University of Groningen Pregnancy-related thrombosis and fetal loss in women with thrombophilia Folkeringa, Nienke

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Pregnancy-related thrombosis and fetal loss in women with thrombophilia Folkeringa, Nienke

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Pregnancy-related thrombosis and fetal loss

in women with thrombophilia

Nienke Folkeringa

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Financially support by Astellas Pharma, AstraZeneca, Baxter, Bayer Schering Pharma, Boehringer Ingelheim, CSL Behringer, Federatie van Nederlandse Trombosediensten, GlaxoSmithKline, LEO Pharma, Medical Dynamics, Roche Diagnostics, University of Groningen, Sanofi-Aventis and Wyeth for the publication of this thesis is gratefully acknowledged.

Colofon

Folkeringa, N.

Pregnancy-related thrombosis and fetal loss in women with thrombophilia Thesis University Medical Centre Groningen, with summary in Dutch ISBN 978-90-367-3636-7

All rights are reserved. No part of this publication may be reproduced, stored in a re- trieval system, or transmitted, in any form or by any means - electronic, mechanical, photocopy, recording or otherwise - without prior written permission of the author or the corresponding journal.

Cover design and lay-out by Marcel Zinger, Groningen Printed by Gildeprint Drukkerijen, Enschede

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RIJKSUNIVERSITEIT GRONINGEN

Pregnancy-related thrombosis and fetal loss

in women with thrombophilia

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. F. Zwarts

in het openbaar te verdedigen op woensdag 18 maart 2009 om 14.45 uur

door

Nienke Folkeringa geboren op 9 oktober 1975

te Arnhem

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Beoordelingscommissie: Prof. dr. M.H. Prins Prof. dr. H.R. Büller Prof. dr. P.P. van den Berg

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Paranimfen: Evelien Hoven Ingrit Thiele

Loes Bosboom-Arkes

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1 1 Introduction

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Introduction

Haemostasis is a complex process that maintains the integrity of the vessel wall and the fluidity of blood. Plasma proteins, blood platelets, the vessel wall and blood flow velocity are involved. Vessel wall injury is the physiological trigger of haemostasis.

The response is restricted to the site of injury by natural inhibitors of coagulant proteins and platelets. An excess of fibrin is removed by the generation of the proteolytic enzyme plasmin, as soon as it is appropriate. Finally, migration and proliferation of vessel wall cells are induced to repair the vessel wall. Disturbances of this process may result in either bleeding or thrombosis. Pregnancy is a challenge of haemostasis, particularly by the risk of bleeding at time of delivery. It may explain the physiological changes observed during pregnancy.¹These include increasing plasma levels of procoagulant proteins and adhesion proteins (i.e. fibrinogen and von Willebrand factor), decreasing plasma levels of anticoagulant proteins and a decline of fibrinolytic potency.^1-8Moreover, stickiness of platelets increases.^1,2 Although these changes protect against maternal bleeding, they will result in a state of hypercoagulability.⁹As a consequence, pregnancy is associated with an increased risk of thrombosis, still an important cause of maternal morbidity and mortality in the Western world.¹⁰

In pregnant women, the risk of venous thromboembolism is five-to-six fold higher than non-pregnant woman of the same age.¹¹The incidence of venous thromboembolism in the general female population ranges from 0.5-1 in 1000 deliveries.¹¹ Predilection sites of thrombosis during pregnancy are deep leg veins and iliac veins, may be due to venous stasis in addition to hypercoagulability.^12,13However, venous thrombosis occurs more frequently after delivery than in pregnancy.¹⁴This is plausible, considering that it takes time before the abovementioned changes of haemostatic compounds have been recovered, while the risk of bleeding has already returned to its baseline level.

It is likely that pregnancy induced hypercoagulability may also result in thrombosis of placenta vessels and thereby contributes to obstetrical complications, like fetal loss.^15,16Fetal loss occurs frequently. About 20% of conceiving women experience at least one fetal loss, whereas recurrent fetal loss occurs in approximately 5%

of these women.¹⁷Fetal loss is multicausal. Chromosomal abnormalities of either parent(s) or fetus, uterus anomalies, endocrine disorders and infections are known causes.¹⁸Still, approximately 50% of all fetal losses remain unexplained.¹⁹

Women with thrombophilia, an inherited or acquired coagulation defect that is associated with an increased risk of venous thrombosis, are more prone to develop thrombotic events related to pregnancy. Women with multiple thrombophilic defects are likely at higher risk than women with a single defect. Therefore, it is plausible that thromboprophylaxis during pregnancy and/or puerperium may particularly be of benefit in women with thrombophilia in order to reduce the risk of venous thrombosis and maybe fetal loss.

Pregnancy-related thrombosis and fetal loss in women with thrombophilia

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Thrombophilia

After Egeberg introduced the term thrombophilia in 1965, many defects of coagulation and fibrinolysis have been recognized as risk factors for thrombosis.²⁰Of the es- tablish ed inherited thrombophilic defects, deficiencies of antithrombin, protein C and protein S are the strongest risk factors for venous thromboembolism.²¹Factor V Leiden, prothrombin G20210A, as well as often mixed inherited and acquired elevated levels of factor VIII, factor IX, factor XI, thrombin activated fibrinolysis inhibitor (TAFI) and hyperhomocysteinaemia are mild thrombotic risk factors.^22-34Elevated levels of factor VIII or TAFI, and hyperhomocysteinaemia have also been associated with an increased risk of arterial thrombosis.^26,28,32Antiphospholipid antibodies, like lupus anticoagulant and anticardiolipin antibodies, are acquired thrombophilic defects. The clinical entity that consists of these antibodies, venous and/or arterial thrombosis and/or obstetrical complications is known as the antiphospholipid syndrome.³⁵Together, thrombophilic defects have been reported in at least 15% of the Western population and in up to 50% of patients with venous thrombosis.¹⁶

Women with thrombophilia are at higher risk of pregnancy related venous thrombosis. Approximately 50% of these events have been associated with thrombophilic defects.³⁶The reported risks for individual thrombophilic defects are summarized in Table 1. Women with combinations of thrombophilic defects may be at higher risk than women with a single defect, but there is hardly data available on this issue.

Since 1996, several studies have been performed, that addressed the association of fetal loss with thrombophilia.^42,44-49In a number of these studies, early and late fetal loss rates were separately reported, considering that there are differences in patho- physiology between early and late fetal loss. Relative risks of early and late fetal loss in women with various thrombophilic defects compared to controls are presented in Table 2. It should be noted that different definitions of early and late fetal loss were used in these studies. The cut-off gestational age at time of fetal loss ranged from 10 weeks to 28 weeks, whereas fetal loss at gestational age less than 20-22 weeks and Table 1. Incidence of pregnancy related venous thrombosis in women with thrombophilia

%

Antithrombin deficiency 3-47 ^37-40

Protein C deficiency 2-19 ^37-40

Protein S deficiency 2-27 ^37-40

Factor V Leiden (heterozygous) 2.1 ⁴¹

Prothrombin G20210A (heterozygous) 2.4 ⁴²

Elevated factor VIII levels (>150 IU/dL) 1.3 ²⁶

Mild hyperhomocysteinaemia 0.45 ⁴³

Elevated TAFI levels unknown

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more than 20-22 weeks are classified early and late, respectively, according to the criteria of the World Health Organization.¹⁷Although one might expect a higher risk of fetal loss in women with multiple thrombophilic defects than in women with a single thrombophilic defect, as shown in one study,⁴⁶this assumption has not been established yet.

Thromboprophylaxis

It is still a matter of debate whether thromboprophylaxis should be applied during pregnancy and/or puerperium in women with prior venous thromboembolism, either related to pregnancy or not. Whether thromboprophylaxis during pregnancy and/or puerperium should be considered in women with symptomatic or even asymptomatic thrombophilia is also a controversial issue. Anticoagulant treatment in order to reduce the risk of fetal loss in women with thrombophilia has been suggested, although available evidence is not convincing. The major concerns about the use of anticoagulant drugs during pregnancy are efficacy of the regimes, maternal and fetal risks, and management around the time of delivery.

Anticoagulant therapy during pregnancy can reduce the risk of venous thrombosis.⁵⁰Although this risk is low, venous thrombosis is the second cause of maternal mortality in the Netherlands⁵¹and can cause serious morbidity due to deep venous insufficiency (i.e. post-thrombotic syndrome).⁵²Furthermore, the recurrence rate of venous thromboembolism during pregnancy in women with previous idiopathic venous thrombosis is 2.7%-5.6% and 2.2%-4.5% in women with previous pregnancy related thrombosis.^{53, 54}Current guidelines for primary and secondary thromboprophylaxis in pregnant women are mainly based on experts’ opinions. Issues to be re- solved, apart from efficacy and safety, include the dosage of anticoagulant drugs, the Pregnancy-related thrombosis and fetal loss in women with thrombophilia

Table 2. Relative risk of early and late fetal loss in women with thrombophilia

Early fetal loss Late fetal loss

Antithrombin deficiency 1.5-1.7 ^44,46 5.2-7.6 ^44,46,48

Protein C deficiency 1.4 -2.3 ^44,46,48 2.3-3.1 ^44,46,48

Protein S deficiency 1.3-3.6 ^44,46,48 3.3-20.1 ^44,46,48

Factor V Leiden (heterozygous) 1.0-2.0 ^45,47,48 1.4-3.3 ^45,47,48

Prothrombin G20210A (heterozygous) 1.3-2.5 ^42,48 2.3-2.7 ⁴⁸

Elevated factor VIII levels (>150 IU/dL) 1.2 ⁴⁹ unknown

Hyperhomocysteinaemia 0.8-6.3 ^48,49 1.0-1.3 ⁴⁸

Elevated TAFI levels unknown unknown

Combined defects 0.8 ⁴⁶ 14.3 ⁴⁶

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need for dose adjustments, stratification for risk of venous thrombosis, and start and duration of thromboprophylaxis. An individualized approach may be preferred.^55,56 Considering uteroplacental thrombosis as a cause of fetal loss, it is rational to use anticoagulant drugs for its prevention. This is already common practice in pregnant women with the antiphospholipid syndrome.⁵⁷Some studies suggested a benefit of thromboprophylaxis in women with thrombophilia and (recurrent) fetal loss, but most of these studies had major methodological limitations. As fetal loss is relatively common, the need for treatment also depends on the prognosis of a successful pregnancy outcome after a first fetal loss in women with thrombophilia. Because available evidence from previous studies is not convincing, it does not justify anti coagulant routine treatment in these women.^58,59,60

Aim of this thesis

This thesis addresses the absolute risk of pregnancy related venous thrombo - embolism and fetal loss in women with thrombophilic defects, either single or multiple. The presented studies were performed to assess:

1 The absolute risk of venous thromboembolism during pregnancy and puerperium in women with hereditary deficiencies of antithrombin, protein C or protein S, and the contribution of other concomitant thrombophilic defects to this risk.

2 The absolute risk of fetal loss in women with hereditary deficiencies of either antithrombin, protein C or protein S, and the contribution of other concomitant thrombophilic defects to this risk.

3 The effects of thromboprophylaxis during pregnancy on fetal loss rates in women with hereditary deficiencies of either antithrombin, protein C or protein S.

4 The risk of a second fetal loss in carriers and non-carriers of either factor V Lei- den or prothrombin G20210A after a first unexplained fetal loss.

5 The absolute risk of venous and arterial thromboembolism associated with high TAFI levels, and the contribution of other concomitant thrombophilic defects to this risk.

6 The absolute risk of fetal loss in women with high TAFI levels.

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41 Middeldorp S, Henkens CMA, Koopman MMW, van Pampus ECM, Hamulyák K, van der Meer J, Prins MH, Buller HR. The incidence of venous thromboembolism in family members of patients with factor V Leiden mutation and venous thrombosis. Ann Intern Med 1998; 128: 15-20.

42 Bank I, Libourel EJ, Middeldorp S, van Pampus ECM, Koopman MMW, Hamulyák K, Prins MH, van der Meer J, Büller HR. Prothrombin G20210A mutation: a mild risk factor for venous thromboembolism but not for arterial thrombotic disease and pregnancy-related complications in a family study. Arch Intern Med 2004; 164: 1932-1937.

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45 Meinardi JR, Middeldorp S, de Kam PJ, Koopman MM, van Pampus ECM, Hamulyák K, Prins MH, Büller HR, van der Meer J. Increased risk for fetal loss in carriers of the factor V Leiden mutation. Ann Intern Med 1999; 130: 736-739.

46 Preston FE, Rosendaal FR, walker ID, Briet E, Berntorp E, Conard J, Fontcuberta J, Makris M, Mariani G, Noteboom W, Pabinger I, Legnani C, Scharrer I, Schulman S, van der Meer FJM. Increased fetal loss in women with heritable thrombophilia. Lancet 1996; 348: 913-916.

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49 Middeldorp S, van der Poel MH, Bank I, Hamulyák K, Libourel EJ, Koopman MMW, Prins MH, van der Meer J, Buller HR. Unselected women with elevated levels of factor VIII:c or homocysteine are not at increased risk for obstetric complications. Thromb Haemost 2004; 92: 787-790.

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55 Bates SM, Greer IA, Pabinger I, Sofaer S, Hirsh J. Venous thromboembolism, thrombophilia, antithrombotic therapy, and pregnancy: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest 2008; 133: 844S-886S.

56 NVOG. Diepveneuze trombose, longembolie en zwangerschap. Richtlijn 2003.

57 Bates SM, Greer IA, Pabinger I, Sofaer S, Hirsh J. Venous thromboembolism, thrombophilia, anti - thrombotic therapy, and pregnancy: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest 2008; 133: 844S-886S.

58 Rai R, Backos M, Elgaddal S, Shlebak A, Regan L. Factor V Leiden and recurrent miscarriage- prospective outcome of untreated pregnancies. Hum Reprod 2002; 17: 442-445.

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2 2 High risk of pregnancy-related venous thrombo embolism in women with multiple

thrombophilic defects

N. Folkeringa J.L.P. Brouwer F.J. Korteweg N.J.G.M. Veeger J.J.H.M. Erwich J. van der Meer

British Journal of Haematology 2007; 138: 110-116

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Abstract

Pregnancy is associated with an increased risk of venous thromboembolism, which probably varies according to the presence of single or multiple thrombophilic defects.

This retrospective family cohort study assessed the risk of venous thromboembolism during pregnancy and puerperium, and the contribution of concomitant thrombophilic defects in families with hereditary antithrombin, protein C or protein S deficiencies. Probands were excluded. Of 222 female relatives, 101 were deficient and 121 nondeficient. Annual incidences of venous thromboembolism were 1-76% in deficient women versus 0.19% in non-deficient women [adjusted relative risk (RR) 11-9;

95% confidence interval (CI), 3.9 – 36.2]. Other single and multiple thrombophilic defects increased the risk in deficient women from 1.55% to 2.14% and 2.92%, and in non-deficient women from 0.16% to 0.09% and 0.54% respectively. Deficient women were at lower risk (1.37%; 0.80 – 2.19) than deficient women that had never been pregnant (2.96%; 1.53 – 5.18); RR 0-5 (0.2 – 0.99). This difference was due to the predomi- nance of events related to oral contraceptives in deficient women that had never been pregnant (75%), while 71% of events in deficient women that had had at least one pregnancy were pregnancy-related. In conclusion, women with hereditary deficiencies of antithrombin, protein C or protein S are at high risk of pregnancy-related venous thromboembolism. This risk is increased by multiple additional thrombophilic defects.

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Introduction

Venous thromboembolism in healthy women during pregnancy or puerperium is rare, but it is still an important cause of maternal morbidity and mortality in the Western world.¹Its incidence in the general female population ranges from 0-5 to 1 in 1000 deliveries.^2,3The risk of venous thromboembolism in pregnant women is five times higher than in non-pregnant woman of the same age. It predominantly occurs in the third trimester and puerperium.⁴

It is plausible that the increased risk of venous thromboembolism is related to venous stasis and a state of hypercoagulability, as a result of physiological changes in blood coagulation and fibrinolysis during pregnancy.⁵Consequently, a higher risk has been found in pregnant women with pre-existent thrombophilic defects.^6-8Of these, hereditary deficiencies of antithrombin, protein C and protein S are strong risk factors for venous thromboembolism.^9-12Recently, we demonstrated that the risk in deficient subjects depends on the frequently observed concomitance of other, milder thrombophilic defects.¹³It is likely that multiple deficiencies or defects, in addition to physiological changes, will particularly enhance the risk of venous thromboembolism during pregnancy and puerperium. To test this hypothesis, a sufficient number of deficient women is required to assess interactions between rare deficiencies and more prevalent thrombophilic defects. Therefore, we analysed data from a retrospective family cohort study, which was designed to assess the contribution of currently known thrombophilic defects to the absolute risk of venous thromboembolism associated with hereditary deficiencies of antithrombin, protein C or protein S.

Materials and methods

Study population

The original study comprised three cohorts of families with hereditary deficiencies of antithrombin, protein C and protein S, respectively.¹³Probands were consecutive patients with documented venous thromboembolism in whom one of these deficiencies was demonstrated. They were referred with clinically suspected venous thrombo embolism to the thrombosis out-patient clinic of our hospital over a period of 12 years. First-degree relatives that were 15 years of age or older were identified by pedigree analysis. As the number of antithrombin-deficient probands was small, second degree relatives from a deficient parent were also identified. Written informed consent was obtained from all participants. Detailed information about episodes of venous thromboembolism, exposure to external risk factors for thrombosis, obstetric history and anticoagulant treatment was collected by the physicians of our out-patient clinic using a validated questionnaire and reviewing medical records.¹⁴Blood samples for thrombophila testing were taken after clinical data had been collected.

Thrombophilia and pregnancy-related thrombosis

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In addition to the index deficiency, tests included prothrombin G20210A, factor V Lei- den, increased levels of factors VIII, IX and XI, lupus anticoagulant and hyperhomocysteinaemia. For convenience, additional abnormalities are termed hereafter as

‘thrombophilic defects’. The study was approved by the institutional review board of our hospital. The present study analysed the overall risk of venous thromboembolism, particularly the risk of pregnancy-related venous thromboembolism in female relatives of reproductive age.

Definitions

Venous thromboembolism was considered established if proximal deep vein thrombosis was confirmed by compression ultrasound or venography (not in pregnancy), and pulmonary embolism by ventilation/perfusion lung scanning, spiral computed tomography (CT) scanning or pulmonary angiography (not in pregnancy), or when the patient had received full dose unfractionated heparin and a vitamin K antagonist for at least 3 months without objective testing at a time when current techniques were not available. Venous thromboembolism was classified as provoked if it had occurred within 3 months of exposure to one or more external risk factors, including surgery, trauma, immobilisation for more than 7 d, use of oral contraceptives, pregnancy, puerperium and malignancy. In the absence of these risk factors, it was considered spontaneous.

Laboratory studies

Antithrombin activity (Coatest; Chromogenix, Mo¨lndal, Sweden) and protein C activity (Berichrom Protein C; Dade Behring, Marburg, Germany) were measured by chromogenic substrate assays. Protein C and total protein S and antigen levels were measured by enzyme-linked immunosorbent assay (ELISA) (DAKO, Glostrup, Den- mark). Normal ranges (mean ± 2 SD) were determined in 393 healthy blood donors, who had no (family) history of venous thromboembolism and were not pregnant, and had not used oral contraceptives for at least three months. Antithrombin deficiency was defined by levels of antithrombin activity below the lower limit of its normal range (<74 IU/dl), protein C deficiency type I and type II were defined by reduced levels of either protein C antigen (<63 IU/dl) and/or activity (<64 IU/dl). Protein S deficiency type I was defined by lowered total protein S antigen levels (<67 IU/dl).

Deficiencies were considered inherited if they were confirmed by measuring a second sample that was collected 3 months later and were demonstrated in at least two family members. Relatives with acquired conditions were excluded. If there was a discrepancy between the results of the two tests, a third sample was tested. A deficiency was considered acquired through use of oral contraceptives or pregnancy, unless it was confirmed at least 3 months after withdrawal of oral contraceptives or delivery, respectively. Relatives with abnormal liver function tests were only evaluable if deficiencies were established at repeated measurements after recovery.

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Factor V Leiden and the prothrombin G20210A mutation were demonstrated by poly- merase chain reaction.^15,16Coagulant activity of Factors VIII (FVIII:C), IX (FIX:C) and XI (FXI:C) were measured by one-stage clotting assays (Amelung GmbH, Lemgo, Ger- many) and were considered increased at levels above 150 IU/dl.^17-19Fasting and post- methionine-loading levels of homocysteine were measured by high performance liquid chromatography.²⁰Hyperhomocysteinaemia was defined as a fasting level above 18.5 µmol/l and/or a post-loading level above 58.8 µmol/l, as described in the Dutch population.²¹In probands and relatives with venous thromboembolism, blood samples were collected at least 3 months after this event had occurred. If, at that time, subjects that were still receiving acenocoumarol, a short acting vitamin K anta gonist, samples were taken after this therapy had been interrupted for at least 2 weeks, meanwhile nadroparin was given subcutaneously.

Statistical analysis

We first compared the overall absolute risk of the first episode of venous thromboembolism in deficient and non-deficient female relatives at reproductive age (15–

45 years). Probands were excluded from analysis to avoid bias. Annual incidences were calculated by dividing the number of symptomatic relatives by the total number of observation years. Observation time was defined as the fertile period (from age 15 until 45 years), or from age 15 years until the first venous thromboembolic episode, or until the end of study. Second, we compared the risk of venous thromboembolism in deficient and non-deficient relatives that had had at least one pregnancy versus those who had never been pregnant. Third, we compared the incidence of pregnancy- related venous thromboembolism in deficient and non-deficient relatives. In these analyses, pregnancies after a prior episode of venous thromboembolism were not counted and women were considered as never having been pregnant when their first pregnancy occurred after a prior episode of venous thromboembolism, because the outcome of these pregnancies might have been influenced by thromboprophylaxis.

Pregnancies that ended in fetal loss or termination, or were ectopic, were not excluded from analysis.

The relative risk of venous thromboembolism was calculated from annual incidences. The 95% confidence intervals (95% CI) and P-values were computed using small sample statistics based on mid-P-value functions from the binomial proba- bility model.²²Random effects logistic regression was used to adjust for clustering of women in families. Continuous variables were expressed as mean values and standard deviations, and categorical data as counts and percentages. Differences between groups were evaluated by the Student’s t-test or Mann–Whitney U-test, depending on the normality of data for continuous data, and by Fisher exact test for categorical data. A two-tailed P-value of less than 0.05 was considered to indicate statistical sig- nificance. Statistical analyses were performed using SAS software, version 9.1 (SASInstitute inc., Cary, NC, USA).

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Results

Our family cohort consisted of 12 probands with antithrombin deficiency and their 185 relatives, 40 probands with protein C deficiency and their 277 relatives, and 39 probands with protein S deficiency and their 262 relatives. Of all 326 female relatives, 234 were alive and aged 15 years or older. Consent was refused or could not be obtained for geographic reasons in 12 of these relatives. The remaining 222 women were eligible, of whom 101 were deficient and 121 nondeficient. Their characteristics are summarised in Table 1. Of 222 relatives, 133 had been pregnant of whom 54 were deficient (162 pregnancies) and 79 were non-deficient (243 pregnancies). Mean parity was comparable in deficient and non-deficient women.

Twenty-nine of 101 deficient women (29%) and five of 121 non-deficient women (4%) had experienced venous thromboembolism before 45 years of age (Table 1). Mean age at time of the first episode of venous thromboembolism was 27 years in each group. Of 29 first episodes of venous thromboembolism in deficient women, 12 (41%) were related to pregnancy and 11 (38%) to use of oral contraceptives, compared to one of five and three of five episodes in non-deficient women respectively. Eight of 12 pregnancy-related episodes of venous thromboembolism in deficient women occurred during puerperium, as did the only episode of venous thromboembolism in nondeficient relatives.

Concomitant thrombophilic defects were demonstrated in 57% of deficient and in 70% of non-deficient relatives (Table 2). The prevalence’s of these defects were higher in both deficient and non-deficient women than reported in the normal population, except for high levels of factor IX and factor XI in deficient relatives. Lupus anticoagulant was not found. Differences between deficient and non-deficient women were not statistically significant.

The overall annual incidences of venous thromboembolism were 1.76 per 100 person-years (95% CI, 1.18–2.53) in deficient women and 0.19 per 100 person-years (95% CI, 0.06–0.45) in non-deficient women; the relative risk adjusted for clustering of women in families was 11.9 (95% CI, 3.9–36.2) (Table 3). In deficient women, these were 1.56 per 100 person-years (95% CI, 0.67–3.07) in the absence of concomitant thrombophilic defects, compared to 2.14 (95% CI, 1.14–3.66) and 2.92 per 100 person- years (95% CI, 1.17–6.01) when one, and more than one concomitant thrombophilic defects were present, respectively. Annual incidences were 0.16 (95% CI, 0.004–0.87), 0.09 (95% CI, 0.002–0.53) and 0.54 (95% CI, 0.11–1.58) per 100 person-years in nondeficient women with none, one, and more than one other defect respectively.

Deficient women that had had at least one pregnancy had a lower absolute risk of venous thromboembolism (1.37 per 100 person-years; 95% CI, 0.80–2.19) than deficient women that had never been pregnant (2.96; 95% CI, 1.53–5.18); relative risk 0.5 (95% CI, 0.2–0.99) (Table 4). A similar difference was observed in non-deficient women. Of 54 deficient women that had had at least one pregnancy, 24 had never used oral contraceptives, of whom eight (33%) had pregnancy-related thrombosis.

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Table 2. Prevalence of concomitant thrombophilic defects in deficient and non-deficient women.

Deficient Non-deficient

% (n tested) (n = 101) (n = 121) P-value

Prothrombin G20210A 6 (89) 9 (109) 0.42

Factor V Leiden 13 (90) 15 (110) 0.84

Factor VIII >150 IU/dl 36 (84) 42 (102) 0.45

Factor IX >150 IU/dl 9 (68) 13 (104) 0.47

Factor XI >150 IU/dl 9 (88) 16 (105) 0.20

Hyperhomocysteinaemia 16 (83) 19 (96) 0.69

No concomitant defect 43 (86) 30 (101)

1 concomitant defect 38 (86) 49 (101) 0.18

>1 concomitant defect 19 (86) 21 (101)

Expected prevalences in the normal population: prothrombin G20210A, 1–2%; factor V Leiden, 5%;

increased factor VIII level, 25%; increased factor IX level, factor XI level and hyperhomocystei - naemia, 5% each.

Table 1. Characteristics of deficient and non-deficient women.

(n = 101) (n = 121) P-value

Age at enrolment, mean (SD), years 47 (19) 49 (18) 0.26

Observation period, mean (SD), years 16 (10) 21 (11) <0.01

Women with at least one pregnancy, n (%) 54 (53) 79 (65) 0.08

Pregnancies prior to first venous thromboembolism, n 162 245 0.06

Parity, mean (SD) 1.6 (2) 2.0 (2) 0.06

Use of oral contraceptives at any time, n (%) 56 (55) 78 (64) 0.21

Women with venous thromboembolism, n (%) 29 (29) 5 (4) <0.01

Age at time of venous thromboembolism, mean (SD), years 27 (6) 27 (9) 0.75 Provoked, by

Pregnancy, n (%) 4* (14) 0 1.0

First trimester, n 1 0

Second trimester, n 1 0

Third trimester, n 1 0

Puerperium, n (%) 8 (27) 1 (20) 1.0

Oral contraceptives, n (%) 11 (38) 3 (60) 0.63

Surgery, trauma or immobility, n (%) 3 (10) 1 (20) 0.49

Spontaneous, n (%) 3 (10) 0 1.0

Protein levels, mean (SD), IU/dl

Antithrombin activity 52 (8) 101 (16)

Protein C activity 52 (19) 48 (12)

Protein S antigen 48 (13) 104 (23)

SD, standard deviation. *Trimester unknown in one woman.

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Twenty-seven of 47 deficient women that had never been pregnant had used oral contraceptives and in nine of them (33%) venous thrombo embolism was related to oral contraceptives. Table 5 shows the distribution of pregnancy-related venous thromboembolism in the separate antithrombin, protein C and protein S cohorts. Of all the

Table 3. Absolute risk of venous thromboembolism in deficient and non-deficient fertile women.

All women 101 121

Women with event, n 29 5

Observation period, years 1645 2598

Annual incidence, % (95% CI) 1.76 (1.18–2.53) 0.19 (0.06–0.45)

Adjusted relative risk (95% CI)* 11.9 (3.9–36.2)

No concomitant thrombophilic defect, n 37 31

1 concomitant thrombophilic defect, n 33 50

>1 concomitant thrombophilic defect, n 16 22

*Adjusted for clustering of women in families.

Table 4. Venous thromboembolism in women with at least one pregnancy versus women that had never been pregnant.

At least one Never At least one Never

pregnancy pregnant pregnancy pregnant

(n=54) (n=47) (n=79) (n=42)

Women with event, n 17 12 2 3

Observation period, years 1241 405 2169 430

Annual incidence, % (95% CI) 1.37 (0.80-2.19) 2.96 (1.53-5.18) 0.09 (0.01-0.33) 0.70 (0.14-2.04) Relative Risk (95% CI) <——— 0.5 (0.2-0.99) ———> <——— 0.1 (0.02-0.9) ———>

<——————— 14.9 (4.0–95.1) ———————>

<——————-— 4.2 (1.3–18.8) ———-————>

Pregnancy-related events, n (%) 12 (71) 0 1 (50) 0

Oral contraceptives related events, n (%) 2 (12) 9 (75) 0 3 (100)

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deficient women that had had at least one pregnancy, 22% experienced pregnancy- related mvenous thromboembolism, corresponding to 7% of pregnancies. In non-deficient women that had had at least one pregnancy, 1% suffered a pregnancy-related venous thromboembolism and 0.4% of these pregnancies were associated with this complication.

Discussion

This study showed a high absolute risk of venous thromboembolism in deficient women (1.76 per 100 person-years). It was 59-fold higher than reported in the normal female population of comparable age (0.03 per 100 person-years).^23,24Even female non-deficient relatives had a sixfold higher risk (0.19 vs. 0.03 per 100 person-years).

The main risk modulators for venous thromboembolism were the presence of other thrombophilic defects, pregnancy and the use of oral contraceptives.

Concomitance of other single and multiple thrombophilic defects increased the absolute risk of venous thrombosis from 1.55 to 2.14 and 2.92 per 100 person-years, respectively, in deficient women. In non-deficient women, the absolute risk was 0.16 in the absence of any thrombophilic defect, compared to 0.09 and 0.54 per 100 per- sons-years when a single other defect and other multiple defects were demonstrated respectively. This finding suggests that the risk in non-deficient women was also increased by other multiple thrombophilic defects. Why other thrombophilic defects aggregated in our families with hereditary deficiencies of anti thrombin, protein C or Thrombophilia and pregnancy-related thrombosis

Table 5. Pregnancy-related venous thromboembolism in antithrombin, protein C and protein S deficient and non-deficient women.

Total Antithrombin Protein C Protein S

D ND D ND D ND D ND

(n=101) (n=121) (n=33) (n=30) (n=38) (n=50) (n=30) (n=41) Women with at least one

pregnancy, n (%) 54 (53) 79 (65) 18 (55) 19 (63) 19 (50) 32 (64) 17 (57) 28 (68) Pregnancy-related event,

n (%) 12 (22) 1 (1) 8 (24) 0 3 (16) 0 1 (6) 1 (2)

Total pregnancies, n 162 245 45 62 63 109 54 74

Pregnancy-related event,

n (%) 12 (7) 1 (0.4) 8 (18) 0 3 (5) 0 1 (2) 1 (1)

Venous thromboembolism

during pregnancy, n 4 0 3 0 1 0 0 0

Venous thromboembolism

during puerperium, n 8 1 5 0 2 0 1 1

D: Deficient, ND: Non-deficient

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protein S remains to be clarified. Previously, we observed a similar aggregation in families with factor V Leiden.²⁵ It may be that aggregation contributes to the thrombophilic phenotype of symptomatic subjects. However, such selection bias is unavoidable, considering that asymptomatic subjects will not be tested for thrombophilic deficiencies or defects neither in studies, except family studies (with a symptomatic proband), nor in clinical practice.

Remarkably, women that had had a least one pregnancy were at lower risk of venous thromboembolism than women that had never been pregnant, whether they were deficient (1.37 vs. 2.96 per 100 person-years) or non-deficient (0.09 vs. 0.70 per 100 person-years). These differences can be attributed to the use of oral contraceptives. Of the first episodes of venous thromboembolism in deficient women that had had a least one pregnancy, 71% were related to pregnancy, and 12% were related to the use of oral contraceptives, while 75% of these episodes in deficient women that had never been pregnant were related to the use of oral contraceptives. This finding is explained by the planning of pregnancies. Deficient women who used oral contraceptives had mostly already suffered their first episode of venous thromboembolism before they became pregnant. Pregnancy-related venous thromboembolism pre- dominated in deficient women who did not use oral contraceptives. Unfortunately, small numbers did not enable the proper analysis of the contribution of other thrombophilic defects in these subgroups of women.

Pregnancy-related venous thromboembolism in 22% of deficient women corre- sponded to 7% of pregnancies, compared with 1% of non-deficient women, which cor- responded to 0.4% of pregnancies. Considering that 0.5–1 of 1000 pregnancies in the general female population is complicated by venous thromboembolism, deficient women had a 70- to 140-fold higher risk and non-deficient women a four- to eightfold higher risk. The highest risk was demonstrated in antithrombin deficient women (18% of pregnancies), compared with protein C deficient women (5%) and protein S deficient women (2%).

The risk of pregnancy-related venous thromboembolism in our study was less pronounced than in three of four previous studies addressing this issue.^9-12Venous thromboembolism was reported in 37–47% of pregnancies in antithrombin-deficient women, 12–19% in protein C-deficient women and 13–27% in protein S-deficient women.^9,10,12Differences in risk between our study and previous studies are probably due to differences in design and analysis. The higher risk in previous studies can be explained by the inclusion of probands, recurrences of venous thromboembolism and superficial phlebitis. The fourth study had a design comparable to our study and reported venous thromboembolism in 3.0%, 1.7% and 6.6% of the pregnancies in women with deficiencies of antithrombin, protein C and protein S respectively.¹¹

The present study addressed the multicausality of pregnancy-induced venous thromboembolism, including all currently known thrombophilic deficiencies and defects. It comprised families with hereditary deficiencies of antithrombin, protein C and protein S and, frequently, concomitance of other thrombophilic defects. Hence,

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we had the opportunity to assess interactions between rare deficiencies that are recognised as strong risk factors for venous thromboembolism, and more prevalent but mild thrombophilic defects. However, as a result of numerous combinations of deficiencies and defects, the numbers of women were too small to obtain reliable risk estimates for specific combinations. Therefore, deficient and non-deficient women were classified according to the numbers of additional thrombophilic defects. It should be noticed that risk estimates were unstable and confidence intervals were wide, due to the small size of the subgroups in this approach. Moreover, a number of subjects were not tested for all thrombophilic defects, because of insufficient plasma samples. Our study had several other limitations because of its retrospective design.

In a number of subjects, venous thromboembolism was not established by objective techniques because these were not available at the time of occurrence. Of 34 events, six episodes of proximal deep vein thrombosis (five in deficient women, one in a non- deficient woman) had occurred before 1974 and were not objectively confirmed. These were not excluded from analysis because five of six women experienced a recurrence, which was considered as indirect confirmation of the first episode or could have been classified as the first, confirmed event. Referral bias cannot be excluded, considering the university hospital setting of the study, but this was probably limited by testing all patients with venous thromboembolism for hereditary deficiencies. Of 1600 tested patients, 91 were deficient, corresponding to a prevalence of 5.7%, which agrees with previous reports on unselected patients.²⁶Selection bias was reduced by testing consecutive patients with venous thromboembolism and by an extraordinarily high response rate (95%) of eligible female relatives. Recall bias was reduced as much as possible by collecting clinical data prior to laboratory testing for thrombophilic deficiencies and defects. Inherent in its design as a family cohort study, controls (i.e. non- deficient relatives) were at higher risk of venous thromboembolism than the general population. Therefore, relative risks may have been underestimated. For this reason, we also compared absolute risks in deficient and non-deficient relatives with available data from previous population studies.

Our data supports the concept of a multicausal pathogenesis of venous thromboembolism. Both pregnancy and puerperium, and the use of oral contraceptives were shown to be the main determinants of venous thromboembolism in deficient women of reproductive age. A possible clinical implication of our findings might be that thromboprophylaxis could be considered during pregnancy and puerperium, and oral contraceptives discouraged in these women.

In conclusion, women of reproductive age with hereditary deficiencies of antithrombin, protein C or protein S are at high risk of pregnancy-related venous thromboembolism. Our data provides evidence that this risk is increased by multiple additional thrombophilic defects.

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63: 319-320.

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11 Friederich PW, Sanson BJ, Simioni P, Zanardi S, Huisman MV, Kindt I, Prandoni P, Büller HR, Girolami A, Prins MH. Frequency of pregnancy-related venous thromboembolism in anti coagulant factor- deficient women: implications for prophylaxis. Ann Intern Med. 1996; 125: 955-960.

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742-748.

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19 Bank I, Libourel EJ, Middeldorp S, Hamulyák K, van Pampus EC, Koopman MM,Prins MH, van der Meer J, Büller HR. Elevated levels of FVIII:C within families are associated with an increased risk for venous and arterial thrombosis. J Thromb Haemost. 2005; 3: 79-84.

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3 3 Fetal loss in women with hereditary deficiencies of antithrombin, protein C or

protein S, and the contribution of cosegregation of other

thrombophilic defects

F.J. Korteweg N. Folkeringa J.L.P. Brouwer N.J.G.M. Veeger J.J.H.M. Erwich J.P. Holm J. van der Meer

Submitted

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Abstract

Hereditary deficiencies of antithrombin (AT), protein C (PC) and protein S (PS) are strong risk factors for venous thromboembolism (VTE). The risk is reinforced by cosegregation of other thrombophilic defects. Single deficiencies and cosegregation may similarly increase the risk of fetal loss due to placental thrombosis. In a retrospective family cohort study, we assessed the absolute risk of fetal loss, comparing deficient women to non-deficient relatives. Of 630 women, 317 were evaluable, who had 987 pregnancies (582 in 185 deficient women). Total fetal loss rates were 47% (AT deficient), 45% (PC deficient), 21% (PS type I deficient) and 30% (PS type III deficient), compared to 32%, 28%, 29% and 27% in non-deficient women, respectively. Adjusted relative risks were 2.3 (95% CI, 0.9-6.1), 2.1 (0.9-4.7), 0.7 (0.2-1.8) and 1.1 (0.6-2.0). Dif- ferences were mainly due to higher late fetal loss rates in AT deficient (adjusted relative risk 11.3; 95% CI, 3.0-42.0) and PC deficient women (4.7; 1.3-17.4). Cosegregation of factor V Leiden and prothrombin G20210A did not increase the risk, neither in deficient women (29% vs. 34%), nor in non-deficient women (24% vs. 28%). This was explained by excluding pregnancies after prior VTE. In excluded women, total fetal loss rates were 39% in deficient women and 0% in non-deficient women. Hereditary AT and PC deficiencies were associated with a high absolute risk of fetal loss. An additional effect of cosegregation was not demonstrated, may be due to the exclusion of women at highest risk of VTE.

University of Groningen Pregnancy-related thrombosis and fetal loss in women with thrombophilia Folkeringa, Nienke

Pregnancy-related thrombosis and fetal loss in women with thrombophilia Folkeringa, Nienke

Pregnancy-related thrombosis and fetal loss

in women with thrombophilia

Nienke Folkeringa

Pregnancy-related thrombosis and fetal loss

in women with thrombophilia

Contents

1

1 Introduction

Introduction

Thrombophilia

Thromboprophylaxis

Aim of this thesis

2

2 High risk of pregnancy-related venous thrombo embolism in women with multiple

thrombophilic defects

N. Folkeringa J.L.P. Brouwer F.J. Korteweg N.J.G.M. Veeger J.J.H.M. Erwich J. van der Meer

Abstract

Introduction

Materials and methods

Results

Discussion

3

3 Fetal loss in women with hereditary deficiencies of antithrombin, protein C or

protein S, and the contribution of cosegregation of other

thrombophilic defects

F.J. Korteweg N. Folkeringa J.L.P. Brouwer N.J.G.M. Veeger J.J.H.M. Erwich J.P. Holm J. van der Meer

Abstract