Prevalence of genetic markers for thrombophilia in recurrent pregnancy loss

Howard Carp1,2,4, Ophira Salomon3, Daniel Seidman1, Rima Dardik3, Nurith Rosenberg3 and Aida Inbal3

1 Department of Obstetrics and Gynecology, Sheba Medical Center, Tel Hashomer, 2 Department of Embryology, Tel Aviv University and 3 Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine, Tel Aviv University, Israel


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The genetic predispositions to venous thrombosis such as factor V Leiden (FVL) mutation (Arg 506 Gln), prothrombin (FII) gene mutation (G20210A), and mutation of the methylenetetrahydrofolate reductase (MTHFR) gene (C677T) have been reported to be associated with recurrent pregnancy loss. This paper examines the prevalence of markers for genetic thrombophilias in women with recurrent miscarriage. METHODS: The prevalence of FVL, FII G20210A and MTHFR C677T was compared in 108 women with three or more pregnancy losses either exclusively in the first trimester, or mixed first and second trimester losses, with the prevalence found in 82 fertile parous control women without miscarriages. Markers for the thrombophilias were assessed by PCR analysis. RESULTS: Twenty-three of the 108 patients (21.3%), had thrombophilia markers, which was similar to the proportion of patients in the control group (20.7%) with these markers. The prevalences of FVL and FII G20210A were lower in the study group than in the control group (3.7 versus 6.1% for FVL and 4.6 versus 6.1% for FII respectively); however, the difference was not statistically significant. In contrast, the prevalence of MTHFR C677T was higher in the study group than the control population (13 versus 8.5% respectively), but this difference was not statistically significant. There was no statistically significant prevalence of any particular thrombophilia in patients with previous first and second trimester pregnancy losses compared with patients with first trimester losses alone. CONCLUSION: Thrombophilia was not found to be associated with recurrent pregnancy loss.

Key words: habitual abortion/recurrent pregnancy loss/thrombophilia


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recurrent miscarriage is usually defined as the loss of three or more consecutive pregnancies prior to 20 or even 28 weeks of pregnancy (Salat-Baroux, 1988Go; Crosignani and Rubin, 1991Go). However, within this definition is a large and heterogeneous group of patients with many different causes of miscarriage. Thrombosis in decidual vessels is believed to be one such cause, leading to intrauterine growth retardation, fetal death, and possibly recurrent miscarriage. Pregnancy itself is a hypercoagulable state associated with increased levels of procoagulant factors (Stirling et al., 1984Go) and decreased levels of naturally occurring anticoagulants such as protein S (Comp et al., 1986Go). Prothrombotic antibodies such as lupus anticoagulant and anticardiolipin antibodies have been suggested as aetiological agents leading to pregnancy loss (De Wolf et al., 1982Go). Recently, it has been reported that genetic tendencies to thrombosis (hereditary thrombophilias) may also be associated with late pregnancy loss (Preston et al., 1996Go; Rai et al., 1996Go; Grandone et al., 1997Go; Martinelli et al., 2000Go).

The three most common genetic thrombophilias known to predispose to venous thrombosis are: factor V Leiden (FVL), methylenetetrahydrofolate reductase mutation (MTHFR, C677T) (Arruda et al., 1997Go; Nelen et al., 1998Go) and prothrombin gene mutation (FII, G20210) (Poort et al., 1996Go). In FVL, arginine is substituted by glutamine at amino acid residue 506 in coagulation factor V (Bertina et al., 1994Go). Due to this substitution, factor Va becomes resistant to degradation by activated protein C, increasing the risk of venous thromboembolism 3–5-fold in heterozygous individuals (Dahlback, 1995Go). In FII G20210A, a G to A transition at position 20210 of the 3' untranslated region of the factor II gene, has been found to be associated with increased prothrombin levels and a 3-fold increased risk for venous thrombosis in heterozygotes (Poort et al., 1996Go). The homozygous state for the C to T transition at position 677 of MTHFR gene, is associated with hyperhomocysteinaemia which predisposes to thrombosis (Arruda et al., 1996; Guttormsen et al., 1996Go).

Several studies have reported an association between FVL and late pregnancy loss (Preston et al., 1996Go; Grandone et al., 1997Go; Martinelli et al., 2000Go). However, the role of FVL in early pregnancy loss is unclear. It was reported that there is no increased prevalence of FVL, FII G20210A and MTHFR C677T in recurrent early pregnancy loss (Kutteh et al., 1999Go). Hence, the relationship between thrombophilia and recurrent miscarriage is unclear. However, the presumed relationship between thrombophilia and recurrent pregnancy loss has become sufficient to allow the presence of thrombophilas to be an indication for treatment with anticoagulant drugs (Younis et al., 1997Go).

The above-quoted papers refer to the pregnancy loss rate in the presence of thrombophilias. As it is conceivable that thrombophilias were diagnosed in these series due to thromboembolic phenomena, we considered it necessary to assess the prevalence of thrombophilias in patients with recurrent miscarriage and no previous thromboembolic phenomena. In the present study, we assessed the prevalence of the above thrombophilia markers in a cohort of women with unselected recurrent pregnancy loss as a whole, and separately for first and second trimester abortions, as well as whether the patients were primary, secondary or tertiary aborters (primary aborters are women with no previous live births, secondary if there was a live birth followed by miscarriages). We also classified a third group of patients whom we have termed tertiary aborters (Carp, 1994Go). They had miscarriages followed by a live birth and at least three subsequent miscarriages. The results were compared with those of a control group of fertile parous women.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Patients
The study was performed in 108 women out of 169 patients presented to our service with recurrent miscarriage from 1997 to 2000. The other 61 women were lost to follow-up. The clinical features of each patient and her miscarriages were recorded, paying particular attention to whether the previous miscarriages occurred in the first or second trimesters (fetal death between 13 and 26 weeks), and whether the patients had a previous live birth. The diagnosis of trimester of fetal death was made according to the last menstruation, after confirmation by ultrasound scanning. Patients were excluded from this study if there was a previous history of thrombosis, pregnancy at the time of investigation, or if using oral contraceptives. Patients were only included after other presumptive aetiological factors were found to be normal: karyotype of both parents, glucose tolerance test, toxoplasmosis serology, hysterosalpingogram, thereby excluding anatomical abnormalities, intrauterine adhesions and cervical incompetence, thyroid function, serum prolactin levels, normal luteal phase of >=12 days and plasma progesterone >24.8 ng/ml, absence of antinuclear factor, or antiphospholipid antibodies.

The control group consisted of 82 women with no miscarriages who were matched for ethnic origin. Fifty-three of the 82 women had two or more children, whereas 29 women had one child. The Human Investigation Review Board of the Sheba Medical Center approved the study. Each patient was required to sign an informed consent form prior to enrolment in the study.

Laboratory testing
DNA was extracted from EDTA-anticoagulant blood samples using standard methods (Miller et al., 1988Go). FVL was detected by PCR amplification of a 267 bp fragment and MnII digestion, as previously described (Salomon et al., 1999Go). The C677T substitution in the MTHFR gene was identified using Hinfl cleavage of a 198 bp PCR-amplified product (Frosst et al., 1995Go). For identification of the G20210A substitution in the factor II gene, a slight modification of a published method (Poort et al., 1996Go) was used. A 253 bp fragment of the 3' untranslated region of the gene was amplified by PCR using the same primers as described (Salomon et al., 1999Go) and digested simultaneously with HindIII and MspI. The A20210 and G20210 alleles were discernible by this procedure since the A20210 allele bears a restriction site for both enzymes, whereas the G20210 allele bears restriction site only for MspI.

Statistical analysis
Univariate odds ratios (OR) and 95% CI were estimated separately for each polymorphism. The prevalence of each polymorphism was compared between patients and controls with the use of the two-tailed Fisher's exact test. P < 0.05 was taken to be statistically significant.


    Results
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 Materials and methods
 Results
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 References
 
The median age of the study group was lower than that of the control group (median 31, range 23–42 versus median 36, range 21–55 respectively). The study group was not different from the control group with respect to ethnic origin; 53% of the study group were of European–American origin; 41% were of Asian–African origin, and 6% were of mixed origin. The corresponding figures in the control group were 54, 40 and 6% respectively. The 108 patients in the study group had 440 previous pregnancy losses (mean: 4.07 ± 1.75). Twenty-three (21.3%) were diagnosed as having at least one thrombophilia marker, whereas 85 (78.7%) had no thrombophilia (Table IGo). None of the study or control group was homozygous for FVL or FII G20210A mutations. The prevalence of each of the thrombophilias assessed in comparison with the control population is shown in Table IGo. There was no statistically significant difference in the prevalences of either FVL or FII G20210A between study and control groups, although both were more common in the controls. The prevalence of homozygous MTHFR 677T was higher in the study group, but the difference did not reach statistical significance.


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Table I. Prevalence of thrombophilia polymorphisms in women with recurrent pregnancy loss and controls
 
The 22 patients with thrombophilia and 86 patients without thrombophilia were matched for age and number of pregnancy losses (Table IIGo). Of the entire study group of 108 women, 55 were primary aborters, whereas 53 were secondary or tertiary aborters. Among the 22 women with thrombophilia, 14 were primary aborters (64%) whereas eight were secondary or tertiary aborters (36%). Of the 55 patients who were primary aborters, 14 had thrombophilia (25.5%) compared with eight of 53 who were secondary and tertiary aborters (15.1%); however, the difference was not statistically significant.


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Table II. Details of patients with and without thrombophilia in the study group
 
Ten of the 22 women with thrombophilia (45%) had only experienced first trimester miscarriages, whereas 12 had also experienced second trimester abortions (55%). In total, 70 of the 108 patients had first trimester miscarriages alone, compared with 38 who also had second trimester losses. All the second trimester losses were fetal deaths. There were no abortions of live fetuses in this series, nor premature ruptures of the membranes. When the patients with first trimester miscarriages and those with first and second trimester losses were analysed separately, 14.3% of women with first trimester miscarriages were found to have thrombophilia compared with 31.6% of women with first and second trimester losses (Table IIIGo). The small number of patients with each particular thrombophilia precluded the numbers from reaching statistical significance for each particular thrombophilia. However, when the results were pooled, this difference reached statistical significance (P = 0.03).


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Table III. Prevalence of thrombophilia according to trimester of pregnancy loss
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this series of 108 patients, 22 (20.3%) were found to have hereditary thrombophilia. This prevalence was not higher than that found in the control fertile, parous population. In addition, there was no statistically significant prevalence of any particular thrombophilia in patients with previous second trimester pregnancy losses compared with patients with first trimester losses alone. When the results of all three markers for thrombophilia were pooled, there was a significantly higher prevalence of thrombophilia in patients with at least one second trimester loss (31.6%) compared with the prevalence in patients with only first trimester abortions (14.3%). However, the number of patients with second trimester losses was relatively small.

These results show that the hereditary thrombophilias studied seem to be associated with second trimester rather than first trimester miscarriages. The association of thrombophilia with second trimester losses rather than first trimester losses concurs with most of the reports in the literature (Preston et al., 1996Go; Rai et al., 1996Go; Grandone et al., 1997Go; Martinelli et al., 2000Go). There are few reports associating hereditary thrombophilias with first as well as second trimester miscarriages (Brenner et al., 1997Go; Tal et al., 1999Go; Younis et al., 2000Go). In contrast, a recent large series (Rai et al., 2001Go) found that the prevalence of FVL was not higher than in controls for both first and second trimester abortions. Analysis of the literature with regard to recurrent pregnancy loss shows a conflicting picture: one study (Kutteh et al., 1999Go) did not find a higher prevalence than the general population when FVL, FII G20210A and MTHFR C677T were assessed, neither did another study (Dizon-Townson et al., 1997Go) when assessing FVL. However, FVL was found in significantly more patients than in the control population (Brenner et al., 1997Go). This discrepancy may stem from the different study design.

As thrombophilias are genetic mutations, they should be absolute causes of pregnancy loss. Although in the present study the patients with no live births (primary aborters) had a higher prevalence of thrombophilia, this difference was not statistically significant. Additionally, the eight patients with thrombophilias and previous live births (secondary and tertiary aborters) had 14 previous deliveries without treatment. This represents a prior 16.7% live birth rate, indicating that genetic thrombophilias are not absolute causes of miscarriage. This live birth rate is similar to that in patients presenting without thrombophilia (23.5%). Therefore, if thrombosis associated with thrombophilias leads to pregnancy loss, it is necessary to determine which patients will have pregnancy loss, and which will have live births. In the case of antiphospholipid syndrome, which is also presumed to cause pregnancy loss by thrombosis, Wilson et al. have defined the pregnancy loss as being either three or more pregnancy losses prior to the tenth week or one or more fetal deaths after 10 weeks (Wilson et al., 1999Go). However, a typical clinical presentation has been described (Lockshin et al., 1992Go). Pregnancies start normally, and a fetal heart is detected early in the first trimester. Fetal growth slows in mid pregnancy, oligohydramnios becomes apparent and fetal death eventually ensues. A high incidence of second and third trimester losses with antiphospholipid antibodies has also been reported (Carp et al., 1993Go).

In the case of first trimester miscarriages, there is a significant confounding factor. A large number of recurrent first trimester miscarriages may be lost due to chromosomal aberrations in the fetus. The incidence has been reported to vary between 29 and 60% (Stern et al., 1996Go; Ogasawara et al., 2000Go; Carp et al., 2001Go). In one series (Carp et al., 2001Go), chromosome 16 trisomy and triploidy were relatively common in first trimester losses. These aberrations will invariably cause abortion, irrespective of the presence of thrombophilia. Unfortunately, few centres karyotype the abortus, although fetal karyotyping is an official recommendation of the Royal College of Obstetricians and Gynaecologists (1998). Another confounding factor is the role of lymphokine-activated natural killer cells, which have also been associated with recurrent miscarriage (Aoki et al., 1995Go; Coulam and Beaman, 1995Go; Kwak et al., 1995Go; Clifford et al., 1999Go). It is conceivable that if the karyotypically abnormal losses and the patients with increased natural killer cell activity could have been excluded from this series, there may have been a different prevalence in cases of early pregnancy losses. The figures may also have been skewed by our centre being a tertiary referral centre for patients with pregnancy loss. Selection bias might have occurred as primary care physicians may have diagnosed and treated thrombophilia after two pregnancy losses, (particularly if these were late losses). These patients might not have been referred to our service, leaving us with only the patients with large numbers of first trimester miscarriages and no thrombophilias. It appears that further studies are necessary on a larger cohort of patients with acquired as well as hereditary thrombophilias. Patients losing genetically aberrant embryos should be excluded from studies that purport to study mechanisms causing fetal demise of normal embryos due to thrombosis of decidual blood vessels. Until then, thrombophilias will remain genetic tendencies to thrombosis, not necessarily causes of early pregnancy loss.


    Notes
 
4 To whom correspondence should be addressed at: Department of Obstetrics and Gyneology, Sheba Medical Center, Sheba Medical Center, Tel Hashomer 52621, Israel. E-mail: carp{at}netvision.net.il Back


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Submitted on July 9, 2001; resubmitted on September 9, 2001; accepted on December 6, 2001.