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REGULAR ARTICLE

An unfavorable combination of factor V Leiden withage, weight, and blood group causes high risk ofpregnancy-associated venous thrombosis—apopulation-based nested case-control study

Leena Hiltunen a,1, Anna Rautanen b,c,1, Vesa Rasi a,*, Risto Kaaja d,Juha Kere b,e, Tom Krusius a, Elina Vahtera a, Mikko Paunio f,g

a Department of Hemostasis, Finnish Red Cross Blood Service, Kivihaantie 7, FIN-00310 Helsinki, Finlandb Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Helsinki, Finlandc Finnish Genome Center, University of Helsinki, Helsinki, Finlandd Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, Helsinki, Finlande Department of Biosciences at Novum and Clinical Research Centre,Karolinska Institutet, Huddinge, Swedenf The World Bank, Washington DC, USAg Department of Public Health, University of Helsinki, Helsinki, Finland

Received 4 January 2006; received in revised form 15 March 2006; accepted 17 April 2006Available online 12 June 2006

0049-3848/$ - see front matter D 200doi:10.1016/j.thromres.2006.04.001

Abbreviations: FVL, factor V Leidetissue factor pathway inhibitor; DVT, dattributable risk; PAR%, population att* Corresponding author. Tel.: +358 9E-mail address: vesa.rasi@fimnet.f

1 These authors contributed equally

KEYWORDSABO blood group;Age;Body mass index;Factor V Leiden;Pregnancy;Venous thrombosis

Abstract

Introduction: Hereditary and acquired risk factors increase the risk for thrombosisamong pregnant women. Few risk estimates are, however, well established. The aimof the present study was to assess risk for pregnancy-associated venous thrombosis offactor V Leiden (FVL), FII G20210A, FV A4070G, MTHFR C677T, TFPI C536T, PROCT38853G, FXIII V34L, blood group, age, and body mass index (BMI), and theirinteractions and public health impact.

Thrombosis Research (2007) 119, 423—432

6 Elsevier Ltd. All rights reserved.

n; BMI, body mass index; MTHFR, methylenetetrahydrofolate reductase; PROC, protein C; TFPI,eep venous thrombosis; AR, attributable risk; AR%, attributable risk proportion; PAR, populationributable risk proportion.5801 265; fax: +358 9 5801 484.i (V. Rasi).to this work.

L. Hiltunen et al.424

Materials and methods: Study design is a population-based nested case—controlstudy of 100,000 consecutive pregnancies in Finland. Cases and controls wereidentified by combining national registers. Thirty four cases with objectivelydiagnosed venous thrombosis and 641 controls were studied.Results: FVL (OR 11.6, 95% CI 3.6—33.6), age N35 vs. b25 (OR 6.3, 95% CI 1.7—23.1),and BMI N30 vs. b25 (OR 5.6, 95% CI 2.3—13.9) were associated with thrombosis.Overall absolute risk of a FVL carrier was 1 in 314. FVL interacted with age, BMI, andblood group. Population attributable risk proportion was 19% for FVL, 23% for ageN35, 33% for BMI N25, and 35% for non-O blood group. Unexpectedly, the prevalenceof FVL increased with age in controls.Conclusions: FVL appeared as a strong risk factor for pregnancy-associated venousthrombosis. Especially in elderly overweight mothers, FVL may cause a substantialthrombosis risk. Further studies are needed to confirm the increased prevalence ofFVL in elderly mothers with normal pregnancies.D 2006 Elsevier Ltd. All rights reserved.

Introduction

Venous thromboembolism associated with pregnan-cy causes marked morbidity [1—3] and is one of theleading causes of maternal death in westerncountries [1,2,4]. Estimates for its incidence inrecent studies vary from 71 [5] to 85 [6,7] per100,000 deliveries. Venous thrombosis is a multi-factorial disease which occurs when a sufficientnumber of genetic and acquired risk factors arepresent simultaneously [8]. Pregnant women withgenetic risk factors are increasingly identified, forinstance as members of thrombophilia families.Also, childbearing age and prevalence of over-weight are increasing in western countries. There-fore, population-based estimates of compound riskswould be valuable.

Factor V Leiden (FVL) (G1691A) causes activat-ed protein C resistance and increases the risk forvenous thrombosis [9,10], also in association withpregnancy [5,11,12]. FII G20210A polymorphism inprothrombin gene is associated with venousthrombosis [13], but evidence of its role inpregnancy is scarce [11,12,14]. R2 allele in thefactor V gene (A4070G) [15], a common polymor-phism in the methylenetetrahydrofolate reductase(MTHFR) gene (C677T) [16,17], and point muta-tions in protein C (PROC) gene (T8853G) [18] andin the tissue factor pathway inhibitor (TFPI) gene(C536T) [19] have all been inconsistently associ-ated with venous thrombosis. In contrast, thecommon polymorphism in the factor XIII gene(FXIII V34L) [20] has been variably associated witha decreased risk for venous thrombosis [21—23].Non-O blood group and acquired risk factors suchas body mass index (BMI), age, smoking, highparity, multiple pregnancy, and cesarean sectionhave been associated with pregnancy-associatedvenous thrombosis [2,7,24—26].

This report concerns risk factors for venousthrombosis based on a nationwide population-basednested case-control study of various pregnancycomplications. We assessed the risks caused by sixmutations (FVL, FII G20210A, FV A4070G, MTHFRC677T, PROC T8853G, and TFPI C536T) for venousthrombosis during pregnancy and puerperium. Westudied also whether FXIII V34L polymorphism isprotective against thrombosis. We focused on therisks caused by combinations of thrombophilicmutations and other hereditary and acquiredfactors. The study design allowed us to estimatenot only relative but also absolute and attributablerisks and their percentages on individual andpopulation level. Our results are based on Finnishwomen with pregnancies continuing beyond 8 to 12weeks of pregnancy.

Materials and methods

Subjects

In Finland (population 5.2 million) practically allpregnant women contact Maternity Welfare Clinics,which is a prerequisite for benefits offered by TheSocial Insurance Institution of Finland. After con-tacting their local clinic, usually during the 8th to12th week of pregnancy, they are registered in theNational Register of the Blood Groups and BloodGroup Antibodies of Pregnant Women, maintainedby the Finnish Red Cross Blood Service in Helsinki.Data for 100,000 consecutive pregnancies during1997—1998 were obtained from the register. Onlythe first pregnancy of each woman during the timeperiod (index pregnancy) was evaluated.

The National Research and Development Centrefor Welfare and Health (STAKES) maintains theNational Hospital Discharge Register with diagnoses

Factor V Leiden with age, weight, and blood group causes venous thrombosis 425

classified according to International Classificationof Diseases (ICD-10 since 1996). The two registerswere linked by ID number. In the cohort of 100,000pregnancies, 95 women had an ICD-code for venousthrombosis registered during 1997—1999. Of those,88 fulfilled the invitation criteria (alive, mothertongue Finnish or Swedish, residence in Finland)and 70 (80%) were willing to participate. Themedical records of all those were checked. In 34women (cases) an objectively diagnosed distal (21)or proximal (11) deep venous thrombosis (DVT),pulmonary embolism (1), or cerebral vein throm-bosis (1) had occurred during the index pregnancyor puerperium (10 weeks after delivery). One casewas on prophylaxis during pregnancy. The throm-bosis had been verified with ultrasound, venogra-phy, magnetic resonance imaging, or computedtomography. Of the 36 excluded women, twelvehad superficial venous thrombosis, in four thethrombosis was not diagnosed objectively, and intwenty women the venous thrombosis had occurredbefore the index pregnancy or more than 10 weeksafter delivery.

Controls were sampled randomly (every 46thpregnancy) from the 93,935 women without com-plications (miscarriage, premature birth, birth of asmall-for-gestational-age child, pre-eclampsia,stillbirth, or venous or arterial thrombosis) in theHospital Discharge Register. Of the 2043 women,1930 fulfilling the same invitation criteria thancases were invited and 843 (44%) participated.After checking the medical records, 641 wereeligible (no thrombosis prophylaxis, no pregnancycomplications in the index pregnancy, and noprevious venous or arterial thrombosis, pre-eclampsia, or stillbirth). The prevalence of thepolymorphisms in the Finnish population was de-fined from 644 first-time blood donors.

The cases and controls were invited by lettersand reminders. They filled out questionnaires tosupplement information from the medical recordsand provided blood samples. All data were collect-ed onto standardized forms, blinded to genotypes.All participants gave written informed consent. Thestudy was approved by the ethics committee of theFinnish Red Cross Blood Service and the Ministry ofSocial Affairs and Health.

Laboratory methods

Genomic DNA was isolated from blood samplescollected in EDTA tubes by using a commercial kit(QIAampR DNA Blood Mini Kit, Qiagen, Hilden,Germany). Cases, controls, and blood donors weregenotyped for the seven polymorphisms. Genotyp-ing was based on restriction enzyme digestions

after PCR. Assays were performed multiplexed in atotal volume of 5 Ml in the conditions describedelsewhere [27]. A fluorescent label (6-FAM, NED, orHEX) was included in the primer. (Informationabout primer sequences and restriction enzymesis available upon request). An additional restrictionsite worked as an internal control for the digestionreaction. If a natural digestion site was lacking,primer-induced restriction analysis was used. PCRproducts were digested with 1 U of specificrestriction enzyme (New England Biolabs, Beverly,MA, USA) and then separated with the ABI PRISM377 (PE Applied Biosystems, Foster City, CA, USA)DNA sequencer. The data were analyzed usingGenescan 3.1 and Genotyper 2.5 software (PEApplied Biosystems). Samples with missing geno-type result in FVL or FII G20210A assays wereanalyzed with another method (factor V Leiden Kitand Factor II (Prothrombin) G20210A Kit withLightCyclerR Instrument, Roche Diagnostics GmbH,Mannheim, Germany). In addition, positive findingsof FVL and FII G20210A mutations were checkedwith the latter method. All results were concor-dant. All markers obeyed the Hardy—Weinbergequilibrium.

Statistical analysis

Differences between cases and controls wereevaluated by using chi-square test or exact methodfor proportions for discrete variables, and t-testfor continuous variables (SPSS, version 12.0.1). AP-value of b0.05 was considered statisticallysignificant.

Because only FVL showed positive association inthe preliminary crude stratified analyses, wesystematically evaluated demographic factors,other mutations, and personal variables onlyagainst FVL to study two-way interactions andsources of confounding. To make consideration forstatistical power, the lowest detectable odds ratio(OR) was interactively calculated by assuming 80%power, 0.05 type I error, and 1:19 case-controlratio (Epi-info, version 6 and its interactiveprogram Statcalc).

We performed logistic regression analyses withthe dummy variable strategy to reveal two-wayinteractions and to present meaningful uncon-founded association in the logistic models (SPSS,version 12.0.1). Due to the relatively small caseseries, only adjusted models with one or two mostapparent confounders are presented. In Table 2,the 95% confidence intervals were calculated usingan exact method [28].

As age distribution and blood group data of thecohort of 100,000 pregnancies were known, Poisson

L. Hiltunen et al.426

regression analyses were performed to directlycalculate relative risks (RR) with a program calledR [29] (Table 5). Offsets in the Poisson regressionmodels, i.e. the populations at risk, were obtaineddirectly (age, blood group) or estimated usinginformation from controls (FVL, BMI). For eachstrata, the crude relative risks and their 95%confidence intervals were calculated according tomethod by Breslow and Day [30].

Attributable risk (AR) was calculated as follows:AR= Ie� I0 [31], Ie being the incidence of venousthrombosis in FVL carriers and I0 the incidence ofvenous thrombosis in non-carriers. AR indicates therisk for pregnancy-associated venous thrombosisamong FVL carriers attributable to the FVL itself.Attributable risk proportion (AR%) was calculatedusing formula AR%=(AR/ Ie)�100 [31]. AR% stateswhat proportion of pregnancy-associated venousthrombosis among FVL carriers can be attributed tothe FVL. Population attributable risk (PAR) wascalculated as follows: PAR= IT� I0 [31], IT being theincidence rate of venous thrombosis in the wholestudy cohort or in the subgroups. PAR indicates therisk for pregnancy-associated venous thrombosisattributable to the FVL in the population of womenwith pregnancies not ending during the firsttrimester. Population attributable risk proportion(PAR%) was calculated using formula PAR%=(PAR/IT)�100 [31]. PAR% states the proportion of preg-nancy-associated venous thrombosis in the popula-tion that can be attributed to the FVL. Likewise,estimates of RR, AR, AR%, PAR, and PAR% werecalculated for age, BMI, and blood group.

In Fig. 1, expected incidences according to an addi-tive model of interaction were calculated using for-

1

10

100

1000

10000

Age<25

Age 25

-35

Age>35

BMI<

25

BMI 2

5-30

BMI>

30

Blood g

Blood

Ab

solu

te r

isk

per

100

000,

log

arit

hm

ic s

cale

Figure 1 Estimates of absolute risks for pregnancy-associcarriers. Expected risks calculated according to additivecomparison. Abbreviation: BMI, body mass index.

mula: Baseline I(FVL�/risk factor�)+[I(FVL+/risk factor�)�Baseline I]+[I(FVL�/risk factor+)�Baseline I]. Expectedincidences according to a multiplicative model ofinteraction were calculated using formula: BaselineI +{[RR(FVL)�RR(age, BMI or blood group)]�Baseline I}.

Results

The characteristics of cases and controls aresummarized in Table 1. Prevalence of blood groupsO, A, B, and AB was 20.6%, 52.9%, 17.6%, and 8.8%for cases and 32.3%, 43.4%, 18.3%, and 6.1% forcontrols.

Cases experienced 21 distal DVT, 11 proximalDVT (vena femoralis 4, vena iliaca 6, vena cava 1),one pulmonary embolism without DVT, and onecerebral vein thrombosis. Eight cases (24%) had ahistory of DVT 4 to 15 years earlier during use oforal contraceptives (4), during pregnancy (3), orafter knee surgery (1). Of them, four were carriersof FVL (one homozygous).

PROC T8853G was monomorphic. Only oneheterozygote for TFPI C536T was observed incontrols and one in blood donors. Table 2 showsgenotype frequencies for the other polymorphismsin cases, controls, and in blood donors. Only FVLwas significantly associated with venous thrombo-sis. FVL, MTHFR, FVR2, or FXIII polymorphisms didnot interact significantly with each other (data notshown).

FVL was associated with pregnancy-associatedvenous thrombosis with an OR of 11.6 (95% CI 3.6—33.6). If only cases with their first venous throm-bosis associated with the index pregnancy were

roup O

gro

up non-O

FV Leiden carriers

FV Leiden non-carriers

Multiplicative model in FVLeiden carriers

Additive model in FVLeiden carriers

ated venous thrombosis in FV Leiden carriers and non-and multiplicative model of interaction are shown for

Table 2 Genotype frequencies and association of genotypes with pregnancy-associated venous thrombosis

Polymorphism No. (%) of cases(n =34)

No. (%) of controls(n =641)

No. (%) of blooddonors (n =644)

OR (95% CI)a Lowestdetectable ORb

FV LeidenGG 27 627 602 11.6 (3.6—33.6) 7.6GA 6 (17.6) 14 (2.2) 15 (2.4)AA 1 (2.9) 0 0

FII G20210AGG 33 635 634 3.2 (0.07—27.6) 14.0GA 1 (2.9) 6 (0.9) 3 (0.5)AA 0 0 0

FV A4070Gc

AA 25 506 516 1.3 (0.5—3.2) 3.0AG 8 (24.2) 116 (18.5) 96 (15.5)GG 0 4 (0.6) 7 (1.1)

MTHFR C677Tc

CC 20 359 374 0.9 (0.4—1.9) 2.9CT 13 (38.2) 244 (38.4) 231 (36.4)TT 1 (2.9) 32 (5.0) 30 (4.7)

FXIII V34Lc

GG 21 417 393 1.2 (0.4—1.9) 0.2GT 11 (33.3) 166 (26.9) 187 (31.3)TT 1 (3.0) 34 (5.5) 17 (2.8)

Abbreviations: P-value, probability value; OR, odds ratio; CI, confidence interval; N.A., not assessed.a Cases versus controls; carriers of the polymorphism (hetero- and homozygotes) versus wild-type.b Lowest OR that would have been possible to detect with a current sample size. For FXIII V34L lowest detectable OR for

protective effect.c Some genotype results are missing.

Table 1 Characteristics of cases and controls

Characteristics Cases (n =34) Controls (n =641) P-value

Place of residence: town, n (%) 16 (47) 394 (62)a 0.10Place of residence: rural area, n (%) 18 (53) 243 (38)a

Education level 1, n (%)b 9 (26) 42 (7) 0.001Education level 2, n (%)b 20 (59) 469 (74)Education level 3, n (%)b 5 (15) 122 (19)Age, mean (range), years 31.8 (18—40) 28.8 (16—45) 0.001Height, mean (range), cm 165.0 (142—178) 165.8 (148—183) 0.44Weight before pregnancy, mean (range), kg 71.3 (47—108) 63.6 (41—113) b0.001Body mass index before pregnancy, mean (range) 26.1 (18.8—37.5) 23.1 (16.4—41.9) b0.001Current smoker, n (%) 6 (17.6) 70 (10.9) 0.26Number of pregnancies, mean (range) 2.5 (1—6) 2.4 (1—15) 0.75First pregnancy, n (%) 12 (35.3) 223 (34.8) 0.99Twin pregnancy, n (%) 1 (2.9) 7 (1.1) 0.34Cesarean section, n (%) 7 (20.6) 79 (12) 0.18Bleeding during labor, mean (range), ml 463 (50—2300) 434 (50—3500) 0.67Birth weight of the childc, mean (range), g 3411 (2242—4187) 3609 (2602—4972) 0.009Chronic disease, n (%) 5 (14.7) 64 (10) 0.38Varicose veins, n (%) 8 (23.5) 103 (16) 0.24History of migraine, n (%) 14 (41.2) 107 (17) 0.001Past malignancy, n (%) 0 3 (0.5) 0.99Blood group O (%) 7 (20.6) 207 (32.3) 0.19Blood group non-O (%) 27 (79.4) 434 (67.7)

Abbreviations: P-value, probability value.a Place of residence not specified in four controls.b Education level 1: comprehensive school; level 2: upper secondary school, vocational and professional education; level 3:

university. Education level not specified in eight controls.c Adjusted for gestational age and gender by linear regression, twins excluded.

Factor V Leiden with age, weight, and blood group causes venous thrombosis 427

Table 3 Risk of FV Leiden carriers for venous thrombosis by phase of pregnancy and in puerperium

1st trimester no. (%) 2nd trimester no. (%) 3rd trimester no. (%) Puerperium no. (%)

FVL carrier (n =7) 4 (57) 1 (14) 0 2 (29)FVL non-carrier (n =27) 3 (11) 2 (7) 7 (26) 15 (56)

Definitions: 1st trimester: week b14, 2nd trimester: weekz14 and b27, 3rd trimester: week z27.

L. Hiltunen et al.428

considered, OR was 5.8 (95% CI 1.6—21.8), and ifonly primigravidae were analyzed, OR was 8.7(95% CI 1.5—50.6). Most thromboses in FVL car-riers occurred in early pregnancy while in non-carriers most occurred during puerperium ( P =0.037) (Table 3).

Age over 35 compared with age under 25 (OR6.3, 95% CI 1.7—23.1) and BMI over 30 comparedwith BMI under 25 (OR 5.6, 95% CI 2.3—13.9) wereassociated with increased risk for pregnancy-asso-ciated venous thrombosis. Table 4 shows interac-tion of FVL with age and BMI.

Non-O blood group tended to increase the riskfor thrombosis. In the FVL carriers, 6 of the 7 cases,but only 8 of the 14 controls, had non-O bloodgroup. The effect of blood group itself was notsignificant (OR 1.8, 95% CI 0.8—4.3), but in FVLcarriers non-O blood group was associated with 25-fold risk for venous thrombosis compared with non-carriers (Table 4).

Unexpectedly, the prevalence of FVL in controlsaged over 35 was 7.7% (6/78) and 1.4% (8/563) inyounger controls (P =0.009). After adjusting forage (under and over 35 years), neither parity nornumber of pregnancies affected FVL prevalence(analyses not shown). Thus, age turned out to be a

Table 4 Interaction of FV Leiden with other risk factors f

Risk factor FVL Cases (n =34) no. (%) Controls

Age b25 � 2 (5.9) 131 (20.25—35 � 18 (52.9) 424 (66.N35 � 7 (20.6) 72 (11.b25 + 1 (2.9) 2 (0.325—35 + 2 (5.9) 6 (0.9N35 + 4 (11.8) 6 (0.9

BMI b25 � 15 (44.1) 469 (73.25—30 � 7 (20.6) 119 (18.N30 � 5 (14.7) 39 (6.1b25 + 2 (5.9) 11 (1.725—30 + 2 (5.9) 2 (0.3N30 + 3 (8.8) 1 (0.2

Blood group O � 6 (17.6) 201 (31.Non-O � 21 (61.8) 426 (66.O + 1 (2.9) 6 (0.9Non-O + 6 (17.6) 8 (1.2

Abbreviations: OR, odds ratio; CI, confidence interval; BMI, body ma Reference group.b Adjusted for BMI.c Adjusted for age.d Adjusted for age and BMI.

positive confounder. Accordingly, in the logisticmodel, when BMI and blood group categories wereadjusted for age, somewhat weaker effectsemerged for FVL. Counter-intuitively, thecorresponding associations with FVL and ageslightly increased when adjusted for BMI (Table 4).

The point estimates of relative risks of pregnan-cy-associated deep venous thrombosis for age over35 (vs. b35), BMI over 25 (vs. b25), and non-O bloodgroup (vs. O) were 3.5, 3.0, and 1.8 and the pointestimates of population attributable risk propor-tions were 23%, 33%, and 35% in the cohort of100,000 pregnant women.

The estimated absolute risks (incidence rates)for pregnancy-associated venous thrombosis in FVLcarriers and non-carriers are shown in Table 5. InFVL carriers, the overall estimated absolute riskwas 318 per 100,000, and the highest absolute riskwas in women with BMI over 30 and the lowest inwomen with blood group O. In FVL non-carriers therisk increased with age and BMI, the absolute riskbeing highest among women with BMI over 30 andlowest in women younger than 25. Fig. 1 shows thatthe interaction of FVL with age was compatiblewith additive model, whereas the interaction ofBMI and non-O blood group with FVL seemed to be

or pregnancy-associated venous thrombosis

(n =641) no. (%) OR (95% CI) Adjusted OR (95% CI)

4) 1a 1a

1) 2.8 (0.6—12.1) 2.6 (0.6—11.2)b

2) 6.4 (1.3—31.5) 5.0 (1.0—25.5)b

) 32.8 (2.0—526.4) 34.7 (1.5—792.1)b

) 21.8 (2.6—182.6) 24.9 (1.3—480.5)b

) 43.7 (6.6—287.4) 58.1 (4.2—799.7)b

2) 1a 1a

6) 1.8 (0.7—4.6) 1.7 (0.7—4.3)c

) 4.0 (1.4—11.6) 3.7 (1.3—10.9)c

) 5.7 (1.2—27.9) 5.2 (1.0—26.2)c

) 31.3 (4.1—237.2) 18.8 (2.3—156.3)c

) 93.8 (9.2—955.3) 75.5 (6.7—846.5)c

4) 1a 1a

5) 1.7 (0.7—4.2) 1.6 (0.6—4.0)d

) 5.6 (0.6—53.9) 5.8 (0.6—58.7)d

) 25.1 (6.6—95.4) 16.3 (3.9—68.5)d

ass index.

Table 5 Crude risk estimates of FV Leiden for venous thrombosis in the cohort of 100,000 pregnancies

Incidence rateper 100,000FVL carriersa

Incidence rateper 100,000 FVLnon-carriersa

RR (95% CI) AR per 100,000(95% CI)

AR% (95% CI) PAR per100,000(95% CI)

PAR%(95% CI)

All 318 (7/2200) 28 (27/97,800) 11.5 (5.0—26.5) 290 (111—703) 92 (80—96) 6 (2—16) 19 (16—20)Age

b25b 322 (1/311) 10 (2/20,389) 32.8 (3.0—361.5) 312 (19—3536) 97 (66—100) 5 (0.3—53) 32 (22—33)25—35b 213 (2/939) 27 (18/66,161) 7.8 (1.8—33.7) 186 (22—891) 87 (45—97) 3 (0.3—13) 9 (4—10)N35b 426 (4/939) 62 (7/11,261) 6.9 (2.0—23.4) 364 (63—1393) 85 (50—96) 28 (5—107) 31 (18—35)

BMIb25c 116 (2/1723) 21 (15/73,177) 5.7 (1.3—24.8) 96 (6—487) 82 (23—96) 2 (0.1—11) 10 (3—11)25—30c 623 (2/321) 38 (7/18,579) 16.5 (3.4—79.6) 585 (92—2962) 94 (71—99) 10 (2—50) 21 (16—22)N30c 1936 (3/155) 83 (5/6045) 23.4 (5.6—97.9) 1853 (380—8016) 96 (82—99) 46 (10—200) 36 (31—37)

Blood groupOb 108 (1/924) 19 (6/30,950) 5.6 (0.7—46.4) 89 (�6—880) 82 (�49—98) 3 (�0.2—26) 12 (�7—14)Non-Ob 464 (6/1294) 31 (21/66,832) 14.8 (6.0—36.6) 432 (156—1117) 93 (83—97) 8 (3—20) 21 (18—22)

Abbreviations: RR, relative risk; AR, attributable risk; AR%, attributable risk proportion; PAR, population attributable risk; PAR%,population attributable risk proportion; CI, confidence interval; BMI, body mass index.a Number of FVL carriers in each subgroup estimated from its prevalence among controls in the same subgroup. In parentheses:

numerator, actual number of endpoints; denominator, population at risk.b Proportion of women in each age and blood group category based on data from the 100,000 consecutive pregnant women.c Proportion of women in each BMI category is estimated by using BMI of controls.

Factor V Leiden with age, weight, and blood group causes venous thrombosis 429

even beyond what would be expected by a multi-plicative model.

In FVL carriers, 92% of the thromboses werecaused by this polymorphism (AR%). In the wholestudy population, FVL caused 19% of the thrombo-ses (PAR%). The attributable risk, population at-tributable risk, and population attributable riskproportion for FVL were highest in the subgroup ofwomen with BMI over 30 (Table 5). The unadjustedparameters in Table 5 somewhat overestimate theassociations of FVL, because age (and BMI) was apositive confounder as indicated earlier.

None of the polymorphisms, including FVL, wasassociated with bleeding during delivery or birthweight of the child. Smoking was not significantlyassociated with risk for thrombosis (OR 1.7, 95% CI0.7—4.4).

Discussion

This nationwide population-based nested case-control study of 100,000 consecutive pregnanciesconfirms FVL to be a major risk factor for pregnan-cy-associated venous thrombosis, increasing therisk 11.6-fold. The overall estimated absolute riskof a FVL carrier for thrombosis was 318 per 100,000(i.e. 1 in 314). On the population level FVL wasresponsible for 19% of thrombosis whereas thepopulation attributable risk proportions of age over35, BMI over 25, and non-O blood group were 23%,33%, and 35%. The interaction of FVL was additivewith age and more than additive with BMI and non-O blood group.

The incidence of objectively verified venousthrombosis was 34 per 100,000 pregnancies. Anincidence of 71—85 per 100,000 deliveries has beenreported in recent studies [5—7], those being 66—71 per 100,000 [6,7] if only objectively verifiedthromboses are assessed. Our incidence was basedon women who contacted maternity units, whereasin the referred studies the incidences were basedon deliveries and miscarriages were not counted. Asa result, our denominator is bigger, which partlyexplains the lower incidence. Due to the identifi-cation method of study population, we have missedonly thromboses associated with very early miscar-riages. Also, because of the 80% participation rate,nine true cases are missing if thromboses in theindex pregnancies appeared at the same frequencyin participants and non-participants. It seemsunlikely that DVT, pulmonary emboli, or cerebralvein thrombosis would be considerably underreported in the Hospital Discharge Register or thatrisk factors for thrombosis would have affected thereporting rate causing detection bias. Informationbias was avoided by collecting data to standardizedforms blinded for laboratory results. At the start ofthe study, FVL was known in no more than fourhundred women of childbearing age in the country.If known, thrombosis prophylaxis would not havebeen advocated antenatally in women with nopersonal history of thrombosis. Therefore, we donot think that prophylaxis because of FVL wouldhave caused marked bias either in cases orcontrols. Missing cases might bias absolute riskestimates, but they would not materially influencerelative risks.

L. Hiltunen et al.430

The laborious strategy to check medical recordsand not to rely blindly on the ICD-codes, allowed usto exclude wrong positive and negative findings.This proved crucial, because 28% of the diagnosesof venous thrombosis in the Hospital DischargeRegister were actually not associated with theindex pregnancy. The ICD-10 classification doesnot include a separate code for past thrombosis(status post thrombosis), which is often recorded asdiagnosis when prophylactic anticoagulation isstarted. The bias due to non-validated registry-based diagnoses is a known source of error inepidemiological studies [32].

High BMI and increasing age were independentlyassociated with increased risk for thrombosis, asreported earlier [7,25,33]. They also interactedwith FVL in pregnant women. Non-O blood group isassociated with an increased risk for venousthrombosis [32,34,35]. This was also the trend inour study, but the association was not significant.The blood group distribution in the cohort of100,000 pregnant women was the same as inFinnish population, i.e. O 31%, A 44%, B 17%, andAB 8%. However, the proportion of blood group Owas 32% in controls and only 21% in cases.Furthermore, FVL interacted with non-O bloodgroup, the compound risk being even beyondmultiplicative. Our findings agree with studiesreporting this interaction [32,36,37]. Age, BMI,and non-O blood group in combination with FVL,increased the risk for thrombosis maximally 43-,93-, and 25-fold.

On the individual level, FVL was more importantthan BMI or age when considering OR, absolute risk,AR, or AR%, increasing the risk for pregnancy-associated venous thrombosis 11.6-fold. Otherstudies have reported 4.5- to 16.3-fold risks[11,12,14,16]. Also the reported incidence ofthrombosis by trimester varies substantially[5,38]. In this study, FVL increased the risk forthrombosis especially during the first trimester.The overall absolute risk of a FVL carrier forpregnancy-associated venous thrombosis, 1 in 314,was slightly higher than the 1 in 437 reportedearlier [5]. We found the highest absolute risk, 1 in52, in FVL carriers with BMI over 30. On thepopulation level, age over 35, BMI higher than 25,and non-O blood group were important risk factorsbecause of their high prevalence. In the subgroupsof women with BMI over 30, or age over 35, FVL wasattributable for a third of thromboses.

It was recently concluded in the prospectivestudy of 4885 pregnancies that FVL does notincrease the risk for thrombosis [39]. This wasbased on the observation that none of the 134 FVLcarriers experienced thrombosis. The thrombosis-

free proportion of FVL carriers (134/4881; 2.7%)was very similar to that in our material (14/641;2.2%). However, the number of cases with throm-bosis (four) in their material was too small to revealthe influence of FVL, if the prevalence of FVLamong cases is expected to be the same that weobserved (7/34; i.e. less than 1 in 4).

The prevalence of FVL was the same in controlsas in blood donors (2.2% vs. 2.4%), which is lowerthan in many other countries [40], but comparablewith other Finnish materials (2.1—2.9% in controls)[41,42]. Unexpectedly, the frequency of carrierswas multiple in controls aged over 35 comparedwith younger controls (7.7% vs. 1.4%; P =0.009).The result was the same, if not only the eligible butall participating controls (843) were considered(8.7% vs. 2.3%, P =0.002). Neither parity nor thenumber of previous pregnancies had confounding ormodifying effect on the sudden increase of FVLprevalence after the age of 35 years. It is unlikelythat FVL status would have affected the participa-tion rate of controls, because prior knowledge ofFVL was rare at the time of the study. Nor can wethink of any other viable selection force to thecontrol series according to FVL status. Further-more, participation rate was the same in every agegroup and women with prior venous thrombosiswere excluded. This post hoc finding clearly needsto be studied further. Interestingly, the prevalenceof FII G20210A did not vary according to age.

Our number of cases was too small to detectsignificant OR for FII G20210A for pregnancy-associated venous thrombosis. The lowest OR forFII G20210A that would have been possible todetect with the current sample size was 14.0. Inother studies the OR has varied from 2.9 to 15.2[11,12,14,16]. We would have had a reasonablepower to detect fairly modest increases/decreasesin ORs concerning FV A4070G, MTHFR C677T, andFXIII V34L polymorphisms because of their highminor allele frequencies, but associations withvenous thrombosis were not significant. The riskassociated with PROC T8853G and TFPI C536Tpolymorphisms could not be assessed due to theirrarity.

Strengths of our study include a large number ofconsecutive pregnancies within a short time period,objective evidence of venous thrombosis, cases andcontrols from the same cohort of geneticallyhomogenous population, and the population-basednested case-control study design which allowed usto estimate absolute and attributable risks. Limi-tations of the study are the relatively small numberof cases, and that family history as a risk factorcould not be reliably assessed. We considered first-time and recurrent venous thrombosis as a mani-

Factor V Leiden with age, weight, and blood group causes venous thrombosis 431

festation of the same thrombotic disease, andtherefore included recurrent thromboses. However,if only first venous thromboses were considered,the risk associated with FVL would still have beensignificant (5.8-fold). It is noteworthy that treatingphysicians were unaware of the FVL status of caseswith recurrent thrombosis, because all earlierthromboses had occurred before the mutation wasdiscovered.

This population-based nested case-control studyconfirms FVL to be a strong risk factor for pregnan-cy-associated venous thrombosis on individual lev-el. Our results suggest that FVL interacts withcommon risk factors and increases thrombosis riskespecially in elderly overweight mothers. On thepopulation level, the impact of advanced age,overweight, and non-O blood group on thrombosisrisk was stronger than that of FVL due to their highprevalence. Further studies are needed to confirmthe unpredicted increased prevalence of FVL inelderly mothers with normal pregnancies.

Acknowledgments

This work was supported by Research Fund ofFRCBS, Research Foundation of Orion Corporation,Aarne and Aili Turunen Foundation, Finnish-Norwe-gian Medical Foundation, and the Sigrid Juseliusfoundation.

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