1 Department of Reproductive Science, Faculty of Medicine, Imperial College of Science, Technology and Medicine and 2 Department of Haematology, St Mary's Hospital NHS Trust, London, UK
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Abstract |
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Key words: Factor V Leiden/pregnancy outcome/prospective study/recurrent miscarriage
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Introduction |
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Existing data on the association between Factor V Leiden and pregnancy outcome is weak in three important aspects. Firstly, the prevalence of Factor V Leiden amongst women with recurrent miscarriage has been variously reported to be either increased or similar to that amongst parous controls (Balasch et al., 1997; Brenner et al., 1997
; Dizon-Townson et al., 1997
; Grandone et al., 1997
; Metz et al., 1997
; Kutteh et al., 1998
; Ridker et al., 1998
; Souza et al., 1999
; Tal et al., 1999
; Rai et al., 2001
). This discrepancy may partly be explained by selection bias and the small numbers of women that have been included in some studies. Secondly, whilst placental thrombosis in association with Factor V Leiden and other thrombophilic defects has been reported, this is neither a universal nor a specific finding (Mousa and Alfirevic, 2000
) and finally, there are no data on the prospective outcome of untreated pregnancies amongst women with Factor V Leiden and a history of pregnancy loss. Despite these limitations, some pregnant women with Factor V Leiden and a history of recurrent miscarriage have been subjected to heparin thromboprophylaxis, together with its attendant risks, in an attempt to improve their chance of a successful pregnancy (Brenner et al., 2000
; Younis et al., 2000
).
The aim of this prospective observational study was to determine the pregnancy outcome of women with recurrent miscarriage who carry the Factor V Leiden allele and who receive no pharmacological treatment, except for folic acid, during pregnancy.
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Materials and methods |
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Antiphospholipid antibody assays
All women were screened for aPL on at least two occasions >6 weeks apart prior to pregnancy. Lupus anticoagulant (LA) was detected using the dilute Russell's viper venom time (dRVVT) together with a platelet neutralization procedure. Patient samples with a dRVVT ratio (test/control) of 1.1 were retested with a platelet neutralization procedure. A decrease of
10% in the ratio was considered to be positive for LA (Lupus Anticoagulant Working Party on behalf of the BCSH Haemostasis and Thrombosis Taskforce, 1991
). Anticardiolipin antibodies (aCL) were identified using a standardized enzyme-linked immunosorbent assay. An IgG aCL level
5 GPL units and an IgM aCL
3 MPL units was considered to be positive (Khamashta and Hughes, 1993
). Women with a positive test for LA or a positive aCL titre had a confirmatory test performed on a second sample taken at least 6 weeks after the initial sample. Women with persistently positive tests for either LA or aCL were considered to have the antiphospholipid syndrome and were treated with aspirin and heparin during pregnancy (Rai et al., 1997
).
Factor V Leiden
Genomic DNA was extracted from EDTA whole blood using standard techniques. PCR using known primers was used to amplify exon 10 of the Factor V gene, which contains the GA mutation at nucleotide position 1691 (Bertina et al., 1994
). Following amplification, a 20 ml aliquot of the product was digested overnight with 5 IU of the enzyme MnlI (New England Biolabs, Hitchin, Herts, UK) at 37°C. Samples of the digested and undigested PCR product were separated electrophoretically in a 3% agarose gel and the bands visualized using ethidium bromide. The undigested PCR product measures 223 base-pairs (bp) in size. Following cleavage with MnlI, a normal allele produces bands of 37, 82 and 104 bp. A mutant allele produces bands of 82 and 141 bp due to loss of one MnlI cleavage site. Controls on each gel included a known heterozygote, a normal control known not to possess the Factor V Leiden allele and a water blank containing no input DNA.
Management during pregnancy
No woman received pharmacological treatment during pregnancy except for folic acid (400 mg/day) as prophylaxis against neural tube defects. All women attended a dedicated early pregnancy clinic on a weekly basis from 5 weeks of amenorrhoea until 14 weeks gestation. At these visits, ultrasound scans were performed to confirm fetal viability and to assess fetal growth.
Statistical analysis
Discrete variables were analysed using the 2-test and continuous variables analysed using the MannWhitney U-test. Yates's correction was used for cell values of
5. Survival data was analysed using the logrank test. P-values < 0.05 were taken as statistically significant.
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Results |
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No woman in this study had a symptomatic venous thrombosis during the antenatal, intra-partum or post-partum periods.
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Discussion |
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Pregnancy complications associated with Factor V Leiden and other genetic thrombophilic mutations (e.g. prothrombin G2021A and methylenetetrahydrofolate reductase C677T) are thought to be due to thrombosis of the uteroplacental vasculature. However, the relative risk for the development of systemic thrombosis associated with these mutations is much lower (heterozygous Factor V Leiden: 5-fold; heterozygous Factor II G20210A: 2-fold; homozygous MTHFR C677T: 2-fold) than that of previously identified haemostatic defects, such as heterozygous antithrombin deficiency (2050-fold) (Cattaneo et al., 1997; Alhenc-Gelas et al., 1999
; Lane and Grant, 2000
). There has consequently been a shift in emphasis from the restrictive concept of single, dominant causes of thrombosis to emphasizing the potential role of multiple, inherited risk factors. This concept of the aetiology of systemic thrombosis is also likely to be applicable to placental thrombosis. Kupferminc et al. reported that whilst 52% (57/110) of women with an obstetric complication (pre-eclampsia, placental abruption or intrauterine growth restriction) carried one or more of three thrombophilic mutations (Factor V Leiden, prothrombin G2021A or methylenetetrahydrofolate reductase C677T) these mutations were also present in 17% of women with normal pregnancies (Kupferminc et al., 1999
). Clearly not all women who carry a thrombophilic mutation suffer a pregnancy loss and perhaps it is those who carry multiple thrombophilic defects who are at greatest risk. This is supported by the results of a retrospective study which reported that the odds ratio for stillbirth was significantly higher amongst women with combined thrombophilic defects (14.3-fold) compared with those with single defects (between 2.0- and 5.2-fold) (Preston et al., 1996
).
The contribution of the fetal genotype in determining pregnancy outcome demands further investigation. The placenta receives two arterial supplies (one maternal and one fetal) and placental infarction should only occur if both vascular supplies are compromised. The importance of this concept was illustrated in an earlier study which reported that placental infarction was significantly more often seen when the fetus carried the Factor V Leiden allele compared with when it had a normal Factor V genotype (Dizon et al., 1997).
Two small studies, from the same group of investigators, have reported that heparin thromboprophylaxis during pregnancy leads to a high live birth rate amongst women with a history of adverse pregnancy outcome and a thrombophilic defect (Brenner et al., 2000; Younis et al., 2000
). However, both studies were uncontrolled and the results must therefore be interpreted with caution.
The results of our study suggest that attention should be directed at screening women with recurrent miscarriage associated with placental thrombosis for Factor V Leiden and a policy of targeted thromboprophylaxis during future pregnancies should be assessed in the form of a randomized controlled trial.
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Notes |
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Submitted on May 29, 2001; resubmitted on July 30, 2001
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References |
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accepted on September 18, 2001.