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Inherited thrombophilia and preeclampsia within a multicenter cohort: the Montreal Preeclampsia Study

Published:December 15, 2008DOI:https://doi.org/10.1016/j.ajog.2008.09.023

      Objective

      We sought to evaluate the association between inherited thrombophilia and preeclampsia.

      Study Design

      From a multicenter cohort of 5337 pregnant women, we prospectively identified 113 women who developed preeclampsia and selected 443 control subjects who did not have preeclampsia or nonproteinuric gestational hypertension. Blood samples were tested for DNA polymorphisms affecting thrombophilia (factor V Leiden mutation, prothrombin G20210A mutation, methylenetetrahydrofolate reductase C677T polymorphism), homocysteine, and folate levels, and placentae underwent pathological evaluation.

      Results

      Thrombophilia was present in 14% of patients and 21% of control subjects (adjusted logistic regression odds ratio, 0.6; 95% confidence interval, 0.3-1.3). Placental underperfusion was present in 63% of patients vs 46% of control subjects (P < .001) and was more frequent in women with folate levels in the lowest quartile (P = .04), but was not associated with thrombophilia.

      Conclusion

      We did not find evidence to support an association between inherited thrombophilia and increased risk of preeclampsia. Placental underperfusion is associated with preeclampsia, but this does not appear to be consequent to thrombophilia.

      Key words

      Preeclampsia is a hypertensive disorder that affects 2-7% of all pregnancies and is an important cause of maternal and perinatal morbidity and mortality worldwide.
      • Sibai B.
      • Dekker G.
      • Kupferminc M.
      Pre-eclampsia.
      National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy
      Report of the national high blood pressure education program working group on high blood pressure in pregnancy.
      • Kaunitz A.M.
      • Hughes J.M.
      • Grimes D.A.
      • et al.
      Causes of maternal mortality in the United States, 1979-1986.
      • Duley L.
      Maternal mortality associated with hypertensive disorders of pregnancy in Africa, Asia, Latin America and the Caribbean.
      Risk factors associated with preeclampsia include first pregnancy, multiple pregnancy, personal or family history of preeclampsia, high body mass index (BMI), smoking (protective), chronic hypertension, and diabetes.
      • Duckitt K.
      • Harrington D.
      Risk factors for pre-eclampsia at antenatal booking: systematic review of controlled studies.
      However, underlying etiologic mechanisms remain poorly understood.
      • Sibai B.
      • Dekker G.
      • Kupferminc M.
      Pre-eclampsia.
      • Roberts J.M.
      • Cooper D.W.
      Pathogenesis and genetics of pre-eclampsia.
      For Editors' Commentary, see Table of Contents
      See related editorial, page 121
      See Journal Club, page 212
      During the last decade, the identification of several gene polymorphisms that are associated with hypercoagulability (inherited thrombophilia) has advanced understanding of etiologic mechanisms of thrombosis. Point mutations in the genes encoding coagulation factor V (G1691A; factor V Leiden) and prothrombin (G20210A), which in heterozygous form affect ∼5% and 2% of the population, are associated with an elevated risk of venous thromboembolism.
      • Rosendaal F.R.
      • Koster T.
      • Vanderbroucke J.P.
      • Reitsma P.H.
      High risk of thrombosis in patients homozygous for factor V Leiden.
      • Poort S.R.
      • Rosendaal F.R.
      • Reitsma P.H.
      • Bertina R.M.
      A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated prothrombin levels and an increase in venous thrombosis.
      The C677T variant of the gene encoding 5,10-methyleneterahydrofolate reductase (MTHFR) is found in homozygous form in 10-12% of the population.
      • Frost P.
      • Bloom H.J.
      • Milos R.
      • et al.
      A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase.
      This variant can lead to hyperhomocysteinemia (particularly when folate levels are low
      • Jacques P.F.
      • Bostom A.G.
      • Williams R.R.
      • et al.
      Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations.
      ), which is associated with an increased risk of venous thromboembolism
      • den Heijer M.
      • Blom H.J.
      • Gerrits W.B.J.
      • et al.
      Is hyperhomocysteinemia a risk factor for recurrent venous thrombosis?.
      • Ridker P.M.
      • Hennekens C.H.
      • Selhub J.
      • Miletich J.P.
      • Malinow M.R.
      • Stampfer M.J.
      Interrelation of hyperhomocyst(e)inemia, factor V Leiden, and risk of future venous thromboembolism.
      and coronary heart disease.
      • Eikelbloom J.W.
      • Lonn E.
      • Genest J.
      • Hankey G.
      • Yusuf S.
      Homocyst(e)ine and cardiovascular disease: a critical review of epidemiologic evidence.
      • Welch G.N.
      • Loscalzo J.
      Homocysteine and atherothrombosis.
      In recent years, some studies have suggested that inherited thrombophilia may be associated with preeclampsia.
      • Kupferminc M.J.
      • Eldor A.
      • Steinman N.
      • et al.
      Increased frequency of genetic thrombophilia in women with complications of pregnancy.
      • Dizon-Townson D.S.
      • Helson L.N.
      • Easton K.
      • Ward K.
      The factor V Leiden mutation may predispose women to severe preeclampsia.
      • Grandone E.
      • Margaglione M.
      • Colaizzo D.
      • et al.
      Factor V Leiden, C>T MTHFR polymorphism and genetic susceptibility to preeclampsia.
      Proposed underlying mechanisms include interference with trophoblast differentiation, inadequate placentation, or thrombosis of placental vasculature, with consequent reduced placental perfusion, oxidative stress, and maternal endothelial dysfunction that is believed to trigger the hallmark biological and clinical manifestations of preeclampsia.
      • Roberts J.M.
      • Cooper D.W.
      Pathogenesis and genetics of pre-eclampsia.
      • Hubel C.A.
      Oxidative stress in the pathogenesis of preeclampsia.
      • Middeldorp S.
      Thrombophilia and pregnancy complications: cause or association?.
      To further elucidate these proposed associations, we undertook a prospective, multicenter study of the association between inherited thrombophilia and preeclampsia.

      Materials and Methods

      The Montreal Preeclampsia Study is a case-control study nested within a prospectively recruited cohort of pregnant women who were participants in a large multicenter study on causal pathways of preterm birth.
      • Kramer M.S.
      • Goulet L.
      • Lydon J.
      • et al.
      Socio-economic disparities in preterm birth: causal pathways and mechanisms.
      At the time of providing informed consent for the preterm birth study, the preeclampsia study was explained and women were asked to sign a separate consent form. Both studies were approved by the research ethics committees of the 4 participating hospital centers before the studies were started.

      Recruitment of Cohort and Follow-up to Time of Delivery

      Patients and study setting

      Trained research assistants approached consecutive pregnant women at the time of presentation for routine ultrasound examination (16-18 weeks) (most subjects), prenatal blood drawing (usually 8-12 weeks), or at first- or second-trimester (until 24 weeks) obstetric clinic visits at 4 large maternity hospitals affiliated with McGill University and l'Université de Montréal in Montréal, QC, Canada. Women aged ≥ 18 years at the expected date of delivery who spoke and understood French or English and had a singleton fetus were eligible for enrollment, provided they did not have severe maternal chronic disease (other than hypertension, asthma, or diabetes), known cervical incompetence in a previous pregnancy, placenta previa, or a known major fetal anomaly in the current pregnancy.

      Study visit at 24-26 weeks

      Women who consented to participate were asked to return for a study visit at 24-26 weeks of gestation. Those in whom placenta previa or a major fetal anomaly had been diagnosed since recruitment were excluded. A questionnaire was administered in person that addressed sociodemographic characteristics; medical, obstetric, and family history; and cigarette use, and height and weight were measured. Nonfasting blood samples obtained by venipuncture were immediately placed on ice, centrifuged, and aliquoted into cryovials containing plasma and buffy coat. Cryovials were frozen at -80°C.

      Follow-up of study subjects

      Cohort women were followed up until the time of delivery. To ensure complete follow-up, study staff monitored the delivery wards of the 4 study hospitals daily, a regularly updated list of study participants at each study hospital was posted in each delivery ward directing nurses to call the study staff when a study woman was admitted, and subjects were instructed to ask the ward nurses to contact the study staff at the time of admission for delivery.

      Identification of cases of preeclampsia

      During the admission for delivery, each study woman identified as having had hypertension or preeclampsia during pregnancy or after delivery underwent a detailed interview and chart review to assess whether diagnostic criteria for preeclampsia were met. Preeclampsia was diagnosed using the 1997 Canadian Hypertension Society Consensus Conference criteria
      • Helewa M.E.
      • Burrows R.F.
      • Smith J.
      • Williams K.
      • Brain P.
      • Rabkin S.W.
      1: definitions, evaluation and classification of hypertensive disorders in pregnancy.
      and was further classified as severe or early onset
      • Dekker G.A.
      • de Vries J.I.P.
      • Doelitzsch P.M.
      • et al.
      Underlying disorders associated with severe early-onset preeclampsia.
      (detailed definitions are shown in Table 1). To ensure consistency and accuracy in the diagnosis of preeclampsia, we developed a detailed preeclampsia diagnostic checklist and provided standardized training to study staff on its use. All cases of preeclampsia were independently verified by 2 study investigators (S. R. K., H. M.).
      TABLE 1Classification of preeclampsia among patients (N = 113)
      TotalEarly onset
      Early-onset preeclampsia defined as occurrence at < 34 weeks' gestation;22
      Severe
      Severe preeclampsia defined as presence of any of the following: seizure, HELLP syndrome, proteinuria ≥ 3 g/24 hours, or diastolic blood pressure > 110 mm Hg.22
      Canadian Hypertension Society classification
      Diagnostic criteria used for preeclampsia and nonpreeclamptic hypertensive disorders of pregnancy were adapted from the 1997 Report of the Canadian Hypertension Society Consensus Conference.21 Gestational hypertension was defined as diastolic hypertension (≥ 90 mm Hg) on 2 occasions at least 4-6 hours apart that developed after 20 weeks' gestation. Proteinuria was defined as protein excretion of ≥ 0.3 g/day in 24-hour urine collection or positive dipstick result ≥ 2+. Adverse conditions were defined as convulsions (eclampsia), diastolic pressure > 110 mm Hg, platelet count < 100,000 × 109/L, oliguria, protein excretion ≥ 3 g/day, pulmonary edema, elevated liver enzymes, severe nausea and vomiting, frontal headache, visual disturbances, persistent abdominal pain in right upper quadrant, chest pain or shortness of breath, suggested abruptio placentae, HELLP syndrome, intrauterine growth restriction, oligohydramnios, or absent or reverse umbilical artery end-diastolic flow, as detected by Doppler velocitometry. Chronic hypertension with superimposed preeclampsia was defined as known chronic hypertension associated with further worsening of blood pressure and protein excretion ≥ 3 g/day after 20 weeks' gestation. Nonpreeclamptic hypertensive disorders (criterion for exclusion as patient or control subject) included gestational hypertension, defined as diastolic hypertension (≥ 90 mm Hg) that developed after 20 weeks' gestation but without proteinuria or adverse conditions, and chronic known hypertension, defined as diastolic hypertension (≥ 90 mm Hg) that predated pregnancy or was diagnosed before 20 weeks' gestation, with or without proteinuria;
      Gestational hypertension with proteinuria without adverse conditions12 (11%)3 (8%)0 (0%)
      Gestational hypertension with proteinuria with adverse conditions49 (43%)16 (46%)19 (39%)
      Gestational hypertension without proteinuria with adverse conditions49 (43%)14 (40%)10 (31%)
      Chronic hypertension with superimposed preeclampsia3 (3%)2 (6%)3 (10%)
      Total113 (100%)35 (100%)32 (100%)
      Kahn. Inherited thrombophilia and preeclampsia within a multicenter cohort: the Montreal Preeclampsia Study. Am J Obstet Gynecol 2009.
      a Diagnostic criteria used for preeclampsia and nonpreeclamptic hypertensive disorders of pregnancy were adapted from the 1997 Report of the Canadian Hypertension Society Consensus Conference.
      • Helewa M.E.
      • Burrows R.F.
      • Smith J.
      • Williams K.
      • Brain P.
      • Rabkin S.W.
      1: definitions, evaluation and classification of hypertensive disorders in pregnancy.
      Gestational hypertension was defined as diastolic hypertension (≥ 90 mm Hg) on 2 occasions at least 4-6 hours apart that developed after 20 weeks' gestation. Proteinuria was defined as protein excretion of ≥ 0.3 g/day in 24-hour urine collection or positive dipstick result ≥ 2+. Adverse conditions were defined as convulsions (eclampsia), diastolic pressure > 110 mm Hg, platelet count < 100,000 × 109/L, oliguria, protein excretion ≥ 3 g/day, pulmonary edema, elevated liver enzymes, severe nausea and vomiting, frontal headache, visual disturbances, persistent abdominal pain in right upper quadrant, chest pain or shortness of breath, suggested abruptio placentae, HELLP syndrome, intrauterine growth restriction, oligohydramnios, or absent or reverse umbilical artery end-diastolic flow, as detected by Doppler velocitometry. Chronic hypertension with superimposed preeclampsia was defined as known chronic hypertension associated with further worsening of blood pressure and protein excretion ≥ 3 g/day after 20 weeks' gestation. Nonpreeclamptic hypertensive disorders (criterion for exclusion as patient or control subject) included gestational hypertension, defined as diastolic hypertension (≥ 90 mm Hg) that developed after 20 weeks' gestation but without proteinuria or adverse conditions, and chronic known hypertension, defined as diastolic hypertension (≥ 90 mm Hg) that predated pregnancy or was diagnosed before 20 weeks' gestation, with or without proteinuria;
      b Early-onset preeclampsia defined as occurrence at < 34 weeks' gestation;
      • Dekker G.A.
      • de Vries J.I.P.
      • Doelitzsch P.M.
      • et al.
      Underlying disorders associated with severe early-onset preeclampsia.
      c Severe preeclampsia defined as presence of any of the following: seizure, HELLP syndrome, proteinuria ≥ 3 g/24 hours, or diastolic blood pressure > 110 mm Hg.
      • Dekker G.A.
      • de Vries J.I.P.
      • Doelitzsch P.M.
      • et al.
      Underlying disorders associated with severe early-onset preeclampsia.

      Selection of control subjects

      Control subjects were study women who delivered at the same hospital closest in time to a patient, but who did not have preeclampsia. As control subjects partially overlapped with those of the concurrently running preterm birth study,
      • Kramer M.S.
      • Goulet L.
      • Lydon J.
      • et al.
      Socio-economic disparities in preterm birth: causal pathways and mechanisms.
      the number of control subjects available varied from 3-4 per case of preeclampsia.
      Study women who had hypertension during pregnancy but who did not meet criteria for preeclampsia (as defined in Table 1) were ineligible to be patients or control subjects and were excluded from all analyses.

      Postpartum Procedures for Patients with Preeclampsia and Control Subjects

      Interview and chart review

      Study staff collected data on pregnancy course since the 24- to 26-week study visit by interview and from the mother's and infant's hospital chart, including the occurrence of pregnancy complications and infant birth weight, gestational age, and Apgar scores.

      Laboratory analyses

      Buffy coat and plasma samples of patients and control subjects were retrieved and thawed. Three polymorphisms associated with thrombophilia (factor V Leiden, prothrombin G20210A, and MTHFR C677T) were investigated in the central genetic laboratory using previously described techniques.
      • Rosendaal F.R.
      • Koster T.
      • Vanderbroucke J.P.
      • Reitsma P.H.
      High risk of thrombosis in patients homozygous for factor V Leiden.
      • Poort S.R.
      • Rosendaal F.R.
      • Reitsma P.H.
      • Bertina R.M.
      A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated prothrombin levels and an increase in venous thrombosis.
      • Frost P.
      • Bloom H.J.
      • Milos R.
      • et al.
      A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase.
      Plasma homocysteine and folate were measured in the central biomarker laboratory by IMx (Abbott Diagnostics, Abbott Park, IL) using a fluorescence polarization immunoassay for homocysteine and ion capture technology for folate.
      Placentas of patients and control subjects were refrigerated immediately after delivery. Paraffin blocks were prepared and sent to the central pathology laboratory for reading using a standardized, previously published protocol.
      • Kramer M.S.
      • Goulet L.
      • Lydon J.
      • et al.
      Socio-economic disparities in preterm birth: causal pathways and mechanisms.
      A preplanned substudy to assess the reliability of placenta microscopy readings and statistical clustering of histopathologic features consistent with placental underperfusion in women with preterm birth, women with preeclampsia, and control subjects showed that agreement and clustering were high or moderate for infarction, decidual vasculopathy, and syncytial knotting.
      • Kramer M.S.
      • Chen M.F.
      • Roy I.
      • et al.
      Intra- and interobserver agreement and statistical clustering of placental histopathologic features relevant to preterm birth.
      We also performed exploratory and confirmatory factor analyses (LISREL 8.80; Scientific Software International, Inc, Lincolnwood, IL) that determined that these 3 features (ie, infarction, decidual vasculopathy, and syncytial knotting) loaded together onto a single factor representing underperfusion.
      All laboratory analyses were performed blindly without identifying the source of each specimen as a case or a control.

      Statistical Analysis

      Data collected at the 24- to 26-week study visit in all cohort women were compared in patients and all other cohort (ie, noncase) women (excluding women with nonpreeclamptic gestational hypertension). Data on pregnancy complications, infant outcomes, and laboratory analyses were compared in patients and control subjects. Multiple logistic regression analyses were performed in which the dependent variable was preeclampsia and the independent (explanatory) variable of interest was inherited thrombophilia. We first considered the 3 gene polymorphisms together and then examined individual polymorphisms separately. Homocysteine level (by quartiles) was also included in the model. Other variables of interest for interaction or adjustment were age, parity, low income (defined as family income below Statistics Canada's low-income cut-off

      Statistics Canada. Low income cut-offs. Catalogue 13-551-XIB, Dec. 1999. Ottawa, Canada.

      ), previous preeclampsia, smoking, chronic hypertension, and diabetes. The frequency of placental underperfusion (infarction, decidual vasculopathy, or syncytial knotting) was compared in patients and control subjects, as were underperfusion factor scores. All analyses were performed using software (SAS, Versions 8.2 and 9.1; SAS Institute, Inc, Cary, NC).

      Sample Size and Statistical Power

      The cohort size required to meet the sample size needs of the preterm birth study
      • Kramer M.S.
      • Goulet L.
      • Lydon J.
      • et al.
      Socio-economic disparities in preterm birth: causal pathways and mechanisms.
      predetermined the sample size available for the preeclampsia study. Based on a cohort size of 5000 women, a projected 2.5% incidence of preeclampsia and selection of 3 control subjects per patient, we originally anticipated having 125 patients and 375 control subjects. Assuming a combined prevalence of all gene mutations of about 15% in control subjects, this would have provided 80% power to detect odds ratio (OR) of at least 2.0 associated with the presence of any inherited thrombophilia,
      • Breslow N.E.
      • Day N.E.
      The design and analysis of cohort studies Statistical methods in cancer research. Vol II.
      with a 2-sided α level of 0.05. Although the attained number of patients was slightly lower than projected (n = 113), by increasing the number of control subjects (n = 443), and because there was a higher than projected prevalence of thrombophilia in control subjects, power was 80% to detect OR of at least 1.8 associated with the presence of any thrombophilia. For individual mutations, power was 80% to detect minimum ORs of 1.9, 2.5, and 3.9 for MTHFR C677T, factor V Leiden, and prothrombin G20210A, respectively. The study also provided 80% power to detect an OR of at least 1.75 associated with being in the upper quartile of control population homocysteine levels (corresponding to a homocysteine level of 11.0 μmol/L in control subjects) compared with all other quartiles.

      Results

      In total, of 20,830 women were approached for participation in the study from 1999-2003 (Figure 1), of whom 5337 (31% of eligible women) were recruited, attended the 24- to 26-week study visit and thus comprised the study cohort. Of these, 175 (3.3%) did not deliver at one of the 4 study hospitals and were thus lost to follow-up.
      Figure thumbnail gr1
      FIGURE 1Study flow diagram
      Data on reasons for ineligibility were available in sample of 952 women. Most frequent reasons were lack of fluency in French or English (n = 321), plans to deliver in nonstudy hospital (n = 272), severe chronic disease (eg, epilepsy, Crohn's disease, multiple sclerosis) (n = 90), gestational age at initial contact > 24 weeks (n = 62), twin pregnancy (n = 29), documented incompetent cervix (n = 16), and placenta previa (n = 13).
      Kahn. Inherited thrombophilia and preeclampsia within a multicenter cohort: the Montreal Preeclampsia Study. Am J Obstet Gynecol 2009.
      Of the 5162 study women followed up to delivery, 113 developed preeclampsia (2.2%). No cases of preeclampsia developed postpartum. Table 1 shows preeclampsia subtypes among patients; 31% had early-onset preeclampsia and 28% had severe preeclampsia. Subsequent to identification of patients, 443 control subjects were chosen as described in “Methods” section. In all, 117 (2.3%) women had gestational hypertension without preeclampsia during pregnancy and were not eligible to be patients or control subjects.
      Table 2 compares demographic, socioeconomic, and clinical characteristics in patients and noncase women, including known risk factors for preeclampsia. Patients were of similar age and had comparable levels of education and income as noncase women, but were more likely to have higher BMI, diabetes before pregnancy or before 24 weeks' gestation, chronic hypertension, miscarriage, previous preeclampsia or pregnancy-related hypertension, and family history of preeclampsia, and were less likely to smoke cigarettes. About 92% of patients and noncase women reported at the 24- to 26-week study visit that they were taking prenatal vitamins during pregnancy.
      TABLE 2Comparison of patients with preeclampsia and noncase women: demographic, medical, and obstetric characteristics ascertained at 24- to 26-week study visit
      CharacteristicPatients (n = 113)Noncase women
      Study cohort (n = 5337) excluding patients (n = 113) and women who developed gestational hypertension without preeclampsia (n = 117).
      (n = 5107)
      Control subjects (n = 443)
      Age category (y)
       < 203%3%3%
       20-3478%79%79%
       > 3419%18%18%
      Hospital recruited
       Hôpital St Luc15%22%24%
       Hôpital Maisonneuve Rosemont39%36%37%
       Royal Victoria Hospital22%18%17%
       Jewish General Hospital24%24%22%
      Born outside of Canada29%28%25%
      First language other than English or French35%24%20%
      Did not complete high school10%10%10%
      Low income as per Statistics Canada cut-off

      Statistics Canada. Low income cut-offs. Catalogue 13-551-XIB, Dec. 1999. Ottawa, Canada.

      15%20%20%
      Body mass index before pregnancy, kg/m2
       < 18.52%8%9%
       18.5 to < 2552%63%60%
       25 to < 3027%18%18%
       ≥ 3019%11%13%
      Characteristic
      Primigravida40%35%34%
      Among multigravidas
       No. of earlier pregnancies, mean (SD)1.6 (1.0)2.0 (1.3)2.0 (1.4)
       Number live-born babies, mean (SD)0.5 (0.7)0.9 (0.9)0.8 (0.9)
       Any previous miscarriage27%23%23%
      Use of prenatal vitamins92%92%92%
      Use of cigarettes
       Ever smoker43%50%51%
       Current smoker10%16%16%
      Diabetes before pregnancy or before 24 wks' gestation6%2%1%
      High blood pressure before pregnancy8%4%3%
      Previous preeclampsia or eclampsia (among women with earlier pregnancy lasting > 20 wks)21%3%3%
      High blood pressure during any earlier pregnancy (among women with earlier pregnancies)15%4%5%
      Previous venous thromboembolism4%2%2%
      Family history of preeclampsia, any female relative18%9%9%
      Data for control subjects are also shown.
      Values are percent unless indicated otherwise.
      Kahn. Inherited thrombophilia and preeclampsia within a multicenter cohort: the Montreal Preeclampsia Study. Am J Obstet Gynecol 2009.
      a Study cohort (n = 5337) excluding patients (n = 113) and women who developed gestational hypertension without preeclampsia (n = 117).
      Pregnancy complications and fetal outcomes for the current pregnancy are shown in Table 3. Patients were strikingly more likely than control subjects to have small-for-gestational-age babies, deliver at lower gestational age, require Caesarean delivery, and have gestational diabetes. Babies of preeclamptic mothers had significantly lower Apgar scores and lower birth weights, and were more likely to receive intensive care at birth than babies of control mothers.
      TABLE 3Pregnancy complications and fetal outcomes in patients with preeclampsia and control subjects
      CharacteristicPatients (n = 113)Control subjects (n = 443)P value
      Small for gestational age
      As defined in Kramer et al.43
      (13 missing)
      25 (25%)39 (9%)< .0001
      New onset of diabetes after 24 wks' gestation17 (15%)18 (4%)< .0001
      Gestational age at delivery, wk (9 missing)
      < 328 (7%)1 (0%)< .0001
      32-336 (5%)0 (0%)
      34-3623 (21%)18 (4%)
      ≥ 3776 (67%)415 (96%)
      Mode of delivery (16 missing)
      Cesarean41 (39%)75 (17%)< .0001
      Vaginal64 (61%)360 (83%)
      Newborn Apgar score at 5 minutes (5 missing)
      ≤ 43 (3%)0 (0%)< .0001
      5-65 (5%)4 (1%)
      ≥ 7101 (93%)438 (99%)
      Birth weight at delivery, g (5 missing)
      Mean (SD)2830 (850)3490 (500)< .0001
      Range190, 49901300, 5620
      Newborn received intensive care after birth (9 missing)42 (40%)82 (19%)< .0001
      Malformation present (11 missing)3 (3%)5 (1%).18
      Other health problem in newborn (13 missing)5 (5%)13 (3%).31
      Values are number (percent) unless indicated otherwise.
      Kahn. Inherited thrombophilia and preeclampsia within a multicenter cohort: the Montreal Preeclampsia Study. Am J Obstet Gynecol 2009.
      a As defined in Kramer et al.
      • Kramer M.S.
      • Platt R.W.
      • Wen S.W.
      • et al.
      A new and improved population-based Canadian reference for birth weight for gestational age.
      The frequency of inherited thrombophilia in patients and control subjects is shown in Table 4. Factor V Leiden, prothrombin G20210A, and MTHFR C677T (homozygous) were not more frequent in women with preeclampsia than in control women, whether examined collectively (patients 14.3%, control subjects 20.6%; crude OR, 0.6; 95% confidence interval [CI], 0.4-1.1; adjusted OR, 0.6; 95% CI, 0.3-1.3) or by individual mutation. Similar results were found when analyses were restricted to patients with early-onset or severe preeclampsia (Table 4). Homocysteine levels were lower in patients than in control subjects (mean [standard deviation, SD], 3.4 [0.9] vs 3.7 [0.9] μmol/L; P = .002), and patients were less likely than control subjects to have homocysteine levels in the top quartile of control values (17% vs 25%, P = .05; adjusted OR, 0.8; 95% CI, 0.4-1.4) (Table 4); results were similar for comparisons of patients with early-onset or severe preeclampsia with control subjects. Folate levels were similar in patients and control subjects (mean [SD] 39.6 [32.4] vs 41.1 [36.5] nmol/L, P = .69; percent of values in lowest quartile 25% vs 25%; P = .94).
      TABLE 4DNA mutations associated with inherited thrombophilia and hyperhomocysteinemia in patients with preeclampsia and control subjects
      VariablePatients (n = 113)Control subjects (n = 443)Crude OR (95% CI)Adjusted OR
      Models adjusted for age, body mass index, multigravida vs primigravida, current smoker, chronic hypertension, known diabetes, low income as per Statistics Canada definition,24 history of preeclampsia. Individual mutations not included in same model as combined mutations;
      (95% CI)
      Patients with early-onset or severe preeclampsia (n = 51)Crude OR
      Multivariate analyses not performed because of small numbers of patients with early-onset or severe preeclampsia;
      (95% CI)
      DNA polymorphism
      DNA data missing for 1 patient and 2 control subjects;
      Factor V Leiden or prothrombin G20210A or MTHFR C677T (homozygous)16 (14.3%)91 (20.6%)0.6 (0.4-1.1)0.6 (0.3-1.3)6 (11.8%)0.5 (0.2-1.2)
      Factor V Leiden
      All heterozygous;
      6 (5.4%)22 (5.0%)1.1 (0.4-2.7)1.0 (0.4-3.1)3 (5.9%)1.2 (0.3-4.1)
      Prothrombin G20210A
      Homozygous abnormal in 1 control subject;
      3 (2.7%)8 (1.8%)1.5 (0.4-5.7)1.8 (0.3-4.7)1 (2.0%)1.1 (0.1-8.8)
      MTHFR C677T (homozygous)8 (7.1%)64 (14.5%)0.5 (0.2-1.0)0.5 (0.2-1.1)2 (3.9%)0.2 (0.1-1.0)
      Homocysteine
      Homocysteine data missing for 1 patient and 3 control subjects; cut-off for upper quartile of homocysteine levels in control population: 11.0 μmol/L.
      Homocysteine levels > 75th percentile19 (17.0%)113 (25.7%)0.6 (0.3-1.0)0.8 (0.4-1.4)11 (21.6%)0.7 (0.3-1.3)
      CI, confidence interval; OR, odds ratio.
      Kahn. Inherited thrombophilia and preeclampsia within a multicenter cohort: the Montreal Preeclampsia Study. Am J Obstet Gynecol 2009.
      a Models adjusted for age, body mass index, multigravida vs primigravida, current smoker, chronic hypertension, known diabetes, low income as per Statistics Canada definition,

      Statistics Canada. Low income cut-offs. Catalogue 13-551-XIB, Dec. 1999. Ottawa, Canada.

      history of preeclampsia. Individual mutations not included in same model as combined mutations;
      b Multivariate analyses not performed because of small numbers of patients with early-onset or severe preeclampsia;
      c DNA data missing for 1 patient and 2 control subjects;
      d All heterozygous;
      e Homozygous abnormal in 1 control subject;
      f Homocysteine data missing for 1 patient and 3 control subjects; cut-off for upper quartile of homocysteine levels in control population: 11.0 μmol/L.
      Multivariate analyses confirmed that high BMI (25-30 kg/m2; OR, 4.7; 95% CI, 1.1-22.8; > 30 kg/m2, OR, 4.4; 95% CI, 0.9-22.2; reference category < 18.5 kg/m2), diabetes (OR, 8.8; 95% CI, 2.0-39.3), chronic hypertension (OR, 3.4; 95% CI, 1.3-9.1), and preeclampsia during a previous pregnancy (OR, 4.7; 95% CI, 1.5-15.0) were independently predictive of preeclampsia. Maternal age < 20 years tended to be associated with an increased risk of preeclampsia (OR, 3.9; 95% CI, 0.7-20.6), whereas smoking (OR, 0.5; 95% CI, 0.2-1.1) and being a multigravida (OR, 0.6; 95% CI, 0.4-1.1) tended to be protective. No interactions were observed between inherited thrombophilia and high BMI (P value for interaction = .97), diabetes (P = .98), chronic hypertension (P = .97), having a history of preeclampsia (P = .30), or being a primigravida (P = .75).
      Placental underperfusion was more frequent in patients (106 placentas) than control subjects (426 placentas) (63.2% vs 46.2%, P < .001; OR adjusted for gestational age at delivery, 1.8; 95% CI, 1.1-2.9). Similarly, underperfusion factor scores were higher in patients than in control subjects (mean [SD] score, 0.46 [0.43] vs 0.28 [0.26]; P < .0001); these differences were accentuated for comparisons of patients with early-onset or severe preeclampsia with control subjects (mean score, 0.54 [0.52] vs 0.28 [0.26]; P < .0001). No associations were detected between placental underperfusion and thrombophilia (collectively or by individual mutation) or homocysteine levels (data not shown). Furthermore, these differences were not attributable to differences in the frequency of placental abruption in patients and control subjects, as the prevalence of macroscopic pathological features consistent with abruption was similar in patients and control subjects (data not shown). However, women with folate levels in the lowest quartile of control values had higher underperfusion factor scores than women with values in the remaining quartiles (overall analysis of variance P = .04).

      Comment

      In contrast to some previous studies,
      • Kupferminc M.J.
      • Eldor A.
      • Steinman N.
      • et al.
      Increased frequency of genetic thrombophilia in women with complications of pregnancy.
      • Dizon-Townson D.S.
      • Helson L.N.
      • Easton K.
      • Ward K.
      The factor V Leiden mutation may predispose women to severe preeclampsia.
      • Grandone E.
      • Margaglione M.
      • Colaizzo D.
      • et al.
      Factor V Leiden, C>T MTHFR polymorphism and genetic susceptibility to preeclampsia.
      we did not find an increased risk of preeclampsia, including early-onset or severe preeclampsia, in association with maternal factor V Leiden mutation, prothrombin G20210A mutation, MTHFR C677T polymorphism, or hyperhomocysteinemia. We found that pathological findings consistent with placental underperfusion were more frequent in women with preeclampsia, particularly early-onset or severe preeclampsia, than in control women. This finding did not appear to be a consequence of thrombophilia, but was associated with low plasma folate levels.
      Our study has a number of strengths that lend support to its validity. Cohort women from whom patients and control subjects were drawn were recruited prospectively and consecutively at large maternity hospitals in Montreal, QC, Canada, that serve a wide socioeconomic and demographic spectrum, and are therefore likely to be broadly representative of the general obstetric population. Although the incidence of preeclampsia in our cohort (2.2%) was at the lower end of that previously reported in healthy pregnant women,
      • Sibai B.
      • Dekker G.
      • Kupferminc M.
      Pre-eclampsia.
      • Dizon-Townson D.
      • Miller C.
      • Sibai B.
      • et al.
      The relationship of the factor V Leiden mutation and pregnancy outcomes for mother and fetus.
      • Kocher O.
      • Cirovic C.
      • Malynn E.
      • et al.
      Obstetric complications in patients with hereditary thrombophilia identified using the LCx microparticle enzyme immunoassay: a controlled study of 5,000 patients.
      this likely reflects our use of strict, high-specificity criteria to diagnose preeclampsia. Our success in obtaining “true” cases of preeclampsia is shown by the sizeable differences in frequency of recognized preeclampsia risk factors and indicators of poorer pregnancy outcomes in patients and control subjects.
      There are several potential limitations to our study. First, the cohort size was predetermined by the requirements of the preterm birth study within which our study was nested. Although statistical power to detect clinically meaningful OR of at least 1.8 associated with the presence of any thrombophilia was at least 80%, power was lower for individual thrombophilias, for detection of associations between thrombophilia and preeclampsia subtypes, and for assessment of interactions between individual thrombophilias or with clinical characteristics. Second, in genetic association studies involving single nucleotide polymorphisms, associations may arise (or may be masked) as an artifact of population admixture.
      • March R.E.
      Gene mapping by linkage and association analysis.
      Although we did not perform population stratification based on ethnicity, patients and control subjects were likely to have been at least partly matched on ethnicity by matching by center, as individual hospitals tend to attract and provide care to women of specific ethnic groups. Finally, we did not measure rare thrombophilias such as protein C, protein S, or antithrombin deficiencies or acquired thrombophilic disorders such as the lupus anticoagulant, which have been linked to preeclampsia in some studies.
      • Kupferminc M.J.
      • Eldor A.
      • Steinman N.
      • et al.
      Increased frequency of genetic thrombophilia in women with complications of pregnancy.
      • Robertson L.
      • Wu O.
      • Langhorne P.
      • et al.
      Thrombophilia in pregnancy: a systematic review.
      Some previous studies, mostly retrospective in design, have reported positive associations between preeclampsia and factor V Leiden, prothrombin G20210A, or MTHFR C677T.
      • Kupferminc M.J.
      • Eldor A.
      • Steinman N.
      • et al.
      Increased frequency of genetic thrombophilia in women with complications of pregnancy.
      Results may have been influenced by referral bias or selection bias leading to overrepresentation of unusual cases, as among the few prospectively designed studies
      • Dizon-Townson D.
      • Miller C.
      • Sibai B.
      • et al.
      The relationship of the factor V Leiden mutation and pregnancy outcomes for mother and fetus.
      and a recent population-based case-control study,
      • Morrison E.R.
      • Miedzybrodzka Z.H.
      • Campbell D.M.
      • et al.
      Prothrombotic genotypes are not associated with pre-eclampsia and gestational hypertension: results from a large population-based study and systematic review.
      such associations have not been detected.
      • Kocher O.
      • Cirovic C.
      • Malynn E.
      • et al.
      Obstetric complications in patients with hereditary thrombophilia identified using the LCx microparticle enzyme immunoassay: a controlled study of 5,000 patients.
      • Murphy R.P.
      • Donoghue C.
      • Nallen R.J.
      • et al.
      Prospective evaluation of the risk conferred by factor V Leiden and thermolabile methylenetetrahydrofolate reductase polymorphisms in pregnancy.
      Several recent metaanalyses that have attempted to synthesize these disparate data have reported that pooled OR for associations between inherited thrombophilia and preeclampsia are generally in the 1.0 to 2.5 range, indicating that thrombophilia may be only weakly associated with preeclampsia, if at all.
      • Robertson L.
      • Wu O.
      • Langhorne P.
      • et al.
      Thrombophilia in pregnancy: a systematic review.
      • Morrison E.R.
      • Miedzybrodzka Z.H.
      • Campbell D.M.
      • et al.
      Prothrombotic genotypes are not associated with pre-eclampsia and gestational hypertension: results from a large population-based study and systematic review.
      • Lin J.
      • August P.
      Genetic thrombophilias and preeclampsia: a meta-analysis.
      Findings were similar for pooled analyses restricted to women with severe preeclampsia. Hence, although it has been proposed that severe, early-onset preeclampsia that leads to life-threatening maternal and fetal complications is a different disease entity than preeclampsia characterized by mild hypertension with proteinuria at term,
      • Sibai B.
      • Dekker G.
      • Kupferminc M.
      Pre-eclampsia.
      it appears unlikely that inherited thrombophilia plays a significant causal role in either.
      We did not find evidence that MTHFR C677T or homocysteine levels were associated with an increased risk of preeclampsia. In our study, control women had higher levels of homocysteine than patients, which is consistent with the higher prevalence of MTHFR C677T in control subjects. This finding has been previously observed
      • Lin J.
      • August P.
      Genetic thrombophilias and preeclampsia: a meta-analysis.
      and may be caused, at least in part, by unmeasured differences in ethnicity in patients and control subjects.
      Consistent with previous descriptions of vascular and coagulation-related lesions in preeclamptic placentae,
      • Salafia C.M.
      • Pezzullo J.C.
      • López-Zeno J.A.
      • Simmens S.
      • Minior V.K.
      • Vintzileos A.M.
      Placental pathological features of preterm preeclampsia.
      • Mousa H.A.
      • Alfirevic1 Z.
      Do placental lesions reflect thrombophilia state in women with adverse pregnancy outcome?.
      we found that pathological findings consistent with placental underperfusion were associated with preeclampsia and were more severe in women with severe or early-onset preeclampsia. Decreased placental perfusion is believed to lead to the generation and release of free radical products into the maternal circulation, which initiates the hallmark manifestations of preeclampsia.
      • Hubel C.A.
      Oxidative stress in the pathogenesis of preeclampsia.
      In our study, placental underperfusion was associated with decreased plasma folate concentration, but not with thrombophilia. Growth in a folate-free medium induces apoptosis in human placental trophoblast cells
      • Steegers-Theunissen R.P.M.
      • Smith S.C.
      • Steegers E.A.P.
      • Guilbert L.J.
      • Baker P.N.
      Folate affects apoptosis in human trophoblastic cells.
      and addition of folic acid to homocysteine-exposed trophoblast cells prevents apoptosis,
      • Di Simone N.
      • Riccardi P.
      • Maggiano N.
      • et al.
      Effect of folic acid on homocysteine-induced trophoblast apoptosis.
      suggesting that folate deficiency may be implicated in placenta-mediated pregnancy complications. Indeed, studies of Peruvian women and black African women from Zimbabwe showed that low plasma folate levels were associated with an increased risk of preeclampsia.
      • Sanchez S.E.
      • Zhang C.
      • Malinow M.R.
      • Ware-Jauregui S.
      • Larrabure G.
      • Williams M.A.
      Plasma folate, vitamin B12, and homocyst(e)ine concentrations in preeclamptic and normotensive Peruvian women.
      • Rajkovic A.
      • Mahomed K.
      • Rozen R.
      • Malinow M.R.
      • King I.B.
      • Williams M.A.
      Methylenetetrahydrofolate reductase 677 C → T polymorphism, plasma folate, vitamin b12 concentrations, and risk of preeclampsia among black African women from Zimbabwe.
      In our study, the ability to detect differences in plasma folate levels may have been limited by almost universal use of prenatal vitamins, which is consistent with rates of self-reported vitamin use in a recent Canada-wide maternal experiences survey,
      • Chalmers B.
      • Dzakpasu S.
      • Heaman M.
      • Kaczorowski J.
      The Canadian maternity experiences survey: an overview of findings.
      and ubiquitous food fortification with folate.
      In conclusion, our results do not support the hypothesis that the common inherited thrombophilias factor V Leiden, prothrombin G20210A, and MTHFR C677T are causally associated with preeclampsia in women carriers. Whether thrombophilia acts a cofactor in the pathogenesis of preeclampsia or accelerates its course
      • Gerhardt A.
      • Goecke T.W.
      • Beckmann M.W.
      • et al.
      the G20210A prothrombin-gene mutation and the plasminogen activator inhibitor (PAI-1) 5G/5G genotype are associated with early onset of severe preeclampsia.
      requires further investigation. Heritable aspects of preeclampsia are complex and appear to involve both maternal genes and fetal genes;
      • Skjaerven R.
      • Vatten L.J.
      • Wilcox A.J.
      • Ronning T.
      • Irgens L.M.
      • Lie R.T.
      Recurrence of pre-eclampsia across generations: exploring fetal and maternal genetic components in a population based cohort.
      however, specific modes of inheritance and the maternal or fetal genotype have not been established.
      • Roberts J.M.
      • Cooper D.W.
      Pathogenesis and genetics of pre-eclampsia.
      Our findings challenge the increasingly pervasive practice of screening women with preeclampsia for thrombophilia, especially as randomized controlled trials have not shown that use of anticoagulants during pregnancy prevents preeclampsia in women in whom thrombophilia is detected.
      • Middeldorp S.
      Thrombophilia and pregnancy complications: cause or association?.
      • Rodger M.A.
      • Paidas M.
      • McLintock C.
      • et al.
      Inherited thrombophilia and pregnancy complications revisited.

      Acknowledgments

      The authors gratefully acknowledge the contribution of study coordinator Henriette Gauthier RN, MSc, and the assistance of the study nurses and research assistants in recruiting and enrolling the study subjects.

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      Linked Article

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        American Journal of Obstetrics & GynecologyVol. 200Issue 2
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          The article below summarizes a roundtable discussion of a study published in this issue of the Journal in light of its methodology, relevance to practice, and implications for future research. Article discussed: Kahn SR, Platt R, McNamara H, et al. Inherited thrombophilia and preeclampsia within a multicenter cohort: the Montreal Preeclampsia Study. Am J Obstet Gynecol 2009;200:151.e1-151.e9. The full discussion appears at www.AJOG.org , pages e1-e5.
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      • Inherited thrombophilia and preeclampsia: is the evidence beginning to congeal?
        American Journal of Obstetrics & GynecologyVol. 200Issue 2
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          As part of the routine clinical practice of maternal-fetal medicine, I am often asked to consult with both patients and physicians following an adverse pregnancy outcome. Frequently, an exceptionally thorough evaluation has been initiated by the referring physician, and the patient arrives with a small mountain of paperwork that requires review. Almost invariably, a comprehensive search for inherited thrombophilia is included in the laboratory results.
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      • Confounded thrombophilia studies in preeclampsia
        American Journal of Obstetrics & GynecologyVol. 201Issue 5
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          We compliment Kahn et al on their prospective study of thrombophilia in 5337 pregnant women.1 They identified 113 women who developed preeclampsia and selected 443 controls who had neither preeclampsia nor nonproteinuric gestational hypertension. They tested for the factor V Leiden G1691A mutation, the prothrombin gene G20210A mutation, and the methylenetetrahydrofolate reductase C677T polymorphism. They concluded that they did not find evidence to support an association between these inherited thrombophilias and an increased risk of preeclampsia.
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      • Reply
        American Journal of Obstetrics & GynecologyVol. 201Issue 5
        • Preview
          We thank Drs de Vries, Kist, and Hague for their letter and for bringing to our attention their recently published review, which highlighted the impressive heterogeneity of results of studies on the association between inherited thrombophilia and adverse pregnancy outcomes such as preeclampsia.1 In their review, they suggest that this heterogeneity may be due at least in part to confounding by ethnicity, method of genetic testing, and severity of illness.
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