and Toronto data sets and to ensure smooth transition across areas where the data are sparse. Quantile regression curves are shown on the plot for the median (blue, dashed) and lower 10% (green, dotted) and 5% (red, full). PlGF derived from non-White patients in the Toronto, Canada, cohort were excluded owing to published variation in circulating PlGF at 16 weeks’ gestation across ethnic groups and to align with the ethnic composition of the Cambridge, United Kingdom, cohort.
      • Audette M.C.
      • Levytska K.
      • Lye S.J.
      • Melamed N.
      • Kingdom J.C.
      Parental ethnicity and placental maternal vascular malperfusion pathology in healthy nulliparous women.
      No comparative statistics of the 3 study groups are presented owing to the limited sample size.

      Results

      Placental growth factor reference range

      The gestational age-specific reference range for PlGF levels together with the 50th, 10th, 5th, and 2.5th centiles are presented in Figure 2. Specific cutoff values for each week of gestational age between 12 and 36 weeks are presented in Table 1.
      Figure thumbnail gr2
      Figure 2Gestational age-specific distribution of circulating maternal PlGF levels
      Data are from comparable Cambridge (individual data points at 12, 20, 28, and 36 weeks’ gestation)
      • Sovio U.
      • Gaccioli F.
      • Cook E.
      • Hund M.
      • Charnock-Jones D.S.
      • Smith G.C.
      Prediction of preeclampsia using the Soluble fms-Like tyrosine kinase 1 to placental growth factor Ratio: a Prospective Cohort Study of Unselected Nulliparous Women.
      ,
      • Gaccioli F.
      • Lager S.
      • Sovio U.
      • Charnock-Jones D.S.
      • Smith G.C.S.
      The pregnancy outcome prediction (POP) study: investigating the relationship between serial prenatal ultrasonography, biomarkers, placental phenotype and adverse pregnancy outcomes.
      and Toronto (box plot of median and interquartile range at 16 weeks’ gestation)
      • Wright E.
      • Audette M.C.
      • Ye X.Y.
      • et al.
      Maternal vascular malperfusion and adverse perinatal outcomes in low-risk nulliparous women.
      cohorts of nulliparous patients. Gestational age-specific values at the 2.5th, 5th, 10th, and 50th centiles from 12 to 26 weeks’ gestation are presented in .
      PlGF, placental growth factor.
      McLaughlin et al. Low-molecular-weight heparin in pregnancies at risk of placental dysfunction. Am J Obstet Gynecol 2022.
      Table 1Gestational age-specific values for circulating maternal PlGF (pg/mL) at the 5th, 10th, and 50th centiles derived using quantile regression of merged data from comparable Cambridge
      • Sovio U.
      • Gaccioli F.
      • Cook E.
      • Hund M.
      • Charnock-Jones D.S.
      • Smith G.C.
      Prediction of preeclampsia using the Soluble fms-Like tyrosine kinase 1 to placental growth factor Ratio: a Prospective Cohort Study of Unselected Nulliparous Women.
      ,
      • Gaccioli F.
      • Lager S.
      • Sovio U.
      • Charnock-Jones D.S.
      • Smith G.C.S.
      The pregnancy outcome prediction (POP) study: investigating the relationship between serial prenatal ultrasonography, biomarkers, placental phenotype and adverse pregnancy outcomes.
      and Toronto
      • Wright E.
      • Audette M.C.
      • Ye X.Y.
      • et al.
      Maternal vascular malperfusion and adverse perinatal outcomes in low-risk nulliparous women.
      cohorts of nulliparous patients
      Gestational age (wk)2.5th centile5th centile10th centile50th centile
      1219212540
      1323263049
      1428323863
      1535424984
      16455364110
      17576780139
      18698196166
      198094111191
      2091106126217
      21101118141246
      22110131157280
      23120144175321
      24131159196368
      25140173216420
      26148186234471
      27153194248513
      28153196252539
      29147189246542
      30135175229523
      31119155206489
      32103134179444
      3389116155396
      3478101134349
      357190118305
      366783106267
      All 7 subjects receiving low-molecular-weight heparin had a PlGF value at the <10th centile before administration at <20 weeks’ gestation.
      PlGF, placental growth factor.
      McLaughlin et al. Low-molecular-weight heparin in pregnancies at risk of placental dysfunction. Am J Obstet Gynecol 2022.

      Pregnant patients at risk of placental dysfunction

      The demographic characteristics and obstetrical history are presented in Table 2, with patients stratified into the following 3 groups: patients with low PlGF levels in the early second trimester who initiated LMWH therapy (group 1), patients with low PlGF levels in the early second trimester who did not initiate LMWH therapy (group 2), and patients with low PlGF levels in the late second trimester who did not initiate LMWH therapy and developed early-onset preeclampsia (group 3). Group 3 patients were initially assessed and identified at a later gestational age than patients in groups 1 and 2.
      Table 2Characteristics of pregnant patients at risk of placental dysfunction
      Maternal characteristicsEarly-second-trimester low PlGF, LMWHEarly-second-trimester low PlGF, no LMWHLate-second-trimester low PlGF, early-onset preeclampsia, no LMWH
      n=7n=5n=21
      Demographic characteristics
      Age, y35 (34–38)39 (35–40)33 (29–37)
      Ethnicity
       White5 (71)1 (20)13 (62)
       Black0 (0)0 (0)2 (10)
       East Asian1 (14)1 (20)2 (10)
       South Asian1 (14)3 (60)4 (19)
      BMI, kg/m227 (24–31)26 (21–29)32 (23–39)
      Chronic hypertension0 (0)1 (20)3 (14)
      Preexisting diabetes0 (0)0 (0)2 (10)
      Initial assessment, wk gestation12 (11–13)16 (16–18)22 (20–24)
      Systolic blood pressure at initial assessment, mm Hg
      Blood pressure data missing from 1 patient in group 1.
      118 (92–120)112 (105–143)120 (113–132)
      Diastolic blood pressure at initial assessment, mm Hg
      Blood pressure data missing from 1 patient in group 1.
      66 (59–72)80 (63–89)71 (66–82)
      Obstetrical history
      History of placental complications5 (71)2 (40)6 (29)
      Previous preeclampsia2 (29)2 (40)4 (19)
       Early-onset preeclampsia <34 wk gestation223
       Late-onset preeclampsia ≥34 wk gestation001
      Previous fetal death at ≥20 wk gestation3 (43)2 (40)1 (5)
      Obstetrical characteristics
      Nulliparous2 (29)2 (40)10 (48)
      First trimester aneuploidy screening
       Not performed1 (14)1 (20)7 (33)
       PAPP-A, MoM0.63 (0.39–0.91)0.16 (0.12–0.39)0.70 (0.51–1.48)
       hCG, MoM1.00 (0.98–1.32)2.17 (1.28–3.01)1.62 (1.28–1.86)
      Mean uterine artery PI
       14–20 wk gestation1.99 (1.38–2.49)1.54 (1.42–1.66)
       20–24 wk gestation1.57 (1.36–1.82)1.53 (1.45–1.60)2.09 (1.73–2.54)
       24–28 wk gestation1.54 (1.16–1.78)1.98 (1.80–2.50)
       28–36 wk gestation1.21 (0.90–1.38)1.56 (1.35–2.02)
      Data are presented as median (interquartile range) or number percentage of column).
      BMI, body mass index; hCG, human chorionic gonadotropin; LMWH, low-molecular-weight heparin; MoM, multiples of the median; PAPP-A, pregnancy-associated plasma protein-A; PI, pulsatility index; PlGF, placental growth factor (pg/mL).
      McLaughlin et al. Low-molecular-weight heparin in pregnancies at risk of placental dysfunction. Am J Obstet Gynecol 2022.
      a Blood pressure data missing from 1 patient in group 1.

      Maternal circulating placental growth factor after low-molecular-weight heparin administration

      Individual circulating PlGF results as gestation advanced in patients with severe placental dysfunction are presented in Figure 3. Notably, 5 of 7 patients in group 1 exhibited increases in circulating PlGF following initiation of LMWH, which ultimately decreased as gestational age advanced. In contrast, no patients in group 2 demonstrated a notable rise in circulating PlGF. Patients in group 3, with an initial PlGF test completed at ≥20 weeks’ gestation, exhibited a similar trend as group 2. Longitudinal PlGF values across gestation are presented in Figure 4 by patient group with the superimposed 2.5th, 5th, and 10th centiles of the newly derived PlGF reference curve from Figure 2.
      Figure thumbnail gr3
      Figure 3PlGF levels in pregnant patients at risk of placental dysfunction
      Group 1 (patients with low PlGF levels in the early second trimester who initiated LMWH therapy [n=7]) are presented in dark blue; dark blue open circles indicate pre-LMWH therapy, and dark blue closed circles indicate post-LMWH therapy. Group 2 (patients with low PlGF levels in the early second trimester who did not initiate LMWH therapy [n=5]) are presented in red. Group 3 (patients with low PlGF levels in the late second trimester who did not initiate LMWH therapy and developed early-onset preeclampsia [n=21]) are presented in light blue.
      LMWH, low-molecular-weight heparin; PlGF, placental growth factor.
      McLaughlin et al. Low-molecular-weight heparin in pregnancies at risk of placental dysfunction. Am J Obstet Gynecol 2022.
      Figure thumbnail gr4
      Figure 4PlGF levels in high-risk patients relative to the reference range
      Group 1 (patients with low PlGF levels in the early second trimester who initiated LMWH therapy) are presented in red. Group 2 (patients with low PlGF levels in the early second trimester who did not initiate LMWH therapy) are presented in green. Group 3 (patients with low PlGF levels in the late second trimester who did not initiate LMWH therapy and developed early-onset preeclampsia) are presented in blue.
      LMWH, low-molecular-weight heparin; PlGF, placental growth factor.
      McLaughlin et al. Low-molecular-weight heparin in pregnancies at risk of placental dysfunction. Am J Obstet Gynecol 2022.

      Pregnancy outcomes

      Maternal, fetal, and delivery outcomes together with placental pathology findings are presented in Table 3. Patients in group 1 exhibited a later gestational age at delivery, relative to groups 2 and 3 (36 weeks [33–37] vs 23 weeks [22–26] and 28 weeks [27–31], respectively). Birthweights were higher in group 1 pregnancies than in groups 2 and 3 (1.93 kg [1.1–2.7] vs 0.32 kg [0.19–0.39] and 0.73 kg [0.52–1.03], respectively). The incidence of stillbirth was lowest in group 1, relative to groups 2 and 3 (14% vs 80% and 29%, respectively).
      Table 3Pregnancy outcomes and placental pathology of pregnant patients at risk of severe placental dysfunction
      OutcomesEarly-second-trimester low PlGF, LMWHEarly-second-trimester low PlGF, no LMWHLate-second-trimester early severe preeclampsia, no LMWH
      n=7n=5n=21
      Pregnancy outcome characteristics
      Gestational age at delivery, wk36 (33–37)23 (22–26)28 (27–31)
      Maternal outcome
       Preeclampsia4 (57)0 (0)21 (100)
      Fetal outcome
       Live birth6 (86)1 (20)15 (71)
       Stillbirth1 (14)4 (80)6 (29)
      Antihypertensive medication use3 (43)2 (40)19 (90)
      Birthweight, kg1.93 (1.1–2.7)0.32 (0.19–0.39)0.73 (0.52–1.03)
      Placental pathology
      Placental pathology data missing from 1 patient in group 2.
      Principal placental pathology
       Maternal vascular malperfusion3 (43)2 (40)19 (90)
       Fetal thrombotic vasculopathy0 (0)2 (40)1 (5)
       Fetal vascular malperfusion1 (14)0 (0)0 (0)
       Chronic histiocytic intervillositis1 (14)0 (0)1 (5)
       Perivillous fibrin deposition1 (14)0 (0)0 (0)
       Villitis of unknown etiology1 (14)0 (0)0 (0)
       Massive perivillous fibrin deposition1 (14)0 (0)0 (0)
      Additional pathology features
       Maternal vascular malperfusion2 (29)1 (20)1 (5)
       Chronic histiocytic intervillositis0 (0)1 (20)1 (5)
       Fetal thrombotic vasculopathy1 (14)0 (0)1 (5)
       Weight at the <third centile1 (14)0 (0)0 (0)
       Fetal thrombotic vasculopathy0 (0)0 (0)2 (10)
      Data are presented as median (interquartile range) or number (percentage of column).
      LMWH, low-molecular-weight heparin; PlGF, placental growth factor (pg/mL).
      McLaughlin et al. Low-molecular-weight heparin in pregnancies at risk of placental dysfunction. Am J Obstet Gynecol 2022.
      a Placental pathology data missing from 1 patient in group 2.
      MVM was the predominant principal placental pathology in all patients, with the highest incidence in group 3, relative to groups 1 and 2 (90% vs 43% and 40%, respectively). In group 1, both patients with normal mean uterine artery Doppler were found to each have a rare placental pathology diagnosis (perivillous fibrin deposition and chronic histiocytic intervillositis), whereas the remaining 5 with abnormal mean uterine artery Doppler expressed features of MVM pathology. Mean uterine artery PI across gestation stratified by placental pathology and LMWH therapy is presented in the Supplemental Figure.

      Comment

      Principal findings

      The principal findings of the study are 3-fold. First, we merged data from 2 cohort studies of healthy nulliparous patients to create a reference curve for clinicians to interpret circulating PlGF on a weekly basis between 12 and 36 weeks’ gestation. Second, our approach to the assessment of placental function at 16 weeks’ gestation through PlGF testing identified patients at high risk of placental dysfunction destined to have adverse placenta-mediated pregnancy outcomes despite aspirin prophylaxis. Finally, the administration of prophylactic daily LMWH to a small group of women with low circulating PlGF in the early second trimester resulted in sustained elevations in circulating PlGF.

      Limitations

      We acknowledge that these are preliminary proof of principal findings, limited by sample size and study design. Our data cannot be interpreted as a recommendation for the use of LMWH to improve placental function and therefore clinical outcomes in high-risk pregnancy care. Although all high-risk pregnant patients in our study were identified through serial PlGF testing and ultrasound imaging, we acknowledge that there may have been inherent differences between patients who did and did not consent to initiate LMWH therapy; for example, baseline PlGF levels seem to be higher in patients in group 1 than in patients in group 2, whereas PAPP-A multiple of median values were lowest in group 2.

      Results in the context of what is known

      By applying quantile regression analysis to PlGF data merged from 2 prospective cohorts of nulliparous White women, we established a continuous reference range between 12 and 36 weeks’ gestation. We restricted the construction of the reference range to these 2 cohorts owing to their adequate size and uniformity of assay manufacturer, because assay type is a source of data variability.
      • McCarthy F.P.
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      • Chappell L.C.
      • Shennan A.H.
      Comparison of three commercially available placental growth factor-based tests in women with suspected preterm pre-eclampsia: the COMPARE study.
      Previous studies have grouped reference PlGF values into blocks of time,
      • Agrawal S.
      • Shinar S.
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      • Redman C.
      • Vatish M.
      Predictive performance of PlGF (placental growth factor) for screening preeclampsia in asymptomatic women: a systematic review and meta-analysis.
      ,
      • Levine R.J.
      • Maynard S.E.
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      • et al.
      Circulating angiogenic factors and the risk of preeclampsia.
      • Knudsen U.B.
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      • et al.
      A single rapid point-of-care placental growth factor determination as an aid in the diagnosis of preeclampsia.
      • Verlohren S.
      • Herraiz I.
      • Lapaire O.
      • et al.
      New gestational phase-specific cutoff values for the use of the soluble fms-like tyrosine kinase-1/placental growth factor ratio as a diagnostic test for preeclampsia.
      although one study used quantile regression to derive a reference range for PlGF using serial samples from 180 uncomplicated pregnancies beginning at 12 weeks’ gestation.
      • Chaiworapongsa T.
      • Romero R.
      • Savasan Z.A.
      • et al.
      Maternal plasma concentrations of angiogenic/anti-angiogenic factors are of prognostic value in patients presenting to the obstetrical triage area with the suspicion of preeclampsia.
      Burke et al
      • Burke Ó.
      • Benton S.
      • Szafranski P.
      • et al.
      Extending the scope of pooled analyses of individual patient biomarker data from heterogeneous laboratory platforms and cohorts using merging algorithms.
      merged data derived from 4 test platforms across 22 studies into a continuous reference range; however, this analysis commenced from 20 weeks’ gestation and is therefore of no value in the context of assessing placental function in the early second trimester. In contrast to the use of a pragmatic single cutoff diagnostic value of 100 pg/mL for PlGF at 20 to 36 weeks, when circulating PlGF has achieved a plateau, a continuous reference range resource during the rising phase of PlGF in the early second trimester is a necessity to use serial PlGF to screen high-risk women on aspirin for severe placenta-mediated adverse outcomes.
      • Agrawal S.
      • Shinar S.
      • Cerdeira A.S.
      • Redman C.
      • Vatish M.
      Predictive performance of PlGF (placental growth factor) for screening preeclampsia in asymptomatic women: a systematic review and meta-analysis.
      However, this resource is limited by the use of one test platform and being predominantly derived from White patients. A recent investigation determined that circulating PlGF levels at 16 weeks’ gestation varied considerably by ethnicity
      • Audette M.C.
      • Levytska K.
      • Lye S.J.
      • Melamed N.
      • Kingdom J.C.
      Parental ethnicity and placental maternal vascular malperfusion pathology in healthy nulliparous women.
      ; further studies involving adequate numbers of specific ethnic groups are warranted, especially because some non-White groups are especially vulnerable to developing severe preeclampsia.
      Several investigators have demonstrated an association between the abnormal expression of placenta-derived angiogenic factors and placental disease, principally MVM, which is the most common pathologic finding in this context.
      • Zur R.L.
      • Kingdom J.C.
      • Parks W.T.
      • Hobson S.R.
      The placental basis of fetal growth restriction.
      ,
      • Benton S.J.
      • Hu Y.
      • Xie F.
      • et al.
      Can placental growth factor in maternal circulation identify fetuses with placental intrauterine growth restriction?.
      ,
      • Korzeniewski S.J.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      Maternal plasma angiogenic index-1 (placental growth factor/soluble vascular endothelial growth factor receptor-1) is a biomarker for the burden of placental lesions consistent with uteroplacental underperfusion: a longitudinal case-cohort study.
      Recently, the hypoxic-ischemic features of this disease have been demonstrated in vivo in patients with early-onset preeclampsia and low circulating PlGF using advanced magnetic resonance imaging methods that measure regional tissue oxygenation by T2∗ oximetry.
      • Ho A.E.P.
      • Hutter J.
      • Jackson L.H.
      • et al.
      T2∗ Placental magnetic resonance imaging in preterm preeclampsia: an observational cohort study.
      These observations are consistent with in vitro data in human placental explants demonstrating hypoxia-mediated repression of PlGF synthesis.
      • Khaliq A.
      • Dunk C.
      • Jiang J.
      • et al.
      Hypoxia down-regulates placenta growth factor, whereas fetal growth restriction up-regulates placenta growth factor expression: molecular evidence for “placental hyperoxia” in intrauterine growth restriction.
      Data from the SCOPE consortium, demonstrating that low circulating PlGF combined with abnormal uterine artery Doppler at 16 weeks’ gestation is highly predictive of early-onset preeclampsia,
      • Myers J.E.
      • Kenny L.C.
      • McCowan L.M.
      • et al.
      Angiogenic factors combined with clinical risk factors to predict preterm pre-eclampsia in nulliparous women: a predictive test accuracy study.
      suggest that a strategy of early serial PlGF testing may identify women with markedly impaired early placental development. Up to 15% of women with severe placental dysfunction exhibit diseases other than MVM, especially massive perivillous fibrin deposition and chronic histiocytic intervillositis, in which uteroplacental blood flow is not typically restricted.
      • McLaughlin K.
      • Snelgrove J.W.
      • Audette M.C.
      • et al.
      PlGF (Placental growth factor) testing in clinical practice: evidence From a Canadian Tertiary Maternity Referral Center.
      ,
      • Kingdom J.C.
      • Audette M.C.
      • Hobson S.R.
      • Windrim R.C.
      • Morgen E.
      A placenta clinic approach to the diagnosis and management of fetal growth restriction.
      ,
      • Levytska K.
      • Higgins M.
      • Keating S.
      • et al.
      Placental pathology in relation to uterine artery Doppler findings in pregnancies with severe intrauterine growth restriction and abnormal umbilical artery Doppler changes.
      Therefore, such patients are likely to exhibit normal uterine artery Doppler waveforms, yet may be identified in tandem with a majority who are developing MVM placental disease in the early second trimester by their common deficiency in a failure of PlGF to rise in the 16- to 20-week period. Interestingly, 2 of the 7 patients in group 1 followed this pattern, with normal uterine artery Doppler, a failure to elevate PlGF, and subsequent demonstration of non-MVM placental pathologies.
      The extremely high rates of placental pathology in our subjects, together with their near universal risk of preterm delivery, validated our strategy to identify a niche of patients who remain vulnerable to the effects of severe placental dysfunction despite taking aspirin. The concept of dual screening with circulating angiogenic growth factors and uterine artery Doppler to identify the most common type of underlying placental MVM disease was originally developed by Espinosa et al,
      • Espinoza J.
      • Romero R.
      • Nien J.K.
      • et al.
      Identification of patients at risk for early onset and/or severe preeclampsia with the use of uterine artery Doppler velocimetry and placental growth factor.
      who, in a screening study of 3348 unselected patients in a 22- to 26-week window, noted that a PlGF cutoff value of 280 pg/mL in patients with abnormal uterine artery Doppler predicted 84% of early-onset and 89% of severe preeclampsia. These findings were largely replicated at an earlier gestational age at approximately 16 weeks’ gestation in 3529 healthy nulliparous patients by the SCOPE consortium, which demonstrated a positive likelihood ratio of 9.4 for the prediction of preterm preeclampsia when both uterine artery Doppler and circulating PlGF were abnormal.
      • Myers J.E.
      • Kenny L.C.
      • McCowan L.M.
      • et al.
      Angiogenic factors combined with clinical risk factors to predict preterm pre-eclampsia in nulliparous women: a predictive test accuracy study.
      Common to both studies, patients were not exposed to aspirin, and neither reported placental pathology findings. With the increased use of first trimester screening methods for the prevention of early-onset preeclampsia with aspirin, the high-risk patients in our study were already on aspirin by 12 weeks’ gestation owing to their previous risk factors.
      • Rolnik D.L.
      • Wright D.
      • Poon L.C.
      • et al.
      Aspirin versus Placebo in pregnancies at high risk for preterm preeclampsia.
      • Wright A.
      • Wright D.
      • Syngelaki A.
      • Georgantis A.
      • Nicolaides K.H.
      Two-stage screening for preterm preeclampsia at 11-13 weeks’ gestation.
      • Guy G.P.
      • Leslie K.
      • Diaz Gomez D.
      • et al.
      Implementation of routine first trimester combined screening for pre-eclampsia: a clinical effectiveness study.
      As such, any inference from our findings that LMWH may be of benefit cannot be extended beyond this niche group of vulnerable patients on aspirin therapy, identified in the early second trimester.
      The negative findings from 2 large high-quality trials
      • Groom K.M.
      • McCowan L.M.
      • Mackay L.K.
      • et al.
      Enoxaparin for the prevention of preeclampsia and intrauterine growth restriction in women with a history: a randomized trial.
      ,
      • Haddad B.
      • Winer N.
      • Chitrit Y.
      • et al.
      Enoxaparin and aspirin compared with aspirin alone to prevent placenta-mediated pregnancy complications: a randomized controlled trial.
      testing the hypothesis that prophylactic LMWH can further reduce the risk of preterm delivery in addition to aspirin coupled with evidence that aspirin alone is an effective agent have led clinicians to avoid recommending LMWH in this context.
      • Wright A.
      • Wright D.
      • Syngelaki A.
      • Georgantis A.
      • Nicolaides K.H.
      Two-stage screening for preterm preeclampsia at 11-13 weeks’ gestation.
      ,
      • Guy G.P.
      • Leslie K.
      • Diaz Gomez D.
      • et al.
      Implementation of routine first trimester combined screening for pre-eclampsia: a clinical effectiveness study.
      Both trials recruited women based on their previous obstetrical history and had low rates of serious adverse perinatal outcomes (perinatal death or iatrogenic delivery at <32 weeks) in the control arms, suggesting adequate placental function in a majority of participants. A previous cohort study, involving 212 women of similar risk characteristics, found that a majority (59%) had normal measures of placental function in the early second trimester and had considerably more favorable outcomes than the remainder with one or more placental test abnormalities; for example, 19 of 23 women developing hemolysis, elevated liver enzymes, and low platelet count syndrome and requiring delivery at <34 weeks’ gestation had ≥1 test abnormalities.
      • Toal M.
      • Chan C.
      • Fallah S.
      • et al.
      Usefulness of a placental profile in high-risk pregnancies.
      These observations are relevant in the context of the most updated systematic review on the adjunct role of LMWH that evaluated 15 studies with variable entry criteria, demonstrating that LMWH in addition to low-dose aspirin before 16 weeks’ gestation significantly lowered the risk of early-onset disease (odds ratio, 0.62; 95% confidence interval, 0.41–0.95).
      • Cruz-Lemini M.
      • Vázquez J.C.
      • Ullmo J.
      • Llurba E.
      Low-molecular-weight heparin for prevention of preeclampsia and other placenta-mediated complications: a systematic review and meta-analysis.
      Concentrating future trial recruitment on a subgroup of clinically high-risk women with abnormal measures of placental function in early pregnancy may provide definitive answers on the role of LMWH in this niche group of women.
      The potential mechanisms of action of LMWH in the context of prevention of early-onset preeclampsia are varied and do not necessarily involve anticoagulation of maternal blood within the placenta.
      • Kingdom J.C.
      • Drewlo S.
      Is heparin a placental anticoagulant in high-risk pregnancies?.
      Several non-anticoagulant actions of LMWH may be pertinent in the context of preventing early-onset preeclampsia associated with MVM of the placenta.
      • McLaughlin K.
      • Scholten R.R.
      • Parker J.D.
      • Ferrazzi E.
      • Kingdom J.C.P.
      Low molecular weight heparin for the prevention of severe preeclampsia: where next?.
      These include suppression of leukocyte activation and complement activity alongside promotion of angiogenesis.
      • Wat J.M.
      • Hawrylyshyn K.
      • Baczyk D.
      • Greig I.R.
      • Kingdom J.C.
      Effects of glycol-split low molecular weight heparin on placental, endothelial, and anti-inflammatory pathways relevant to preeclampsia.
      In in vivo studies of pregnant subjects, LMWH acutely elevated suboptimal circulating PlGF by 1.5-fold, and larger increases in circulating PlGF levels have been found in patients receiving therapeutic levels of LMWH.
      • McLaughlin K.
      • Baczyk D.
      • Potts A.
      • Hladunewich M.
      • Parker J.D.
      • Kingdom J.C.
      Low molecular weight heparin improves endothelial function in pregnant women at high risk of preeclampsia.
      ,
      • Yinon Y.
      • Ben Meir E.
      • Margolis L.
      • et al.
      Low molecular weight heparin therapy during pregnancy is associated with elevated circulatory levels of placental growth factor.
      A subsequent publication from the Heparin-Preeclampsia (HEPEPE) trial group focused on circulating angiogenic growth factor profiles in high-risk pregnant patients, demonstrating no differences in circulating PlGF across gestation between patients randomized to receive aspirin alone and aspirin with LMWH therapy.
      • Lecarpentier E.
      • Gris J.C.
      • Cochery-Nouvellon E.
      • et al.
      Angiogenic factor profiles in pregnant women with a history of early-onset severe preeclampsia receiving low-molecular-weight heparin prophylaxis.
      Very few women in this randomized trial had low circulating PlGF levels before 18 weeks’ gestation, few had severe adverse perinatal outcomes, and the trajectory of PlGF in both arms of the trial is similar to the reference range data reported in this study. In contrast, the current observational study recruited high-risk patients (groups 1 and 2) based on low PlGF levels early in the second trimester and was confirmed by both their clinical outcomes and placental pathology testing. Our interpretation is that LMWH therapy could exert beneficial effects in high-risk pregnant patients with considerable placental dysfunction, by reversing a low PlGF phenotype found in the early second trimester. The Enoxaparin for the Prevention of Preeclampsia and Intrauterine Growth Restriction (EPPI) trial had a similar design and overall findings
      • Groom K.M.
      • McCowan L.M.
      • Mackay L.K.
      • et al.
      Enoxaparin for the prevention of preeclampsia and intrauterine growth restriction in women with a history: a randomized trial.
      , and the HEPEPE and EPPI trials have not reported placental pathology data, which would be of interest to elucidating any potential beneficial impacts of LMWH therapy.
      In in vitro studies with human tissues, LMWH augmented the synthesis and release of PlGF by placental villous tissue explants and a similar stimulatory effect by LMWH has recently been demonstrated in endothelial cells.
      • McLaughlin K.
      • Nadeem L.
      • Wat J.
      • Baczyk D.
      • Lye S.J.
      • Kingdom J.C.
      Low molecular weight heparin promotes transcription and release of placental growth factor from endothelial cells.
      ,
      • Drewlo S.
      • Levytska K.
      • Sobel M.
      • Baczyk D.
      • Lye S.J.
      • Kingdom J.C.
      Heparin promotes soluble VEGF receptor expression in human placental villi to impair endothelial VEGF signaling.
      The possibility of inducing an endogenous rise in circulating PlGF via LMWH is preferable to the administration of parenteral exogenous PlGF to achieve the same effect.
      • Spradley F.T.
      • Tan A.Y.
      • Joo W.S.
      • et al.
      Placental growth factor administration abolishes placental ischemia-induced hypertension.
      Collectively, these studies support a plausible mechanism whereby LMWH, administered to a small subset of patients at greatest risk of placenta-mediated early-onset preeclampsia, may be capable of inducing a clinically meaningful sustained rise in their deficient PlGF levels.

      Clinical implications

      For clinicians managing pregnancies at high-risk of placental dysfunction, especially in a maternal-fetal medicine setting, the ability to identify an asymptomatic vulnerable subset in the early second trimester, despite receiving aspirin prophylaxis, is a key strategic goal. The availability of PlGF testing in real time (within 90 minutes) combined with uterine artery Doppler assessment may be one such strategy for clinicians to adopt, as we have done. This approach can focus clinical resources on women who need higher levels of care, and in addition, it may facilitate further research into the adjunct role of additional medical therapies to prevent stillbirth and extreme preterm delivery.
      • McLaughlin K.
      • Snelgrove J.W.
      • Audette M.C.
      • et al.
      PlGF (Placental growth factor) testing in clinical practice: evidence From a Canadian Tertiary Maternity Referral Center.
      ,
      • Espinoza J.
      • Romero R.
      • Nien J.K.
      • et al.
      Identification of patients at risk for early onset and/or severe preeclampsia with the use of uterine artery Doppler velocimetry and placental growth factor.
      ,
      • Dröge L.A.
      • Perschel F.H.
      • Stütz N.
      • et al.
      Prediction of preeclampsia-related adverse outcomes with the sFlt-1 (soluble fms-Like tyrosine kinase 1)/PlGF (placental growth factor)-Ratio in the Clinical Routine: a real-world study.

      Research implications

      Our findings do not support the clinical use of LMWH to prevent placenta-mediated complications, but they may inform the design of new pilot randomized control trials confined to a subgroup of high-risk patients already on aspirin prophylaxis. This approach was taken in a small 2011 trial where LMWH and aspirin were used concurrently that was conducted before the advent of PlGF screening.
      • Kingdom J.C.
      • Walker M.
      • Proctor L.K.
      • et al.
      Unfractionated heparin for second trimester placental insufficiency: a pilot randomized trial.
      At present, we do not know whether any of the observed in vitro effects of LMWH could exert clinically meaningful actions on the placenta such as restoring PlGF release into the maternal circulation, directly acting on the systemic maternal endothelium or on improving the typically restricted uteroplacental circulation that is found in women at highest risk of severe preeclampsia.
      • Espinoza J.
      • Romero R.
      • Nien J.K.
      • et al.
      Identification of patients at risk for early onset and/or severe preeclampsia with the use of uterine artery Doppler velocimetry and placental growth factor.
      ,
      • Myers J.E.
      • Kenny L.C.
      • McCowan L.M.
      • et al.
      Angiogenic factors combined with clinical risk factors to predict preterm pre-eclampsia in nulliparous women: a predictive test accuracy study.
      ,
      • Mello G.
      • Parretti E.
      • Fatini C.
      • et al.
      Low-molecular-weight heparin lowers the recurrence rate of preeclampsia and restores the physiological vascular changes in angiotensin-converting enzyme DD women.
      Therefore, future baseline trial data could incorporate serial urine analysis of the stable C5b fragment that reflects complement activation,
      • Burwick R.M.
      • Fichorova R.N.
      • Dawood H.Y.
      • Yamamoto H.S.
      • Feinberg B.B.
      Urinary excretion of C5b-9 in severe preeclampsia: tipping the balance of complement activation in pregnancy.
      together with serial maternal blood angiogenic growth factors, and incorporate specialist placental pathology blinded to trial allocation.

      Conclusions

      A continuous reference range for maternal circulating PlGF was established between 12 and 36 weeks’ gestation for high-risk pregnancy clinicians to assess the ongoing risk of preterm delivery owing to severe placental dysfunction. Among a small subgroup of pregnancies considered high-risk in this context with low maternal circulating PlGF at 16 to 20 weeks’ gestation, the addition of prophylactic LMWH to aspirin prophylaxis induced a rise in circulating PlGF that was sustained in most patients over several weeks before delivery. Given that the most recent evidence obtained from systematic reviews of relevant trials support the limited use of LMWH in this context, our data may inform future efforts to conduct a pilot randomized controlled trial to further explore the relevant biologic actions of LMWH in this context, which if favorable could inform a subsequent definitive randomized trial with entry criteria that focus on the assessment of placental function in vivo.

      Supplementary Material

      Figure thumbnail fx1
      Supplemental FigureMaternal PlGF levels
      Maternal PlGF levels across gestation in pregnant patients at risk of severe placental dysfunction, stratified by placental pathology diagnosis and LMWH therapy.
      LMWH, low-molecular-weight heparin; MVM, maternal vascular malperfusion; PlGF, placental growth factor.
      McLaughlin et al. Low-molecular-weight heparin in pregnancies at risk of placental dysfunction. Am J Obstet Gynecol 2022.

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