Thrombophilias and adverse pregnancy outcomes - Wiley Online Library

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May 3, 2012 - *Department of Obstetrics and Gynecology, Roskilde Hospital, Roskilde; Department of .... syndrome, was defined according to the American Congress ..... strong causal link between thrombophilias and adverse preg-.
Journal of Thrombosis and Haemostasis, 10: 1320–1325

DOI: 10.1111/j.1538-7836.2012.04773.x

ORIGINAL ARTICLE

Thrombophilias and adverse pregnancy outcomes: results from the Danish National Birth Cohort J. A. LYKKE,*  L. A. BARE,à J. OLSEN,§– R. LAGIER,à A. R. ARELLANO,à C. TONG,à M. J. PAIDAS** and J . L A N G H O F F - R O O S   *Department of Obstetrics and Gynecology, Roskilde Hospital, Roskilde;  Department of Obstetrics, Rigshospitalet, Copenhagen, Denmark; àCelera Diagnostics, Alameda, ; §Department of Epidemiology, School of Public Health, University of California at Los Angeles, Los Angeles, CA, USA; –Department of Epidemiology, Institute of Public Health, University of Aarhus, Denmark; and **Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale Women and ChildrenÕs Center for Blood Disorders, Yale University School of Medicine, New Haven, CT, USA To cite this article: Lykke JA, Bare LA, Olsen J, Lagier R, Arellano AR, Tong C, Paidas MJ, Langhoff-Roos J. Thrombophilias and adverse pregnancy outcomes: results from the Danish National Birth Cohort. J Thromb Haemost 2012; 10: 1320–5.

Introduction Summary. Background: Inherited thrombophilias have inconsistently been linked to adverse pregnancy outcomes. Differences in study design, size and population could explain this heterogeneity. Objective: The aim of the present study was to evaluate if factor (F)V Leiden G1691A, prothrombin mutation G20210A (PTM) and methylenetetrahydrofolate reductase C677T (MTHFR) increased the risk of severe preeclampsia, fetal growth restriction, very preterm delivery, placental abruption and a composite of these outcomes also including stillbirth. Patients and methods: In a nested case– cohort study of pregnant women in Denmark, we genotyped 2032 cases and 1851 random controls. Each of the medical records of the cases was validated. We calculated both genomic and allelic models, and present both models. We also performed sensitivity analyses adjusting for parity, age, smoking, body mass index and socioeconomic status. Results: In the allelic models, FV Leiden increased the risk of the composite outcome (odds ratio [OR] 1.4, 95% confidence interval [CI] 1.1–1.8), severe preeclampsia (OR 1.6, 95% CI 1.1–2.4), fetal growth restriction (OR 1.4, 95% CI 1.1–1.8) and placental abruption (OR=1.7 (95% CI 1.2–2.4). In the sensitivity analyses, adjustment diminished these estimates slightly. PTM was not significantly associated with any of the outcomes, and MTHFR was only significantly associated with severe preeclampsia (OR 1.3, 95% CI 1.1–1.6). Conclusion: FV Leiden predisposes to adverse pregnancy outcomes in a setting of Scandinavian women. Keywords: cardiovascular, complication, hypertension, polymorphism, pregnancy.

Correspondence: Jacob Alexander Lykke, Department of Obstetrics and Gynecology, Roskilde Hospital, 4000 Roskilde, Denmark. Tel.: +45 4732 4200; fax: +45 4732 4200. E-mail: [email protected] Received 8 March 2012, accepted 3 May 2012

Inherited thrombophilias have for a decade been linked to adverse pregnancy outcomes such as preeclampsia, fetal growth restriction, preterm delivery, placental abruption and stillbirth [1,2]. These complications in pregnancy may vary in severity, but the severe cases are potentially life-threatening conditions for the woman and offspring, and may have health consequences later in life for both [3–5]. The proposed thrombophilic constituent of these adverse pregnancy outcomes involves microthrombi formation in the vascular bed of the placenta [6] hereby leading to dysfunctional placenta and overt pregnancy complications [7,8]. From an epidemiological aspect, the thrombophilic contribution is suggested in the link between these adverse pregnancy outcomes and subsequent cardiovascular events in the woman [9–11] as well as in her parents [12]. Many previous studies link factor (F)V Leiden G1691A, prothrombin mutation G20210A (PTM) and methylenetetrahydrofolate reductase C677T (MTHFR) with adverse pregnancy outcomes but with divergent strengths of associations. Chance findings [13] or heterogeneity in study design, size and population [14–16] may explain this variation: most associations from small case-control studies have not been replicated in subsequent cohort studies [14], and differences in severity of the outcomes may affect the strength of the associations [16,17]. Evolutionary migrations are further tied to differences in frequencies of these thrombophilias throughout the world [18]. To elucidate the possible link between these three thrombophilias and adverse pregnancy outcomes, we designed a large nested case–cohort study within the Danish National Birth Cohort consisting of all cases and a randomly selected sample of the cohort. Patients and methods Population

In 1997–2002, about half of all pregnant women in Denmark were invited to participate in the Danish National Birth Cohort  2012 International Society on Thrombosis and Haemostasis

Thrombophilias in pregnancy 1321

(DNBC), and about two-thirds of these gave written, informed consent. The details of this cohort are described elsewhere [19]: a total of 101 042 pregnancies to 91 661 women were followed with telephone interviews. Using the unique Personal Identification Number (PIN) of every citizen in Denmark, we traced all patients in the National Patient Registry (NPR), which collects information on all discharge diagnoses from hospitals and deliveries in Denmark. Maternal blood samples were obtained at the first antenatal visit in the first trimester and again in the second trimester at their general practitioner, sent by mail to the national biobank at ÔStatens Serum InstitutÕ, separated into plasma and buffy coat, and frozen for later analysis. Within the DNBC, we conducted a nested case–cohort study of singleton pregnancies. If a woman had multiple pregnancies in the DNBC, only the first pregnancy was included. By linkage to the NPR, we identified women with severe preeclampsia, fetal growth restriction, very preterm delivery before 34 full weeks of gestation, placental abruption and stillbirth. The reference control cohort from the DNBC was a priori set to include 2000 women, which was the expected number of the largest subgroup of adverse pregnancy outcomes. These women were drawn from the entire DNBC using a computed random process in SAS (SAS Institute Inc., Cary, NC, USA); overlap with the case groups was allowed. Definition of outcomes

Gestational age was based on ultrasound evaluation or last menstruation period if no ultrasound evaluation was performed (< 2%). Fetal growth was assessed as the birthweight standardized for gestational age and gender in a national, ultrasound idealized distribution [20] yielding a z-score; smallfor-gestational age (SGA) was defined as two standard deviations below the mean equivalent to the 3rd percentile. Severe preeclampsia, including eclampsia and HELLP syndrome, was defined according to the American Congress of Obstetricians and Gynecologists definitions from 2002 [21]: preeclampsia (new onset hypertension combined with proteinuria) in the presence of one of the following: a systolic blood pressure ‡ 160 mmHg, diastolic blood pressure ‡ 110 mmHg, severe proteinuria of ‡ 5 g per 24 h or > 2+ on dipstick, severe headaches, thrombocytopenia (platelet count < 100 000 per lL), persistent epigastric/right upper-quadrant pain or abnormal liver enzymes (aspartate aminotransferase [ASAT] ‡ 100 IU L)1 or lactate dehydrogenase [LDH] ‡ 1000 IU L)1); HELLP syndrome was defined as a combination of increased LDH and ASAT, and thrombocytopenia irrespective of hypertension. Eclampsia was diagnosed if convulsions developed in preeclamptic women (n = 18). Placental abruption was diagnosed on the basis of the clinical findings including vaginal bleeding or tender and hypertonic uterus, or both, and confirmed by either the observation of a blood clot behind the placenta or a detached placenta at Cesarean delivery. A stillbirth was defined as fetal demise before labor.  2012 International Society on Thrombosis and Haemostasis

Validation

A trained research midwife validated the gestational age, birth weight and diagnoses of case women identified in the NPR by review of the medical record according to the definitions above. If the diagnosis was not confirmed, the woman was excluded from the specific case group. In contrast, if the woman also had coexisting diagnoses of other adverse pregnancy outcomes, which was not registered in the NPR, these validated diagnoses were included in the respective case groups even although they were not registered as such in the NPR. A priori, we chose to exclude cases with fetal malformations and women with uterine malformations, preexisting medical conditions including hypertension and diabetes, cervical conization and placenta accreta or previa as these conditions affect the risk of adverse pregnancy outcomes. From the questionnaire information in the DNBC, we extracted the pre-pregnancy body mass index (BMI), smoking status in pregnancy and socioeconomic status; the latter was computed as a three-level variable based on educational level and work titles. Analysis

The stored blood samples from the DNBC were shipped to Celera (Alameda, CA, USA). Genotyping of individual DNA samples was done by performing two RT-PCR reactions, using 0.3 ng DNA from each sample and allele-specific primers [22]. Personnel involved in the genotyping were blinded to the clinical outcomes of the women and vice versa. The genotype data were merged with the validated clinical data set for final statistical analysis. Logistic regression models were used to calculate the odds ratio (OR) with 95% confidence interval (95% CI) and twosided P-values. We calculated both genomic and allelic models, and present both models emphasizing the allelic model. We also performed stratified analyzes for parity and smoking status to evaluate possible interactions, and calculated adjusted models controlling for parity, maternal age, body mass index, smoking, and socioeconomic status; these sensitivity analysis are not presented. SAS v9 was used for all calculations. The study was approved by the Danish Research Committee and the Danish National Data Protection Agency; all participants gave written, informed consent to participate in the DNBC. Results We identified cases through NPR yielding 519 cases of severe preeclampsia, 1025 cases of very preterm delivery, 1999 cases of fetal growth restriction and 499 cases of placental abruption. After validation of the medical records, with exclusion and inclusion of cases, there remained 315 cases of severe preeclampsia (61%), 743 cases of very preterm delivery (72%), 1619 cases of fetal growth restriction (81%) and 378 cases of placental abruption (76%). Blood samples were available and genotyping successful in 2032 of 2349 cases

1322 J. A. Lykke et al

(87%) with a composite outcome; 263 of 315 cases (83%) with severe preeclampsia; 1227 of 1415 cases (87%) with fetal growth restriction; 621 of 746 (83%) of cases with very preterm delivery; 308 of 377 cases (82%) with placental abruption; and 1856 of 2000 women (93%) in the control cohort. All three polymorphisms were in Hardy–Weinberg equilibrium. The demographics of the control cohort and each of the case groups are presented in Table 1. Obesity was associated with severe preeclampsia and smoking was associated with all other adverse pregnancy outcomes (APOs) (excluding severe preeclampsia). Low socioeconomic status was associated with all APOs. Nulliparity was associated with increased risk of all APOs (excluding placental abruption). The associations between the thrombophila and APO are presented in Table 2. In the control cohort sample, 6% were heterozygote and 0.4% were homozygote for FV Leiden. In the allelic model, FV Leiden increased the risk for the composite outcome as well as for severe preeclampsia, fetal growth restriction and stillbirth by 40% to 60% increase in risk; adjustment changed the estimates minimally. In the control cohort sample, 2% were heterozygote and 0.1% (n = 1) were homozygote for PTM. In the allelic models, PTM was not associated with any adverse pregnancy outcomes, and the genomic models for the heterozygote paralleled the allelic models. Adjustment only changed the estimates slightly. In the control cohort sample, 43% were heterozygote and 8% were homozygote for MTHFR. In the allelic model, MTHFR was associated with severe preeclampsia (OR 1.27, 1.05–1.55), and adjustment diminished this estimate slightly. None of the other outcomes were associated with MTHFR. Discussion In this case–cohort study, we found FV Leiden associated with several adverse pregnancy outcomes including the composite outcome, but PTM and MTHFR were not consistently associated with any of the case groups we studied. These three thrombophilias have been associated with preeclampsia but with divergent strengths. In a meta-analysis of case–control studies by Lin and August, FV Leiden was associated with severe preeclampsia (OR 2.24; 95% CI 1.28– 3.94), albeit with heterogeneity among the publications. The same study also demonstrated a lack of association between severe preeclampsia and MTHFR and PTM [23]. In contrast, a recent large Swedish case–cohort study reported that FV Leiden carriers were not at increased risk of severe preeclampsia [24]. A systematic review and meta-analysis of prospective cohort studies by Rodger et al. from 2010 also reported no significant association between FV Leiden and PTM and unspecific preeclampsia (FV Leiden OR 1.23, 95% CI 0.89– 1.70) [14]. However, the review of cohort studies by Dudding et al. [15] from 2008, did report a significant association between FV Leiden and preeclampsia (OR 1.49; 95% CI 1.13– 1.96). Interestingly, although the two reviews included only cohort studies, Dudding et al. included a large Norwegian study by Nurk et al. from 2006 [25] reporting a significant

positive association, which was not included by Rodger et al. as they only included prospective cohort designs. HELLP syndrome, a variant of severe preeclampsia, has been investigated previously in a case–control study by Mutze et al. who found FV Leiden associated with HELLP syndrome (OR 4.45, 95% CI 1.31–15.1), but no association with PTM or MTHFR [26]. In aggregate, it seems that the case–control studies of FV Leiden favor a two-fold increase in risk of preeclampsia, whereas the cohort studies in general suggest a weaker or no association; however, the severity and type of the preeclampsia may contribute to this discrepancy. No significant effect of PTM or MTHFR has been found. The associations with fetal growth restriction, mainly defined as the 10th percentile, have been reviewed recently by Facco et al., who also carried out meta-analyzes for each of the three thrombophilias [27]. They found an overall association between FV Leiden and fetal growth restriction (OR 1.23, 95% CI 1.04– 1.44) with higher estimates in case–control studies than in cohort studies, probably as a result of publication bias. They did not find PTM or MTHFR associated with fetal growth restriction. Notably, our definition of fetal growth restriction is more stringent than the above-mentioned studies and this may explain the different estimates. Studies on preterm delivery are sparse. In a large cohort of 4872 women, Kocher et al. [28] found no association with FV Leiden, but with PTM (OR 3.12, 95% CI 1.07–9.13). In contrast, Hiltunen et al. [29] in a Finish case–control study found FV Leiden to be associated with late preterm delivery (gestational age [GA] 32–36 weeks), but not with very preterm delivery (GA < 32 weeks); PTM was not associated with preterm delivery. We did not find evidence of any association with very preterm delivery. Studies on placental abruption are vast. A review by Zdoukopoulos and Zintzaras [30] in 2008 found that FV Leiden and PTM were associated with placental abruption. FV Leiden was associated with the overall risk (OR 3.42, 95% CI 1.42–8.25), and in Caucasian women this estimate was slightly higher. PTM increased the overall risk (OR 6.67, 95% CI 3.21–13.9), and again in Caucasian women this estimate was slightly higher. Our estimates are lower than these, but comparable to the estimates from a Swedish study by Procha´zka et al. [31] who found an OR of 1.5 (95% CI 0.9–2.7) for FV Leiden. The strength of the present study is the sampling from a large cohort in investigating relatively rare outcomes in combination with relatively rare exposures using a nested case–control model. The benefit of this design is the amelioration of the potential selection bias introduced in the control sample. We assessed baseline characteristics of the women and controlled for these in the adjusted models: these variables did not function as confounders, but could potentially function as intermediates only if the genotype influences risk factors for pregnancy complications (pleiotrophy). In the sensitivity analyzes, the adjustment did not significantly alter the estimates, which makes random variation probable.  2012 International Society on Thrombosis and Haemostasis

Thrombophilias in pregnancy 1323 Table 1 Baseline characteristics and outcomes of the population Control cohort (n = 1851)

Maternal age (years) < 25 years 25–30 years 30–35 years > 35 years Body mass index (kg m)2) < 25 25–30 30–35 > 35 Missing Smoking No Yes Missing Socioeconomic status High Middle Low Missing Parity Nulliparae Parous Missing Gender Male Female Missing Gestational age (days) At term, ‡ 37 weeks 34–36 weeks 28–33 weeks < 28 weeks Missing Birthweight (g) > 2500 g 1000–2500 g < 1000 g Missing Fetal growth (Z-score) > )2 £ )2 (SGA) Missing Severe preeclampsia Pla cental abruption Stillbirth Veneous thrombosis

Composite outcome (n = 2032)

Severe preeclampsia (n = 263)

Fetal growth restriction (n = 1227)

Very preterm delivery (n = 621)

Placental abruption (n = 308)

n/mean

%/SD

n/mean

%/SD

n/mean

%/SD

n/mean

%/SD

n/mean

%/SD

n/mean

%/SD

30.3 185 731 674 261 23.5

4.3 10% 39% 36% 14% 4.2

30.2 268 771 654 339 23.6

4.8 13% 38% 32% 17% 4.7

30.2 35 106 82 40 25.6

4.9 13% 40% 31% 15% 4.9

30.2 169 461 398 199 23.4

4.9 14% 38% 32% 16% 4.7

30.1 91 228 199 103 24.0

4.9 15% 37% 32% 17% 4.9

30.4 39 112 98 59 23.2

5.0 13% 36% 32% 19% 4.3

1275 343 119 34 80

69% 19% 6% 2% 4%

1391 363 149 54 75

68% 18% 7% 3% 4%

134 74 32 12 11

51% 28% 12% 5% 4%

859 216 83 33 36

70% 18% 7% 3% 3%

411 104 56 21 29

66% 17% 9% 3% 5%

222 47 16 7 16

72% 15% 5% 2% 5%

1516 303 32

82% 16% 2%

1355 645 32

67% 32% 2%

224 35 4

85% 13% 2%

745 466 16

61% 38% 1%

437 178 6

70% 29% 1%

212 90 6

69% 29% 2%

897 726 174 54

48% 39% 9% 3%

869 833 281 49

43% 41% 14% 2%

127 99 32 5

48% 38% 12% 2%

518 512 173 24

42% 42% 14% 2%

259 248 97 17

42% 40% 16% 3%

129 123 46 10

42% 40% 15% 3%

928 919 4

50% 50% 0%

1345 684 3

66% 34% 0%

197 65 1

75% 25% 0%

859 368 0

70% 30% 0%

410 209 2

66% 34% 0%

153 154 1

50% 50% 0%

958 887 6 279

52% 48% 0% 13

1032 983 17 255

51% 48% 1% 32

131 131 1 247

50% 50% 0% 27

612 615 0 267

50% 50% 0% 26

318 289 14 214

51% 47% 2% 20

175 130 3 247

57% 42% 1% 33

1744

94%

1157

57%

110

42%

913

74%

0

0%

141

46%

75 25 3 4 3558 1771 66 7 7 0.02

4% 1% 0% 0% 589 96% 4% 0% 0% 1.14

254 512 109 0 2268 811 1085 132 4 )1.70

13% 25% 5% 0% 744 40% 53% 6% 0% 1.47

70 71 12 0 2245 98 139 26 0 )1.46

27% 27% 5% 0% 921 37% 53% 10% 0% 1.49

142 151 21 0 2231 498 658 71 0 )2.62

12% 12% 2% 0% 601 41% 54% 6% 0% 0.70

0 512 109 0 1519 24 461 132 4 )1.16

0% 82% 18% 0% 599 4% 74% 21% 1% 1.83

64 75 28 0 2483 161 120 27 0 )0.64

21% 24% 9% 0% 1001 52% 39% 9% 0% 1.43

1788 48 15 14 18 5 0

97% 3% 1% 1% 1% 0% 0%

783 1227 22 263 308 127 10

39% 60% 1% 13% 15% 6% 0.5%

157 104 2 263 11 3 3

60% 40% 1% 100% 4% 1% 1.1%

0 1227 0 104 52 40 6

0% 100% 0% 8% 4% 3% 0.5%

430 172 19 83 103 54 4

69% 28% 3% 13% 17% 9% 0.6%

252 52 4 11 308 14 1

82% 17% 1% 4% 100% 5% 0.3%

SGA, small-for-gestational age

Population stratification may be one explanation for the variation in published estimates for thrombophilia and adverse pregnancy outcomes: differences in allele frequencies within Europe and also across the world can be traced back to  2012 International Society on Thrombosis and Haemostasis

evolutionary migrations [18]. Also, gene–environment and gene–gene interactions may distort the effects, and these are sample size demanding to evaluate. This necessitates attention in evaluating the external validity of any study in this area.

1.60 (0.80–3.20) 1.18 (0.75–1.85) 1.13 (0.88–1.46) 1 (reference) 1.11 (0.92–1.34)

Also, the low strength of these associations do not support a strong causal link between thrombophilias and adverse pregnancy complications, but rather contributors to the development of the complications [13]. In conclusion, we found that FV Leiden, but not PTM and MTHFR, was associated with severe adverse pregnancy outcomes in a population of Scandinavian women, albeit with ORs all lower than two. This makes the clinical usefulness limited, as these ORs will translate into low absolute risk differences. SGA, small-for-gestational age; GA, gestational age; OR, odds ratio; NA, not applicable; PTM, prothrombin mutation G20210A; MTHFR, methylhydrofolate reductase C677T.

26 (8.5%) 139 (45.6%) 140 (45.9%) 1.36 (0.78–2.37) 1.10 (0.78–1.54) 0.93 (0.77–1.13) 1 (reference) 1.00 (0.86–1.15) 54 (8.7%) 254 (41.0%) 311 (50.2%) 0.86 (0.51–1.45) 1.27 (0.98–1.66) 1.02 (0.88–1.19) 1 (reference) 1.08 (0.97–1.21) 117 (9.6%) 521 (42.7%) 582 (47.7%) 1.14 (0.50–2.62) 1.74 (1.13–2.69) 1.19 (0.91–1.57) 1 (reference) 1.27 (1.05–1.55) 31 (11.8%) 118 (45.0%) 113 (43.1%) 0.99 (0.64–1.52) 1.24 (0.98–1.57) 1.00 (0.88–1.15) 1 (reference) 1.07 (0.97–1.18) 191 (9.5%) 856 (42.4%) 974 (48.2%) 143 (7.8%) 793 (43.1%) 906 (49.2%) Allelic model MTHFR TT CT CC Allelic model

1.66 (1.15–2.40) NA 1.73 (0.85–3.54) 1 (reference) 0 (0.0%) 10 (3.2%) 298 (96.8%) 1 (0.1%) 19 (1.6%) 1205 (98.4%) 1 (0.4%) 4 (1.5%) 255 (98.1%) 1 (0.1%) 35 (1.9%) 1809 (98.0%) Allelic model PTM AA AG GG

2 (0.1%) 36 (1.8%) 1986 (98.1%)

1.41 (1.13–1.76) 1.82 (0.17–20.11) 0.94 (0.59–1.50) 1 (reference)

1.63 (1.10–2.42) 7.09 (0.44–113.8) 0.81 (0.29–2.30) 1 (reference)

1.43 (1.12–1.83) 1.50 (0.09–24.02) 0.82 (0.46–1.43) 1 (reference)

1 (0.2%) 15 (2.4%) 599 (97.4%)

1.32 (0.98–1.79) 3.01 (0.19–48.25) 1.29 (0.70–2.39) 1 (reference)

0.91 (0.11–7.39) 1.87 (1.25–2.81) 1 (reference) 1 (0.3%) 34 (11.3%) 266 (88.4%) 6 (0.5%) 111 (9.3%) 1077 (90.2%) 0 (0.0%) 30 (11.7%) 227 (88.3%) 7 (0.4%) 115 (6.4%) 1685 (93.2%) AA AG GG FV Leiden

10 (0.5%) 180 (9.1%) 1789 (90.4%)

1.35 (0.51–3.54) 1.47 (1.16–1.88) 1 (reference)

NA 1.94 (1.27–2.96) 1 (reference)

1.34 (0.45–4.00) 1.51 (1.15–1.98) 1 (reference)

4 (0.7%) 50 (8.2%) 553 (91.1%)

1.74 (0.51–5.97) 1.33 (0.94–1.87) 1 (reference)

Model OR 95% CI Numbers n% Model OR 95% CI Numbers n (%) Model OR 95% CI Numbers n (%) Model OR 95% CI Numbers n (%) Model OR 95% CI Numbers n (%) Numbers n (%) Genotype SNP

Composite outcome Control cohort

Table 2 Associations between polymorphisms and pregnancy outcomes

Severe preeclampsia

Fetal growth restriction (SGA)

Very preterm delivery

Placental abruption

1324 J. A. Lykke et al

Acknowledgements The Danish National Research Foundation established the Danish Epidemiology Science Centre, which initiated and created the Danish National Birth Cohort. Furthermore, the cohort was established with the support of a major grant from this foundation. Additional support for the Danish National Birth Cohort is obtained from the Pharmacy Foundation, the Egmont Foundation, the March of Dimes Birth Defects Foundation, and the Augustinus Foundation. The Research Committee in Denmark approved the data collection in the Danish National Birth Cohort research database. Approval to use data from the birth cohort for the present study on preterm birth was obtained. The Danish Data Protection Agency approved the storage, handling, and linkage of the data. Celera financed the genotyping and shipping of the blood samples.

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