Preeclampsia, fetal growth restriction, stillbirth, and placental abruption are serious pregnancy complications that represent important contributors to both perinatal and maternal morbidity and mortality. Prediction and prevention of these complications remain limited, with timely delivery representing the only effective treatment strategy in cases of preeclampsia, fetal growth restriction, and placental abruption. The subsequent delivery of a preterm neonate may require the investment of significant community and family financial, physical, intellectual, and emotional resources to fulfill their potential. The longer-term sequelae of fetal growth restriction, such as adult-onset diabetes, hypertension, and obesity,1 and the effects of prematurity represent additional burdens.
The precise causes of these conditions are largely idiopathic. A common finding among patients experiencing many of these complications is that of areas of thrombosis on histologic examination of the placenta.2,3 This has prompted the suggestion that disturbances in coagulation may contribute to the etiology of these conditions.
A large number of case–control studies have reported associations between adverse pregnancy outcomes and inherited thrombophilias, but the results have been heterogeneous; there have been few prospective cohort studies. Meta-analyses of these case–control studies have consistently demonstrated positive associations between thrombophilias and adverse pregnancy outcomes, but even these findings may be misleading because of publication bias.4–6 Furthermore, there is limited information about the natural history of pregnancy in women who carry inherited thrombophilias but have not demonstrated any symptoms related to these thrombophilias. In addition, the thrombomodulin C1418T and methylenetetrahydrofolate reductase enzyme (MTHFR) A1298C polymorphisms have the potential to interact with other thrombophilias, including the factor V Leiden and MTHFR C677T polymorphisms.
The aim of this study was to estimate prospectively the risk of adverse pregnancy outcomes in asymptomatic carriers of inherited thrombophilia polymorphisms including factor V Leiden, prothrombin gene mutation, MTHFR C677T and A1298C polymorphisms, and thrombomodulin C1418T polymorphism.
This prospective cohort study was undertaken in Melbourne, Australia, at The Royal Women's Hospital and the Mercy Hospital for Women. These hospitals are tertiary referral hospitals delivering approximately 6,000 and 5,000 patients per annum, respectively. Institutional research and ethics approval was obtained at both centers before commencement of the study. All participants were antenatal patients who spoke sufficient English to provide informed consent and were planning to deliver at either hospital. Pregnant women were approached while attending their first antenatal visit at the hospital if they met the following eligibility criteria:
1. Estimated gestation less than 22 completed weeks (based on last menstrual period dates or ultrasonography)
2. No previous pregnancy that had continued beyond 20 weeks of gestation
3. No personal or family history of venous thromboembolism
4. No personal or family history of thrombophilia, including the antiphospholipid syndrome
Exclusion criteria included multiple pregnancy, major congenital anomalies, essential hypertension, recurrent (three or more) miscarriages, underlying medical probl ems associated with a risk of adverse pregnancy outcomes (eg, systemic lupus erythematosus, renal disease, preexisting diabetes), and chemical dependency (other than cigarettes).
Written consent was obtained from all eligible women who agreed to take part. External validation of the accuracy of the data provided by the patient was not specifically sought (eg, if the patient did not report personal or family history of venous thromboembolism, she was regarded as eligible for the study). Data regarding patients who were deemed ineligible to participate or who declined to participate were not kept.
Venous blood was collected from participants, and genotyping for factor V Leiden mutation, prothrombin gene mutation, MTHFR mutations C677T and A1298C, and thrombomodulin mutation was performed in the Pregnancy Research Centre laboratories at The Royal Women's Hospital in an ABI 7700 Sequencer (Applied Biosystems, Foster City, CA) using previously described techniques.7 These assays were performed after the pregnancy had been completed.
Pregnancy outcome data were recorded for all patients at the time of delivery by the midwife caring for the patients. Blinded to the results of laboratory assays, a single author (J.M.S.) reviewed the medical records of all participants to retrieve and verify this pregnancy data after delivery. In addition to basic demographic, pregnancy, delivery, and neonatal data, the medical record was scrutinized for the presence of specific pregnancy complications, which are defined in Table 1.
The primary outcome was a composite measure of the more severe pregnancy complications, including severe preeclampsia, fetal growth restriction less than the fifth centile for sex and gestation,9 placental abruption, stillbirth, and neonatal death. Secondary outcomes were these complications individually as well as a composite measure of several less severe complications, including mild preeclampsia, gestational hypertension, fetal growth restriction between the 5th and 10th centiles, prematurity, 5-minute Apgar score less than 7, and admission to the neonatal nursery for any reason. Newborns admitted to the neonatal intensive care unit after initial hospital discharge were not included.
We estimated that the frequency of the composite primary outcome would be approximately 7% (fetal growth restriction 5%, severe preeclampsia 1%, placental abruption 0.5%, and stillbirth or neonatal death 0.5%). We calculated that a sample size of 1,630 women would be required to show a twofold increase (odds ratio [OR] 2.0) in the frequency of severe pregnancy complications (as defined by the primary outcome) with 80% power and α=.05. The calculated sample size was increased by approximately 20% to account for miscarriages occurring in the first and second trimesters and loss to follow-up through patients transferring to other hospitals for delivery. Accordingly, a sample size of 2,000 was required to ensure that the study was adequately powered to answer the question of whether an underlying thrombophilia increased the risk of the primary composite adverse outcome.
Logistic regression using Stata 7.0 (Stata Corporation LP, College Station, TX) was used to investigate the relationship between the various thrombophilic markers and adverse pregnancy outcomes. Confounders considered in the analysis were maternal age (at recruitment and delivery), body mass index, ethnicity, smoking status, folate use, number of previous miscarriages or terminations and presence of gestational diabetes. A P value less than .05 was regarded as significant.
A total of 2,034 women were recruited to this study between September 2000 and August 2003, although as explained in Figure 1, a number of women were excluded from the analysis. A total of 1,707 women were included in the final analysis. For logistic reasons, it was not possible to recruit all eligible women consecutively; hence, this represents a convenience sample of eligible women.
Table 2 describes the baseline characteristics of the cohort. Participants were recruited equally from both participating institutions. All patients had ultrasonography performed during the second trimester to confirm dates and assess fetal morphology. All personnel recording these data were blinded to the patient's thrombophilia status.
The prevalence of thrombophilia among the cohort of women with known pregnancy outcome is shown in Table 3.
No differences were observed between the women lost to follow-up and those included in the study with regard to maternal age, ethnicity, number of previous pregnancies, gestation at recruitment or blood sampling, and body mass index. Importantly, there were no significant differences observed in the overall prevalence of the inherited thrombophilias among the women included in the study compared with the women who were lost to follow-up.
The primary composite outcome of severe preeclampsia, fetal growth restriction (birth weight less than the fifth centile for sex and gestation9), placental abruption, or stillbirth or neonatal death occurred in 136 patients (8.0% of the cohort of 1,707 included women). Severe preeclampsia developed in 32 women (1.9% of the cohort), 91 women delivered growth-restricted neonates (5.3%), nine women experienced placental abruption (0.5%), six women had stillbirths (0.4%), and four women had neonatal deaths (0.2%). The proportion of each pregnancy complication adds up to greater than 100% of patients experiencing primary outcomes because six patients experienced more than one primary outcome event. One patient with severe preeclampsia also had fetal growth restriction; one patient delivered a stillborn, growth-restricted fetus. Three patients with placental abruption delivered stillborn fetuses, and one patient with placental abruption delivered a growth-restricted neonate. All patients with stillbirth underwent further investigations to determine underlying etiologic factors, including fetal karyotyping, maternal viral serology, and testing for antiphospholipid syndrome. Two stillbirths also had postmortem investigations performed. The results of these investigations were all normal, which is consistent with a classification of these cases as idiopathic stillbirths.
Logistic regression was used to determine the association between inherited thrombophilia and adverse pregnancy outcomes. Table 4 demonstrates the univariable relationship between the individual thrombophilias tested and the composite primary outcome. A significant association between the composite primary outcome and prothrombin gene mutation was observed, with an OR of 2.45 (95% confidence interval [CI] 1.06–5.64, P<.05). In addition, a significant association was observed between the MTHFR1298 polymorphism and the primary composite outcome (OR 0.44, 95% CI 0.20–0.98). A significant relationship was not observed between any of the other inherited thrombophilias and the primary outcome.
Multivariable logistic regression was used to model the relationship between these variables and the composite primary outcome. This confirmed that prothrombin gene mutation, smoking during pregnancy, and ethnicity are independent predictors of the composite primary outcome (Table 4). In addition, homozygosity for the MTHFR 1298 polymorphism reduced the risk of development of the primary outcome.
To assess the relationship between the composite secondary outcome, patients who experienced the composite primary outcome were excluded. Of the remaining 1,571 women, 336 experienced one of the secondary outcome events. This included 59 women with gestational hypertension (3.8% of the cohort of 1,571 women), 65 women with mild preeclampsia (4.1%), 75 women delivering before 37 weeks (4.8%), 16 women delivering neonates with 5-minute Apgar scores less than 7 (1.0%), 86 women delivering growth-restricted neonates with birth weights between the 5th and 10th centiles for gestation and sex (5.5%), and 108 women with newborns admitted to the neonatal nursery (6.9%). As with women experiencing primary outcome complications, several women experienced more than one secondary outcome complication (eg, premature delivery and neonatal nursery admission) but, for the purposes of analysis, were included only once.
Univariable logistic regression was used to assess the impact of the inherited thrombophilias on the composite secondary outcome (Table 5). None of the inherited thrombophilias were associated with an increased risk of the composite secondary outcome. Analysis of other factors revealed that maternal body mass index and gestational diabetes were independently associated with an increased risk of the composite secondary outcome (data not shown).
We undertook prespecified analyses to investigate the relationship between individual pregnancy complications and inherited thrombophilias (Table 6). Placental abruption was significantly associated with prothrombin gene mutation (OR 12.15, 95% CI 2.45–60.39, P=.002), whereas factor V Leiden conferred an increased risk of stillbirth (OR 8.85, 95% CI 1.60–48.92, P=.01).
Homozygosity for the MTHFR 1298 polymorphism was associated with a significant reduction in the risk of severe fetal growth restriction (birth weight less than fifth centile for sex and gestation) (OR 0.35, 95% CI 0.12–0.97, P=.04). This association remained when the cohort of pregnancies complicated by growth restriction with birth weight less than the 10th centile was examined (OR 0.42, 95% CI 0.21–0.83, P=.01). Even after adjusting for smoking status and ethnicity, this relationship remained for the entire group with birth weight less than the 10th centile (OR 0.23, 95% CI 0.08–0.66, P=.006), as well as the subgroup with birth weight less than the fifth centile (OR 0.11, 95% CI 0.01–0.81, P=.03).
No significant associations were observed between any inherited thrombophilia polymorphisms and preeclampsia (mild and severe), neonatal death, gestational hypertension, premature delivery, low Apgar scores, and neonatal nursery admissions. No patients in this cohort experienced venous thromboembolism.
This large antenatal cohort describes the pregnancy outcomes in asymptomatic women whose thrombophilia status is known. Importantly, women and their caregivers were blinded to the results of thrombophilia testing, making it possible to examine the natural history of women with thrombophilias during pregnancy without introducing the bias of selective treatment.
Our study has demonstrated several important findings. First, we have demonstrated that asymptomatic carriers of prothrombin gene mutation have up to a 3.6-fold increase in the risk of adverse pregnancy outcomes, including severe preeclampsia, fetal growth restriction, placental abruption, or stillbirth. Although patients with this mutation experienced one or more of these complications, the association between prothrombin gene mutation and placental abruption was particularly strong. This finding is consistent with previous case–control studies,11,12 but our study reports this association in a prospective study among asymptomatic women.
Second, we observed a significant reduction in the risk of serious pregnancy complications among homozygous carriers of the MTHFR 1298 polymorphism. Although it is possible that this finding has arisen by chance alone, the larger numbers of patients experiencing fetal growth restriction increase the power to detect a statistically significant result. It should be noted that linkage disequilibrium was observed between the two MTHFR loci. Thus, patients homozygous for the MTHFR 1298 polymorphism were neither heterozygous nor homozygous for the MTHFR 677 polymorphism. As such, patients homozygous for the MTHFR 1298 polymorphism were “protected” from any potential deleterious effects associated with carrying the MTHFR 677 polymorphism. Although we can only speculate further about the mechanism by which homozygosity for the MTHFR 1298 polymorphism exerts its beneficial effects, it is noteworthy that the effect was seen predominantly in protecting pregnancies from severe fetal growth restriction.
A third important finding of this study was the confirmation that most asymptomatic patients with inherited thrombophilias can experience problem free pregnancies. These findings have important implications when considering treatment strategies in thrombophilic patients. It is important to note, however, that this patient cohort was asymptomatic at the commencement of pregnancy. It is plausible that thrombophilic patients who have been detected on the basis of poor past medical, obstetric, or family histories may express a different phenotype and may have higher complication rates than their asymptomatic counterparts.
Although only prothrombin gene mutation was associated with an overall increase in the risk of the composite primary outcome, we did observe an association between factor V Leiden and stillbirth. These data must be regarded with caution because of the small numbers in this patient group (n=6). Nevertheless, this finding is of interest and is certainly consistent with previous case–control studies13–15 and meta-analyses.4,16
Analysis of the composite secondary outcome and the various secondary outcome variables supports the view of previous authors that thrombophilias are not strongly associated with milder pregnancy complications and that, if an association does exist, it is only with the more serious pregnancy complications.
Venous thromboembolism affects approximately 1 in 1,000 pregnant women.17 As such, we would have expected one or two women to have experienced a venous thromboembolic event among this cohort of 1,707 women. Although it is certainly plausible that no one experienced an event during this study, our ascertainment of venous thromboembolic events may have been incomplete, particularly in the postpartum period.
Although the use of a composite primary outcome as an endpoint increases the chance of achieving a statistically significant result within sample size and financial constraints, there are a number of potential disadvantages. Achieving a statistically significant result with a composite endpoint in such a large cohort with only a modest association raises the question of whether it really is important in an overall clinical sense. This study was underpowered to examine the association between individual pregnancy complications and thrombophilias, but several important associations were observed, such as those between prothrombin gene mutation and abruption and factor V Leiden and stillbirth. It is possible that associations with other specific complications and thrombophilias may be seen if a larger cohort is examined.
To date, there have been six other prospective studies on this topic.18–24 Three of these examined factor V Leiden alone prospectively, and all failed to detect a statistically significant difference in the rate of pregnancy complications such as preeclampsia, fetal growth restriction, placental abruption, or stillbirth among carriers of this mutation18,19,21 (although one did show an association between neonatal death and factor V Leiden21). One study examined factor V Leiden and MTHFR 677 and found no significant increase in the risk of adverse pregnancy outcomes, although the small sample size (n=584) and low prevalence of factor V Leiden (2.7%) meant that this cohort was underpowered to detect a significant difference.20 Two studies investigated factor V Leiden, prothrombin gene mutation, and the MTHFR 677 polymorphism.23,24 No significant correlation was seen between any of these thrombophilias and adverse pregnancy outcomes in one study from Israel.23 The second study, in a relatively small Greek cohort of 392 women, reported significant associations between factor V Leiden and MTHFR 677 and placental abruption.24
This study provides important data regarding the natural history of a range of inherited thrombophilias during pregnancy in women who would not otherwise be suspected of carrying a thrombophilia. Although most patients can experience a problem-free pregnancy, there is an increased risk of adverse pregnancy outcomes among prothrombin gene mutation carriers.
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