Inherited thrombophilia and pregnancy loss

Best Practice & Research Clinical Haematology Vol. 16, No. 2, pp. 311 –320, 2003 doi:10.1053/ybeha.2003.250

12 Inherited thrombophilia and pregnancy loss Benjamin Brenner*

MD

Chief Thrombosis and Haemostasis Unit, Department of Haematology, Rambam Medical Centre, P.O. Box 9602, Haifa 31096, Israel

A growing body of evidence obtained during the past 6 years suggests a significant role for inherited thrombophilia in the development of gestational vascular complications. Case – control and cross-sectional studies have demonstrated that thrombophilia is more prevalent in cohorts of women with pregnancy loss. Placental pathological findings in women with thrombophilia are hallmarked by thrombosis and fibrin deposition. Preliminary case– control studies suggest that low-molecular-weight heparins (LMWH) are effective in preventing pregnancy loss in women with thrombophilia and previous fetal wastage. Key words: fetal loss; thrombophilia; low-molecular-weight heparin; placenta; thrombosis.

The successful outcome of pregnancy depends on the development and maintenance of adequate placental circulation. Abnormalities in placental vasculature may result in a number of gestational pathologies, including first- and second-trimester miscarriages, intrauterine growth restriction (IUGR), intrauterine fetal death (IUFD), placental abruption, and pre-eclampsia.1 This chapter reviews recent data concerning inherited thrombophilia and fetal loss, and highlights potential therapeutic modalities for the prevention of placental vascular pathology in order to achieve a successful gestational outcome.

RECURRENT FETAL LOSS About 20% of women experience at least one fetal loss. Recurrent fetal loss (RFL) is a common health problem, with three or more loses affecting 1 –2% and two or more losses affecting up to 5% of women at the reproductive age.2,3 While several causes have been implicated in RFL—including chromosomal translocations and inversions, anatomical alterations of the uterus, endocrinologic abnormalities and autoimmune disorders4,5—until recently the majority of cases of RFL have remained unexplained. The association of acquired thrombophilic states—such as antiphospholipid syndrome * Tel.: þ 972-4-854-2541; Fax: þ972-4-854-2342. E-mail address: [email protected] (B. Brenner). 1521-6926/03/$ - see front matter Q 2003 Elsevier Science Ltd. All rights reserved.

312 B. Brenner

and essential thrombocythaemia—with poor pregnancy outcome is well established and is discussed in other chapters of this issue. More recently, studies in women with inherited thrombophilia have suggested an association with fetal loss. A case –control study in women with the inherited thrombophilia, protein C, protein S and antithrombin deficiencies documented an increased risk for RFL. Forty-two out of 188 pregnancies (22%) in 60 women with thrombophilia resulted in pregnancy loss compared to 23/202 (11%) in controls (OR 2.0; 95% CI 1.2 – 3.3).6 Two studies which examined the association between protein C deficiency and RFL did not reveal a significant association.7,8 However, studies of women with recurrent pregnancy loss demonstrated an association with protein S deficiency.7,8 A high incidence of gestational abnormalities was reported in 15 women with dysfibrinogenaemia associated with thrombosis. Of 64 pregnancies, 39% ended by miscarriage and 9% by IUFD.9

THROMBOPHILIC POLYMORPHISMS Factor V Leiden A number of case – control studies have evaluated the prevalence of factor V Leiden mutation in women with RFL (Table 1). Despite differences in ethnic Caucasian sub-populations and selection criteria for RFL, most studies documented a significantly increased prevalence of factor V Leiden mutation in women with RFL. Ridker et al10 have studied women with RFL without an extensive aetiological work-up except, for ruling out chromosomal abnormalities. They found a 2.3-fold increase in the prevalence of factor V Leiden in women with RFL. Pooled data from 14 studies that excluded other causes for fetal loss revealed a significant association with factor V Leiden.11 In women with RFL of unknown cause—following exclusion of chromosomal abnormalities, infections, anatomical alterations and endocrinological dysfunction – studies by Grandone et al12 and by our group13,14 have suggested that evaluation for factor V Leiden mutation is highly warranted because as a significant percentage of women with RFL are found to be carriers of the mutation. Nevertheless, it should be emphasized that other reports did not document an association between factor V Leiden mutation and RFL.15,16 Activated protein C (APC) resistance and factor V Leiden mutation are particularly associated with late fetal loss.17 However, Younis et al18 have reported that APC resistance and factor V Leiden mutation can be associated with first-as well as second-trimester RFL. A recent study found that pregnancy loss in women with thrombophilia is distributed throughout gestation. However, while the majority of pregnancy loss in the study group occurred at the first trimester, late pregnancy wastage occurred more frequently in women with thrombophilia, compared to women Table 1. Factor V Leiden mutation in women with recurrent pregnancy loss. Study Grandone et al (1997)12 Ridker et al (1998)10 Brenner et al (1999)13 Wrasnsby et al (2000)19

Patients

Controls

OR

95% CI

P

7/43(16%) 9/113(8%) 24/76(32%) 13/84 (15.5%)

5/118(4%) 16/437(3.7%) 11/106(10%) 2/69 (2.9%)

4.4 2.3 4.0 7.2

1.3– 14.7 1.0– 5.2 1.8– 8.8 1.5– 34

0.01 0.05 0.001 0.008

Inherited thrombophilia and pregnancy loss 313

without thrombophilia (160/429 (37%) versus 39/162 (24%), respectively, P ¼ 0.002).14 A study from Sweden documented that women who are primary aborters have a higher frequency of factor V Leiden mutation 10/36 (28%) compared to 2/619 (3%) of controls (P ¼ 0.0003).19 In populations where homozygosity for factor V Leiden is highly prevalent, significant association of this state with RFL can also be demonstrated.13 In fact, the risk for RFL is greater in homozygous carriers than in heterozygous carriers of factor V Leiden.20 Screening of family members is advisable because siblings of thrombophilic women with factor V Leiden mutation are also at an increased risk for RFL.21 Pregnant women with and without factor V Leiden exhibit substantial activation of coagulation, with higher D-dimer levels in the formers.22 APC resistance in pregnancy When analysing the association between RFL and APC resistance it should be emphasized that the APC sensitivity ratio falls progressively throughout normal pregnancy either with or without a correlation to changes in factor VIII, factor V and protein S levels.23,24 Transient APC resistance can be documented during normal gestations, even in women with normal factor V genotype. APC sensitivity ratios may be further reduced during gestation, potentially explaining the higher prevalence of RFL in women with factor V Leiden mutation. Of interest, APC resistance in the absence of factor V Leiden mutation has also been recently associated with pregnancy loss.25,26 Indeed, in a recent prospective case –control study, APC resistance was the most common thrombophilic defect, documented in 39% of women with pregnancy loss compared to only 3% of control group (OR ¼ 18.0, 95% CI: 7.0 –53.6, P , 0.0001).14 Autoantibodies such as anti-b2-glycoprotein-1 are one potential mechanism for acquired APC resistance.27 Factor II G20210A While our recent studies demonstrated that factor II G20210A—as a solitary defect— is not associated with an increased risk for RFL, compared to controls13,14, other studies suggested that factor II G20210A mutation is a mild risk factor for certain types of RFL.28,29 In a recent study from Italy, Martinelli et al have documented that both factor V Leiden and factor II G20210A mutations are associated with IUFD.29 Eleven of the 67 women with late loss (16%) and 13 of the 232 control women (6%) had either the factor V or the prothrombin mutation. The relative risks of late fetal loss in carriers of the factor V and prothrombin mutations were 3.2 (95% confidence interval, 1.0 to 10.9) and 3.3 (95% confidence interval, 1.1 to 10.3), respectively. Thus, both the factor V and the prothrombin mutations were associated with an approximate tripling of the risk of late fetal loss. Pooled analyses reveal a significant relationship between prothrombin mutation and early (, 13 weeks) and late (. 25 weeks) fetal loss.11

HYPERHOMOCYSTEINAEMIA Homocysteine levels decrease in pregnancy by 50%. An association of gestational vascular complications and hyperhomocysteinaemia (HHC) is increasingly reported. HHC was documented in 26% of women with placental abruption, in 11% of the cases

314 B. Brenner

with IUFD and in 38% of women delivering babies whose birth weight was below the fifth percentile compared with an estimated 2– 3% in the general control population.30 Likewise, HHC was documented in 26/84 (31%) women with previous placental infarcts or abruption compared to 4/46 (9%) in controls.31 In addition, hyperhomocysteinemia was found in 6/35 (17%) of patients with recurrent abortions.32 In a recent meta-analysis, Nelen et al33 reviewed 10 case – control studies which examined the association of RFL and hyperhomocysteinemia and reported a 3– 4-fold increased risk, while in six studies the risk for homozygosity for MTHFR was 1.4. Pooled analysis revealed no association between MTHFR 677TT and recurrent or non-recurrent early or late fetal loss.11 These data suggest the HHC is a risk factor for RFL while homozygocity for MTHFR as a solitary thrombophilic defect is not. However, testing for MTHFR 677TT may be of value in women with decreased folate and vitamin b12 as well as for identifying women with combined thrombophilia. Combined thrombophilia It is well established that combinations of inherited or acquired thrombophilic states increase the risk for thrombosis.34,35 Similary, combinations of thrombophilic states may further increase the risk for RFL. For example, coexistence of factor V Leiden and homozygous HHC36 or a combination of factor V Leiden with familial antiphospholipid syndrome37 were reported to result in thrombosis and RFL. It is therefore not surprising that the EPCOT study documented the highest odds ratio for stillbirth (OR ¼ 14.3, 95% CI 2.4 – 86) in patients with combined thrombophilic defects.38 In our recent study, at least one thrombophilic defect was found in 96/145 (66%) of women with RFL group compared to 41/145 (28%) in controls (OR ¼ 5.0, 95% CI: 3.0 – 8.5 P , 0.0001).14 Combined thrombophilic defects were documented in 31/145 (21%) of women with pregnancy loss compared to 8/145 (5.5%) in controls (OR ¼ 5.0, 95% CI: 2.0 – 11.5, P , 0.0001). APC resistance without factor V Leiden was documented in 18% of women with pregnancy loss either as the only defect (9%) or in combination with other thrombophilia (9%). While factor V Leiden was more common in women with pregnancy loss (25 versus 7.6%, OR ¼ 4.0, 95% CI: 1.9 –8.8, P , 0.0001), factor II G20210A and homozygosity for MTHFR C677T contributed to pregnancy loss only when associated with other thrombophilic defects.14 In the NOHA-5 study, placental pathological findings were documented in 88% of women with combined thrombophilia, and in 100% of those with a combination of any thrombophilia and MTHFR C677T.8

PLACENTAL PATHOLOGICAL FINDINGS Mechanisms responsible for the association of inherited thrombophilia with RFL have not been elucidated. Pathological studies of placentae obtained from gestations terminated by fetal loss reveal thrombotic changes and infarcts. These can be observed in the maternal vessels in the placenta in a large proportion of women who experienced stillbirth.39 However, these changes can also be found in a significant proportion of women with IUFD without thrombophilia.8,29,39 The location of placental thrombotic changes can be either at fetal vessels or at maternal vessels. A role for feto-placental thrombosis has been suggested by studies

Inherited thrombophilia and pregnancy loss 315

demonstrating an association between factor V genotype in miscarried fetuses and placental infarction.40 However, the finding of thrombotic changes at the maternal side, and the efficacy of low-molecular-weight heparin(LMWH), which does not cross the placenta, in preventing fetal loss, support the latter. In addition, the NOHA-5 study demonstrated that the spouse’s genotype did not contribute to stillbirth.8 Gene targeting of factor V in mice demonstrate that mice heterozygotic for factor V Leiden are born uneventfully.41 Thus, the role, if any, of fetal factor V genotype in fetal demise is currently not established. As up to 65% of vascular gestational abnormalities can be accounted for by genetic thrombophilias13,42, the implication is to screen for these mutations in all women with vascular gestational abnormalities. Furthermore, this high prevalence of genetic thrombophilias, which is similar to the findings in women with pregnancy-related venous thromboembolism43, and the findings of thrombotic changes in the placentae of the majority of women with thrombophilia and stillbirth8, suggest that antithrombotic drugs may have potential therapeutic benefit in women with gestational vascular complications. Placental pathological findings, including villous infarction and fibrin deposition, are more common in women with thrombophilia44 but can also be found in women with a negative thrombophilia work-up8,29, raising the possibility of yet undetermined prothrombotic risk factors operating in these settings.

THERAPEUTIC REGIMENS Without therapeutic intervention only about 20% of gestations in women with thrombophilia and RFL result in live birth.13,14 Emerging data on the therapy of women with inherited thrombophilia and pregnancy loss is mostly uncontrolled and include small series of patients treated mostly with LMWH. The potential advantages of LMWH over unfractionated heparin are a higher antithrombotic ratio, meaning less bleeding for a better antithrombotic effect, longer half-life with a potential need for only one injection per day, smaller injected volume and less heparin-induced thrombocytopenia. A recent collaborative study has demonstrated the safety of using LMWH during 486 gestations.45 Successful outcome was reported in 83/93 (89%) gestations in women with recurrent pregnancy loss and in all 28 gestations in women with pre-eclampsia in previous pregnancy.45 Administration of the LMWH enoxaparin 20 mg/day to women with primary early RPL and impaired fibrinolytic capacity resulted in normalization of impaired fibrinolysis, conception in 16/20 (80%) and successful live birth in 13/16 (81%).46 We have treated 61 pregnancies in 50 women with thrombophilia who presented with RPL with the LMWH enoxaparin throughout gestation and 4 weeks into the postpartum period. Enoxaparin dosage was 40 mg/day, except for patients with combined thrombophilia or in case of abnormal Doppler velocimetry suggesting decreased placental perfusion, where the dosage was increased to 40 mg bid. In the case of previous thrombosis LMWH therapy was continued for 6 weeks after delivery. Forty-six of the 61 pregnancies (75%) resulted in live birth compared to a success rate of only 20% in these 50 women in prior gestations without antithrombotic therapy.47 These preliminary results are very encouraging. However, the optimal dosage of LMWH is yet unknown and should be determined by prospective randomized trial in order to maximize successful gestational outcome. LIVE-ENOX is a multi-centre prospective randomized trial, conducted in Israel, comparing enoxaparin 40 mg once daily and 80 mg (40 mg bid) in 180 women with

316 B. Brenner

Table 2. Placental vascular complications associated with thrombophilia.

Antithrombin III deficiency Protein C deficiency Protein S deficiency Dysfibrinogenaemia APC resistance Factor V Leiden MTHFR 677tt HHC Factor II G20210a Antiphospholipid syndrome Combined defects

Miscarriages

IUFD

Pre-eclampsia

Placental abruption

þþ þþ þþ þþ þþ þþ þ þ þ þþ þþ

þþ þþ þþ þ þþ þþ þ þ þ þþ þþ

þ þ þ

þ þ

þþ þþ þ þþ þ þþ þþ

þ þþ þ þþ þþ þþ þþ

IUFD, intra-uterine fetal death. Degrees of association: þ, possible association; þ þ , established association.

thrombophilia and RFL. Enrolment was finalized and the results are expected in the first quarter of 2003. Whether monitoring of anti-Xa levels is of value is still unknown. However, levels of 0.2 – 0.6 IU/ml, 3 hours post-injection, are expected in pregnant women who receive a prophylactic dose of LMWH. The role of aspirin, if any, in the setting of thrombophilia and vascular gestational abnormalities remains to be confirmed. In patients with antiphospholipid syndrome, or in those with combined thrombophilia, aspirin is given along with LMWH. However, whether aspirin has an added benefit to heparin or LMWH alone has not been evaluated.

SUMMARY Pregnancy is uneventful in the majority of women with inherited thrombophilia. However, the risks for miscarriage, IUFD, placental abruption and severe IUFD are increased in carriers of thrombophilia. Why certain women with thrombophilia present Practice points † women with recurrent miscarriages should be evaluated for thrombophilia † women with IUFD should be evaluated for thrombophilia † due to physiological changes in coagulation during pregnancy and puerperium, it is advisable to perform thrombophilia testing 3 months after delivery or pregnancy termination † women who—following recurrent pregnancy loss or IUFD—are found to harbour thrombophilia should, on subsequent pregnancy, be offered participation in randomized clinical trials or should be treated with LMWH on an individual basis

Inherited thrombophilia and pregnancy loss 317

Research agenda Current research in this field is already dealing with a number of aspects. † verification of the potential associations of the various genetic thrombophilias with gestational vascular pathologies by case – control, cohort and crosssectional studies is rapidly emerging (Table 2) † as 30 –50% of vascular gestational pathologies cannot be accounted for by thrombophilia, currently under investigation is the question of whether novel genetic and acquired thrombophilia does play a role. Recent observations claim that polymorphisms at the thrombomodulin and endothelial protein C receptor genes48,49, PAI-1 4G/4G polymorphism50, and low levels of factor XII51 and protein Z52 may be associated with RFL. In addition, circulating microparticles derived from platelets, monocytes and endothelial cells can be identified by flow cytometry. Several studies have recently suggested a role for microparticles in women with RFL53 † in view of the potential association of thrombophilia and RFL, and the high prevalence of thrombophilia in Caucasian populations, issues of screening are raised. As thrombophilia work-up is currently elaborate and costly, screening tests are highly warranted. One such potential assay is the protein C global test, which, in a preliminary study, was abnormal in 70% of 61 women with RFL compared to only 11% of 60 controls.54 Furthermore, the test identified 15/29 (52%) of women with RFL who did not have any thrombophilic defect † the pathogenetic mechanisms responsible for placental vascular pathologies in women with thrombophilia have not been fully elucidated. Moreover, it is yet unknown why only some women with thrombophilia express vascular gestational pathologies while others do not. It is possible that this may relate to local factors affecting coagulation, fibrinolysis and vascular tone at the level of placental vessels † currently our group is studying the local expression of tissue factor, tissue factor pathway inhibitor, thrombomodulin and EPC-R in placental trophoblasts from normal pregnancies and from women with gestational vascular pathologies † insight into the mechanism leading to fetal loss has been obtained from gene targeting of coagulation factors in mice (see Chapter 3 in this issue). Emerging data suggest the presence of two mechanisms: one involving coagulation and thrombosis and the other relating to increased apoptosis of placental trophoblasts55 † finally, the role of antithrombotic therapeutic modalities deserves prospective clinical trials—several of which are currently ongoing—in order to improve outcome for a large population of women who currently experience poor gestational outcome † because novel inherited and acquired prothrombotic abnormalities have been identified, the question of prophylaxis in women with RFL who do not have a specific thrombophilic defect is currently often addressed by clinicians and should be answered by prospective clinical trials. A trial comparing low-dose aspirin and enoxaparin 40 mg/day in women with recurrent first- or unexplained second-trimester pregnancy loss without thrombophilia is currently underway

318 B. Brenner

with fetal loss is still unknown but may be related to combination with another inherited or acquired prothrombotic risk, either systemic or operating locally at the placental level. Fibrin deposition and infarction are found in the placentae of women with thrombophilia and poor gestational outcome. Reports on prophylaxis with LMWH and UFH, with or without aspirin, suggest a benefit in women with thrombophilia and previous poor pregnancy outcome. Controlled trials presently underway will hopefully define the role of antithrombotics on gestational outcome in women with thrombophilia.

REFERENCES 1. Salafia CM, Minior VK, Pezzullo JC et al. Intrauterine growth restriction in infants of less than thirty-two weeks’ gestation: associated placental pathologic features. American Journal of Obstetrics and Gynecology 1995; 173: 1049–1057. 2. Cook CL & Pridham DD. Recurrent pregnancy loss. Current Opinion in Obstetrics and Gynecology 1995; 7: 357–366. 3. Clifford K, Rai R, Watson H & Regan L. An informative protocol for the investigation of recurrent miscarriage: preliminary experience of 500 consecutive cases. Human Reproduction 1994; 9: 1328–1332. 4. Hatasaka HH. Recurrent miscarriage: epidemiologic factors, definitions, and incidence. Clinical Obstetrics and Gynecology 1994; 37: 625– 634. 5. Raziel A, Arieli S, Bukovsky I et al. Investigation of the uterine cavity in recurrent aborters. Fertility and Sterility 1994; 62: 1080–1082. 6. Sanson BJ, Friederich PW, Simioni P et al. The risk of abortion and stillbirth in antithrombin, protein C, and protein S-deficient women. Thrombosis and Haemostasis 1996; 75: 387 –388. 7. Raziel A, Komberg Y, Friedler S et al. Hypercoagulable thrombophilic defects and hyperhomocysteinemia in patients with recurrent pregnancy loss. American Journal of Reproductive Immunology 2001; 45: 65– 71. 8. Gris JC, Quere I, Monpeyroux F et al. Case– control study of the frequency of thrombophilic disorders in couples with late feotal-loss and no thrombotic antecedent—the Nimes Obstetricians and Haematologists Study 5 (NOHA5). Thrombosis and Haemostasis 1999; 81: 891–899. 9. Haverkate F & Samama M. Familial dysfibrinogenemia and thrombophilia. Report on a study of the SSC Subcommittee on Fibrinogen. Thrombosis and Haemostasis 1995; 73: 151– 161. 10. Ridker PM, Miletich JP, Buring JE et al. Factor V Leiden mutation as a risk factor for recurrent pregnancy loss. Annals of Internal Medicine 1998; 128: 1000– 1003. 11. Rey E, Kahn SR, David M, & Shrier I. Thrombophilic disorders and fetal loss: a meta-analysis by type and timing of fetal loss. Lancet, in press. 12. Grandone E, Margaglione M, Colaizzo D et al. Factor V Leiden is associated with repeated and recurrent unexplained fetal losses. Thrombosis and Haemostasis 1997; 77: 822–824. 13. Brenner B, Sarig G, Weiner Z et al. Thrombophilic polymorphisms in women with fetal loss. Thrombosis and Haemostasis 1999; 82: 6 –9. 14. Sarig G, Hoffman R & Younis J. Thrombophilia is common in women with pregnancy loss and is associated with late pregnancy wastage. Fertility and Sterility 2002; 77: 342–347. 15. Dizon-Townson DS, Kinney S, Branch DW & Ward K. The factor V Leiden mutation is not a common cause of recurrent miscarriage. Journal of Reproductive Immunology 1997; 34: 217–223. 16. Rai R, Shlebak A, Cohen H et al. Factor V Leiden and acquired activated protein C resistance among 1000 women with recurrent miscarriage. Human Reproduction 2001; 16: 961–965. 17. Roque H, Paidas M, Rebarber A et al. Maternal thrombophilia is associated with second and third trimester fetal death. American Journal of Obstetrics and Gynecology 184; pp S27, abst 0060. 18. Younis JS, Brenner B, Ohel G et al. Activated protein C resistance and factor V Leiden mutation can with first-as well as second-trimester recurrent pregnancy loss. American Journal of Reproductive Immunology 2000; 43: 31–35. 19. Wramsby ML, Sten-Linder M & Bremme K. Primary habitual abortions are associated with high frequency of factor V Leiden mutation. Fertility and Sterility 2000; 74: 987– 991. 20. Meinardi JR, Middeldorp S, de Kam PJ et al. Increased risk for fetal loss in carriers of the factor V Leiden mutation. Annals of Internal Medicine 1999; 130: 736 –739. 21. Tormene D, Simioni P, Prandoni P et al. The risk of fetal loss in family member of probands with factor V Leiden mutation. Thrombosis and Haemostasis 1999; 82: 1237–1239.

Inherited thrombophilia and pregnancy loss 319 22. Eichinger S, Weltermann A, Philipp K et al. Prospective evaluation of hemostatic system activation and thrombin potential in healthy pregnant women with and without factor V Leiden. Thrombosis and Haemostasis 1999; 82: 1232– 1236. 23. Clark P, Brennand J, Conkie JA et al. Activated protein C sensitivity, protein C, protein S and coagulation in normal pregnancy. Thrombosis and Haemostasis 1998; 79: 1166–1170. 24. Kjelberg U, Andersson NE, Rosen S et al. APC resistance and other haemostatic variables during pregnancy and puerperium. Thrombosis and Haemostasis 1999; 81: 527–531. 25. Tal J, Schliamser LM, Leibovitz Z et al. A possible role for activated protein C resistance in patients with first and second trimester pregnancy failure. Human Reproduction 1999; 14: 1624–1627. 26. Brenner B, Mandel H, Lanir N et al. Activated protein C resistance can be associated with recurrent fetal loss. British Journal of Haematology 1997; 97: 551 –554. 27. Mercier E, Quere I, Mares P & Gris JC. Primary recurrent miscarriages: anti-b2-glycoprotein I igG antibodies induces an acquired activated protein C resistance that can be detected by the modified activated protein C resistance test. Blood 1998; 92: 2993– 2994. 28. Souza SS, Ferriani RA, Pontes AG et al. Factor V Leiden and factor II G20210A mutations in patients with recurrent abortion. Human Reproduction 1999; 14: 2448–2450. 29. Martinelli I, Taioli E, Cetin I et al. Mutations in coagulation factors in women with unexplained late fetal loss. New England Journal of Medicine 2000; 343: 1015–1018. 30. de Vries JIP, Dekker GA, Huijgens PC et al. Hyperhomocysteinaemia and protein S deficiency in complicated pregnancies. British Journal of Obstetrics and Gynaecology 1997; 104: 1248–1254. 31. Goddijn-Wessel TA, Wouters MG, van der Molen EF et al. Hyper-homocysteinemia: a risk factor for placental abruption or infarction. European Journal of Obstetrical Gynecology and Reproductive Biology 1996; 66: 23–29. 32. Coumans AB, Huijgens PC, Jakobs C et al. Haemostatic and metabolic abnormalities in women with unexplained recurrent abortion. Human Reproduction 1999; 14: 211 –214. 33. Nelen WLDM, Blom HJ, Steegers EAP et al. Hyperhomocysteinemia and recurrent early pregnancy loss: a meta-analysis. Fertility and Sterility 2000; 74: 1196–1199. 34. Zoller B, Berntsdotter A, Garcia de Frutos P & Dahlback B. Resistance to activated protein C as an additional genetic risk factor in hereditary deficiency of protein S. Blood 1995; 85: 3518–3523. 35. Brenner B, Zivelin A, Lanir N et al. Venous thromboembolism associated with double heterozygosity for R506Q mutation of factor V and for T298M mutation of protein C in a large family of a previously described homozygous protein C deficient newborn with massive thrombosis. Blood 1996; 88: 877– 880. 36. Mandel H, Brenner B, Berant M et al. Coexistence of hereditary homocysteinuria and factor V Leiden— effect on thrombosis. New England Journal of Medicine 1996; 334: 763–768. 37. Brenner B, Vulfsons SL, Lanir N & Nahir M. Coexistence of familial antiphospholipid syndrome and factor V Leiden—impact on thrombotic diathesis. British Journal of Haematology 1996; 94: 166– 167. 38. Preston FE, Rosendaal FR, Walker ID et al. Increased fetal loss in women with heritable thrombophilia. Lancet 1996; 348: 913 –916. 39. Mousa HA & Alfirevic Z. Do placental lesions reflect thrombophilia state in women with adverse pregnancy outcome? Human Reproduction 2000; 15: 1830– 1833. 40. Dizon-Townson DS, Meline L, Nelson LM et al. Fetal carriers of the factor V Leiden mutation are prone to miscarriage and placental infarction. American Journal of Obstetrics and Gynecology 1997; 177: 402–405. 41. Eitzman DT, Westrick RJ, Bi X et al. Lethal perinatal thrombosis in mice resulting from the interaction of tissue factor pathway inhibitor deficiency and factor V Leiden. Circulation 2002; 105: 2139–2142. 42. Kupferminc MJ, Eldor A, Steinman N et al. Increased frequency of genetic thrombophilias in women with complications of pregnancy. New England Journal of Medicine 1999; 340: 9– 13. 43. Grandone E, Margaglione M, Colaizzo D et al. Genetic susceptibility to pregnancy-related venous thromboembolism: roles of factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations. American Journal of Obstetrics and Gynecology 1998; 179: 1324–1328. 44. Many A, Schreiber I, Rosner S et al. Pathologic features of the placenta in women with severe pregnancy complications and thrombophilia. Obstetrics and Gynecology 2001; 98: 1041–1044. 45. Sanson BJ, Lensing AW, Prins MH et al. Safety of low-molecular-weight heparin in pregnancy: a systematic review. Thrombosis and Haemostasis 1999; 81: 668 –672. 46. Gris JC, Neveu S, Tailland ML et al. Use of low-molecular weight heparin (Enoxaparin) or of a phenforminlike substance (Moroxydine Chloride) in primary early recurrent aborters with an impaired fibrinolytic capacity. Thrombosis and Haemostasis 1995; 73: 362 –367. 47. Brenner B, Hoffman R, Blumenfeld Z et al. Gestational outcome in thrombophilic women with recurrent pregnancy loss treated by enoxaparin. Thrombosis and Haemostasis 2000; 83: 693–697. 48. Nakabayashi M, Yamamoto S & Suzuki K. Analysis of thrombomodulin gene polymorphism in women with severe early-onset preeclampsia. Seminars in Thrombosis and Hemostasis 1999; 25: 473–479.

320 B. Brenner 49. Franchi F, Biguzzi E, Cetin I et al. Mutations in the thrombomodulin and endothelial protein C receptor genes in women with late fetal loss. British Journal of Haematology 2001; 114: 641–646. 50. Glueck CJ, Kupferminc MJ, Fontaine RN et al. Genetic hypofibrinolysis in complicated pregnancies. Obstetrics and Gynecology 2001; 97: 44–48. 51. Yamada H, kato EH, Ebina Y et al. Factor XII deficiency in women with recurrent miscarriage. Gynecologic and Obstetric Investigation 2000; 49: 80– 83. 52. Gris JC, Quere I, Dechaud H et al. High frequency of protein Z deficiency in patients with unexplained early fetal loss. Blood 2002; 99: 2606–2608. 53. Laude I, Rongieres-Bertrand C, Boyer-Neumann C et al. Circulating procoagulant microparticles in women with unexplained pregnancy loss: a new insight. Thrombosis and Haemostasis 2001; 85: 18–21. 54. Sarig G, Lanir N, Hoffman R & Brenner B. Protein C global assay in the evaluation of women with idiopathic pregnancy loss. Thrombosis and Haemostasis 2002; 8: 32 –36. 55. Isermann B, Hendrickson SB, Hutley K et al. Tissue-restriced expression of thrombomodulin in the placenta rescues thrombomodulin-deficient mice from early lethality and reveals a secondary developmental block. Development 2001; 128: 827–838.