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ORIGINAL RESEARCH |
From the University Department of Obstetrics and Gynecology, Liverpool Women’s Hospital; and the Department of Hematology, Royal Liverpool University Hospital, Liverpool, United Kingdom.
Address reprint requests to: Zarko Alfirevic, MD, MRCOG University Department of Obstetrics and Gynecology Liverpool Women’s Hospital University of Liverpool Liverpool, L69 3BX United Kingdom E-mail: zarko{at}liv.ac.uk
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Abstract |
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Methods: We studied 102 women who had pregnancy complications and 44 healthy women with uncomplicated pregnancies. All women were tested 10 weeks postpartum for mutations of factor V Leiden, methylenetetrahydrofolate reductase (MTHFR) C677T, and G20210A prothrombin gene; deficiencies of protein C, protein S, and antithrombin III; and the presence of lupus anticoagulant and anticardiolipin antibodies. We aimed to recruit 100 cases and 300 controls to detect a 10% difference in thrombophilia between the groups. However, we were able to recruit only 44 controls.
Results: Abnormal thrombophilia screen was found in 54 women with pregnancy complications (53%) and in 17 women (39%) with normal pregnancies (odds ratio [OR] 1.8; 95% confidence interval [CI] 0.87, 3.67). Mutations encoding for factor V Leiden, G20210A prothrombin gene, and MTHFR C677T (homozygous) were identified in 18% of women with complications compared with 16% of controls (OR 1.1; 95% CI 0.44, 2.94). Activated protein C resistance, not due to factor V Leiden mutation, was the most common thrombophilic defect, found in 26% of women with pregnancy complications compared with 18% of controls
Conclusion: In our cohort of women with pregnancy complications, maternal thrombophilia was less common than previously thought, and multiple thrombophilias were not a major additional risk factor
Maternal thrombophilia has recently been explored as a cause of placental thrombosis,1,2 severe preeclampsia,3,4 placental abruption,5 fetal growth restriction (FGR),6 and stillbirth.7,8 In 1999, Kupferminc et al9 published further evidence of an increased frequency of inherited and acquired thrombophilias in Jewish women with severe pregnancy complications, compared with healthy women.
Data from the European Prospective Cohort on Thrombophilia study suggested that more than one thrombophilic defect may increase the risk even further.10 This prospective observational study found a 14-fold increased risk of stillbirth in women with multiple defects, compared with only a three- to fivefold increase in women with a single abnormality.10 The combination of factor V Leiden mutation with either protein S, protein C, or antithrombin III deficiency was the most common multiple thrombophilia in this cohort. Systematic searching for thrombophilia in women with severe preeclampsia requiring delivery before 34 weeks’ gestation revealed a 25% prevalence of multiple defects.11
The strength of this evidence has prompted clinicians to focus on maternal thrombophilia as the most common identifiable cause of adverse pregnancy outcomes associated with impaired placentation and to consider selective testing.12
The purpose of our study was to examine the frequency of genetic mutation of guanine to adenine at nucleotide 1691 in the factor V gene (factor V Leiden mutation), the mutation of cytosine to thymine at nucleotide 677 in the gene encoding methylenetetrahydrofolate reductase (MTHFR), and the mutation of guanine to adenine at nucleotide 20210 in the prothrombin gene in women with severe pregnancy complications. We also looked for other inherited and acquired thrombophilias, including deficiency of antithrombin III, protein C, or protein S; activated protein C resistance; and the presence of anticardiolipin antibodies, lupus anticoagulant, or hyperhomocysteinemia. In addition, we compared perinatal outcomes between women with multiple thrombophilic defects and women with a single abnormality.
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Materials and Methods |
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The control group included healthy women with no abnormal medical or obstetric history who delivered in the same hospital within 6 months of the index case. The funding was available for 15 months, and we aimed to recruit three controls matched for age, parity, and gestation for each included patient. We intended to recruit 100 cases and 300 controls, which would give us adequate power to detect a 10% difference in thrombophilia between the groups if the background prevalence was approximately 5%. However, matching proved difficult, and we were able to recruit only 44 controls during the study period.
The study was approved by the Regional Ethics Committee. All women were fully informed of the aim of the study, and informed consent was obtained from all participants.
All blood tests were done at least 10 weeks after delivery and included assays of antithrombin III, protein C activity, free and total antigen protein S, activated protein C resistance, anticardiolipin antibodies (immunoglobulin [Ig]G and IgM), lupus anticoagulant by activated partial thromboplastin time (APTT) and dilute Russell viper venom test, and fasting plasma homocysteine. Genetic studies were performed to look for factor V Leiden mutation, mutation in the gene encoding MTHFR, and the prothrombin gene mutation.
Antithrombin III, protein C activity, activated protein C resistance without predilution with factor V–deficient plasma, and lupus clotting screens (prothrombin time, APTT, dilute Russell viper venom test) with the phospholipid neutralization procedure were all measured on the multidiscrete analyzer (MDA-180; Organon-Teknika, Cambridge, United Kingdom) with a variety of reagents, according to the manufacturer’s instructions. Protein S assays (free and total) were performed by an enzyme-linked immunosorbent assay (ELISA) method (Diagnostica Stago, Asniers, France).
Homocysteine was quantified by an ELISA using the Axis Homocysteine EIA (Bio-Rad, Hemel-Hempstead, United Kingdom). Anticardiolipin antibodies were measured with the Cambridge Life Science Melisa kit (Cambridge Life Science, Huntington, United Kingdom). Results were read on a multiscan reader (Titertek Multiscan II MCC/340; Laboratories Ltd, Helsinki, Finland) at 450 nm and calculated using the manufacturer’s software.
Genomic DNA was extracted from 400 µL of whole blood using the Puregene kit (Gentra, Minneapolis, MN) according to the manufacturer’s instructions. DNA sequences of the factor V gene, prothrombin gene, and MTHFR gene were amplified by polymerase chain reaction with the use of primers.14–16.
Results were considered abnormal only if two consecutive tests were outside the reference range for the local population. Specific cutoff points for abnormal dynamic tests have not been given because these tests are subject to wide interlaboratory variation. Multiple thrombophilia was diagnosed when at least two thrombophilia markers were detected.
For comparison of frequencies, we calculated odds ratios (ORs) with 95% confidence intervals (CIs) using Simple Interactive Statistical Analysis, which is available freely on the Internet (http://www.home.clara.net/sisa; Dann Vitenbroek, United Kingdom, 2000). Subgroup analyses of perinatal outcomes in patients with single and multiple thrombophilias were performed with the 
2 test, Fisher exact test, Mann-Whitney test, and Student t test, where appropriate, with P < .05 taken as significant. Statistical analyses were performed with the Statistical Package of Arcus (Quickstat 1.0; Iain Buchan, United Kingdom, 1997).
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Results |
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). Activated protein C resistance was the most common thrombophilic defect, found in 28 of 102 women (29%) with pregnancy complications compared with 11 of 44 women (25%) with normal pregnancies. Only five of 39 women with activated protein C resistance had factor V Leiden mutation (two women in the study group and three in the control group). The prevalence of maternal thrombophilia according to the specific type of perinatal complication is given in Table 3.
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. All women in this group were negative for the factor V Leiden mutation.
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Perinatal outcomes did not differ in subgroup analyses of patients with single and multiple thrombophilias (Table 5).
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Discussion |
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Several studies have shown that the presence of more than one thrombophilic defect is a risk factor for adverse pregnancy outcome.5,9,10 Similar perinatal outcomes in the patients with single and multiple thrombophilias in our study were, therefore, unexpected.
We acknowledge that our series may not be large enough and may therefore be at risk for a false-negative result. However, the observed difference of 14% between the groups (53% versus 39%) would be statistically significant if 140 cases and 420 controls were recruited (power 80%, alpha = .05). It is noteworthy that half of the controls had abnormal activated protein C resistance, but were negative for factor V Leiden mutation, an abnormal test not often included by others.9,18
Acquired activated protein C resistance not due to factor V Leiden mutation, which has recently been identified as an independent risk factor for deep venous thrombosis,19,20 was the most common thrombophilic defect in our series (26%). Our observation is in accordance with the results of Krauss et al,21 who found that 33% of women with a history of the syndrome of hemolysis, elevated liver enzymes, and low platelets had evidence of activated protein C resistance, with factor V Leiden mutation identified in only 19% of cases. In addition, Wiener-Megnagi et al22 reported that among 17 women with placental abruption and activated protein C resistance, only seven had factor V Leiden mutation. The presence of as yet unidentified genetic defects has been suggested.23,24 However, the mechanism of placental-mediated pregnancy complications among women with acquired activated protein C resistance remains to be elucidated.
The factor V Leiden mutation was detected in 2% of women with adverse pregnancy outcomes; this prevalence is similar to that found by van Pampus et al3 and O’Shaughnessy et al,25 but lower than that in other studies.1,9,18,22 Large variations in the prevalence of genetic thrombophilia in reported studies could limit generalization of the results. This issue was highlighted by the results of two recent case-control studies from The Netherlands. Van Pampus et al3 identified the factor V Leiden mutation in 6% of women with a history of severe preeclampsia compared with 1.5% of healthy controls, whereas van der Molen et al5 detected factor V Leiden mutation in 14% of women with a history of placental vasculopathy compared with 5% of healthy controls. The 9% difference in the prevalence of factor V Leiden mutation in the study by van der Molen et al5 was significant, whereas the 4.5% difference found by van Pampus et al3 was not. One must be careful when interpreting these results. Statistical significance (or lack of it) should not be used as definitive proof of clinical significance or causality, particularly if the sample sizes are relatively small and the CIs around the estimated prevalence are large.
Our results confirm previous observations that protein S deficiency may be more important than protein C deficiency in women with adverse pregnancy outcome.1,7,8 The fact that protein S–deficient women are at relatively low risk for thromboembolism during pregnancy26 suggests that different mechanisms may be responsible for these placental perinatal complications. This view is further supported by the apparently lower prevalence of genetic thrombophilic markers in women with severe pregnancy complications compared with women with a history of thromboembolism.8,27 Anticardiolipin antibodies and lupus anticoagulant have been previously reported as risk factors for poor pregnancy outcome, and our data, to some extent, support this view.5,28
Hyperhomocysteinemia may be exacerbated by deficiency of vitamin B12 and folate or abnormalities within the methioninehomocysteine pathway.2 Our study group had ten women (10%) with hyperhomocysteinemia, eight of whom had C677T mutation in the gene encoding for MTHFR (six homozygous and two heterozygous), compared with one woman in the control group. Van der Molen et al5 examined the correlation between hyperhomocysteinemia and placental vasculopathy (infarction or abruption) at different serum cutoff levels. They observed a twofold increase in placental vasculopathy in women with high homocysteine levels compared with matched controls (36% compared with 22%; 95% CI 1.18, 4.39).5 However, only 13% of patients were homozygous for the C677T mutation of the MTHFR gene.5 This is in keeping with the study by Daly et al,29 who described an association between reduced total enzymatic activity and heterogeneous carriers for the C677T mutation of the MTHFR gene.
Positive postnatal screening for thrombophilia creates a real clinical dilemma in subsequent pregnancies and beyond. In our series, the prevalence of thrombophilia was lower than expected, and the difference between the study group and control group did not reach statistical significance for any of the individual tests. Larger numbers could have resulted in statistically significant results. Also, if all abnormal tests are analyzed together, women with severe pregnancy complications may have a statistically higher chance of being screen positive.9 Is this sufficient justification to offer universal postnatal testing for women with an obstetric complication? Currently, there is no evidence to support thromboprophylaxis for women with abnormal thrombophilia screening results and no history of thromboembolism. Large clinical trials are required to prove the effectiveness of aspirin, heparin, or both in reducing perinatal complications and to address the issue of safety for such women. In the absence of such trials, knowledge of positive thrombophilia status is likely to increase anxiety in carriers and offers no health benefit.
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Footnotes |
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Received August 15, 2000. Received in revised form November 28, 2000. Accepted December 15, 2000.
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2. Ray JG, Laskin CA. Folic acid and homocyst(e)ine metabolic defects and the risk of placental abruption, pre-eclampsia and spontaneous pregnancy loss: A systematic review. Placenta 1999; 20:519–29.[Medline]
3. van Pampus MG, Dekker GA, Wolf H, Huijgens PC, Koopman MM, von Blomberg BM, et al. High prevalence of haemostatic abnormalities in women with a history of severe preeclampsia Am J Obstet Gynecol 1999;80:1146–50.
4. Wang J, Trudinger BJ, Duarte N, Wilcken DE, Li Wang X. Elevated circulating homocyst(e)ine levels in placental vascular disease and associated pre-eclampsia. Br J Obstet Gynaecol 2000;107:935–8.
5. van der Molen EF, Verbruggen B, Novakova I, Eskes TKAB, Monnens LAH, Blom HJ. Hyperhomocysteinemia and other thrombotic risk factors in women with placental vasculopathy. Br J Obstet Gynaecol 2000;107:785–91.
6. Yasuda M, Takakuwa K, Tokunaga A, Tanaka K. Prospective studies of the association between anticardiolipin antibodies and outcome of pregnancy. Obstet Gynecol 1995;86:555–9.[Abstract]
7. De Vries JI, Dekker GA, Huijgens PC, Jakobs C, Blomberg BM, van Geijn HP. Hyperhomocysteinaemia and protein S deficiency in complicated pregnancies. Br J Obstet Gynaecol 1997;104:1248–54.[Medline]
8. Gris JC, Quere I, Monpeyroux F, Mecier E, Ripart-Neveu S, Tailland ML, et al. Case-control study of the frequency of thrombophilic disorders in couples with late fetal loss and no thrombotic antecedent. Thromb Haemost 1999;81:891–9.[Medline]
9. Kupferminc MJ, Eldor A, Steinman N, Many A, Bar-Am A, Jaffa A, et al. Increased frequency of genetic thrombophilia in women with complications of pregnancy. N Engl J Med 1999;340:9–13.
10. Preston FE, Rosendaal FR, Walker ID, Briet E, Berntorp E, Conard J, et al. Increased fetal loss in women with heritable thrombophilia Lancet 1996;348:913–6.[Medline]
11. Dekker GA, de Veries JI, Doelitzsch PM, Huijgens PC, von Blomberg BM, Jacobs C, et al. Underlying disorders associated with severe early-onset pre-eclampsia. Am J Obstet Gynecol 1995; 173:1042–8.[Medline]
12. Nelson-Piercy C. Inherited thrombophilia and adverse pregnancy outcome: Has the time come for selective testing? Br J Obstet Gynaecol 1999;106:513–5.[Medline]
13. Davey DA, MacGillivray I. The classification and definition of hypertensive disorders of pregnancy. Am J Obstet Gynecol 1988; 58:892–8.
14. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64–7.[Medline]
15. Frosst P, Blom HJ, Govette P, Sheppard CA, Matthews RG, Boers GJ, et al. A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111–3.[Medline]
16. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3'-unsaturated region of the prothrombin gene was associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;88:3698–703.
17. Chikosi AB, Moodley J, Pegoraro RJ, Lanning PA, Rom L. 5,10 Methylene-tetrahydrofolate reductase polymorphism in black South African women with pre-eclampsia. Br J Obstet Gynaecol 1999;106:1219–20.[Medline]
18. Kupferminc MJ, Fait G, Many A, Gordon D, Eldor A, Lessing JB Severe preeclampsia and high frequency of genetic thrombophilic mutations. Obstet Gynecol 2000;96:45–9.
19. Rodeghiero F, Tosetto A. Activated protein C resistance and factor V Leiden mutation are independent risk factors for venous thromboembolism. Ann Intern Med 1999;130:643–50.
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21. Krauss T, Augustin HG, Osmers R, Meden H, Unterhalt M Activated protein C resistance and factor V Leiden in patients with hemolysis, elevated liver enzymes, low platelets syndrome. Obstet Gynecol 1998;92:457–60.[Abstract]
22. Wiener-Megnagi Z, Ben-Shlomo I, Goldberg Y, Shalev E. Resistance to activated protein C and the Leiden mutation: High prevalence in patients with abruptio placentae. Am J Obstet Gynecol 1998;179:1565–7.[Medline]
23. Dahlback B. Factor V gene mutation causing inherited resistance to activated protein C as a basis for venous thromboembolism J Intern Med 1995;237:221–7.[Medline]
24. Gherman RB, Goodwin TM. Obstetric implications of activated protein C resistance and factor V Leiden mutation. Obstet Gynecol Surv 2000;55:117–22.[Medline]
25. O’Shaughnessy KM, Fu B, Ferraro F, Lewis I, Downing S, Morris NH. Factor V Leiden and thermolabile methylenetetrahydrofolate reductase gene variants in an East Anglian preeclampsia cohort Hypertension 1999;33:1338–41.
26. Walker ID. Inherited coagulation disorders and thrombophilia and pregnancy. In: Bonnar J, ed. Recent advances in obstetrics and gynecology. 1st ed. Edinburgh: Churchill Livingstone, 1998:35–64.
27. Gerhardt A, Scharf RE, Beckmann MW, Struve S, Bender HG, Pillny M, et al. Prothrombin and factor V mutations in women with a history of thrombosis during pregnancy and the puerperium N Engl J Med 2000;342:347–80.
28. Backos M, Rai R, Baxter N, Chilcott IT, Cohen H, Regan L.Pregnancy complications in women with recurrent miscarriage and antiphospholipid antibodies treated with aspirin and heparin. Br J Obstet Gynaecol 1999;106:102–7.[Medline]
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