Hum. Reprod. Advance Access originally published online on January 23, 2006
Human Reproduction 2006 21(5):1161-1165; doi:10.1093/humrep/dei466
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Genetic thrombophilic mutations among couples with recurrent miscarriage
Department of Obstetrics & Gynaecology, Division of Surgery, Oncology, Reproductive Biology and Anaesthetics, Imperial College London, Mint Wing, St Mary’s Hospital, London, UK
1 To whom correspondence should be addressed at: Department of Obstetrics & Gynaecology, Division of Surgery, Oncology, Reproductive Biology and Anaesthetics, Faculty of Medicine, Imperial College London, Mint Wing, St Mary’s Hospital, London W2 1PG, UK. E-mail: r.rai{at}imperial.ac.uk
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Abstract |
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BACKGROUND: Some cases of recurrent first trimester miscarriage (RM)—the loss of three or more consecutive pregnancies at <12 weeks’ gestation—have a thrombotic aetiology. METHODS: We determined (i) the prevalence of three thrombophilic mutations [factor V Leiden (FVL), prothrombin G20210A (PTG) and methylenetetrahydrofolate reductase (MTHFR) C677T] amongst 357 Caucasian couples with RM and 68 parous Caucasian couples with no history of miscarriage and (ii) the prospective outcome of untreated pregnancies amongst couples with RM in which either partner carried a thrombophilic mutation. RESULTS: The allele frequencies of FVL (2%), PTG (2%) and MTHFR C677T (31%) were similar between cases and controls. The prevalence of multiple thrombophilic mutations (greater than one mutation) was also similar between cases and controls. Amongst couples in whom either partner carried greater than one thrombophilic allele, the relative risk of miscarriage in a future untreated pregnancy was 1.9 (95% confidence interval, 1.3–2.8) compared with those couples who carried no thrombophilic mutation. CONCLUSION: The prevalence of thrombophilic mutations is similar in couples with RM and parous controls. In couples with RM, multiple genetic thrombophilic mutations in either partner significantly increases the risk of miscarriage in a subsequent pregnancy.
Key words: factor V Leiden/first trimester miscarriage/methylenetetrahydrofolate reductase C677T/prothrombin G20210A/thrombophilic mutation
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Introduction |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionAcknowledgementsReferences |
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Pregnancy is a hypercoagulable state secondary to (i) an increase in coagulation factors, (ii) a reduction in naturally occurring anticoagulants and (iii) an impairment of fibrinolysis (Stirling et al., 1984
; Clark et al., 1998). The evolutionary advantage of these changes is thought to be stabilization of haemochorial placentation and reduction in post-partum blood loss.
Some cases of recurrent first trimester miscarriage (RM)—the loss of three or more consecutive pregnancies at less than 12 weeks’ gestation—may have a thrombotic aetiology. The evidence for this comes from histological studies reporting microthrombi to be a common finding in the placental vasculature of pregnancies amongst women with RM (Rushton, 1988; Out et al., 1991; Rai et al., 1996) and studies reporting an increased prevalence of thrombophilic mutations [factor V Leiden (FVL), prothrombin G20210A (PTG) and methylenetetrahydrofolate reductase (MTHFR) C677T] amongst women with RM (Rey et al., 2003). In addition, antiphospholipid antibodies (lupus anticoagulant and anticardiolipin antibodies), an acquired thrombophilic defect, are now recognized as an important treatable cause of RM (Kutteh, 1996; Rai et al., 1997).
We have reported that whilst the prevalence of FVL is similar amongst those with RM compared with controls, women with RM who carry the FVL mutation have a significantly higher miscarriage rate in future untreated pregnancies compared with those who have a normal factor V genotype (Rai et al., 2002). It has been reported that the fetal thrombophilia genotype, which will of course be determined by the parental thrombophilia genotype, is important in governing pregnancy outcome (Dizon-Townson et al., 1997). We therefore determined (i) the prevalence of the three thrombophilia mutations in both partners of couples with RM and (ii) the prospective outcome of untreated pregnancies amongst couples in whom either partner carried a thrombophilia mutation.
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Subjects and methods |
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This study was approved by the local research ethics committee of St Mary’s Hospital NHS Trust, London.
Thrombophilic mutation prevalence study
Subjects
Between October 2000 and February 2003, 357 consecutive Caucasian couples attending the Recurrent Miscarriage Clinic at St Mary’s Hospital, London, were recruited to this study. A control group of 68 parous, age-matched Caucasian couples with no history of miscarriage were recruited from post-natal wards. No participant in this study had a personal or family history of venous thromboembolic disease. The demographic details and outcomes of previous pregnancies of these women are summarized in Table I. All individuals gave signed informed consent to participate in this study.
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All couples with RM were investigated according to our protocol (Rai et al., 2001). In particular, all women were screened for antiphospholipid antibodies and both partners had peripheral blood karyotyping performed. Genetic testing for three thrombophilic mutations—FVL, PTG and MTHFR C677T—was performed on genomic DNA extracted from leukocytes from whole blood, using a standard Qiagen kit.
Factor V Leiden
PCR using known primers was used to amplify exon 10 of the factor V gene, which contains the mutation G
A at nucleotide position 1691 (Bertina et al., 1994). The amplified segment of DNA was then digested overnight using the enzyme MnlI (New England BioLabs) at 37°C. Digested segments of DNA were separated by electrophoresis in a 1.8% agarose gel stained with ethidium bromide at a potential difference of 100 V. Digested bands of DNA were examined under UV light. PCR amplification using specific primers generates a 267-bp fragment spanning the mutation site. The G1691A mutation on the factor V gene destroys a MnlI cleavage site. Digestion of the amplified fragment with MnlI generates three fragments—37, 67 and 163 bp in length—in the presence of normal factor V genotype. In the presence of the affected allele, digestion with MnlI generates two fragments—67 and 200 bp in length. A heterozygous sample would therefore generate four bands—37, 67, 163 and 200 bp in length—and a homozygous sample would generate only two bands—67 and 200 bp in length.
Prothrombin G20210A
PCR using specific primers was used to amplify a 345-bp segment, followed by overnight digestion with the restriction endonuclease HindIII at 37°C, followed by gel electrophoresis as described above (Poort et al., 1996). The G20210A mutation in the 3' untranslated region of the prothrombin gene does not disrupt a natural recognition site for any restriction endonuclease. The primers used are designed to introduce a HindIII cleavage site only if the mutant allele is present. Amplification by PCR followed by subsequent digestion with HindIII yields one fragment of 345 bp in a normal individual, two fragments of 322 and 23 bp in a homozygous individual and three fragments of 345, 322 and 23 bp in a heterozygous individual.
Methylenetetrahydrofolate reductase C677T
PCR using specific primers was used to amplify a 198-bp segment, followed by overnight digestion with the restriction endonuclease HinfI at 37°C, followed by gel electrophoresis as described above (Frosst et al., 1995). The MTHFR C677T mutation creates a recognition site for the HinfI restriction endonuclease. Amplification of the MTHFR gene using specific primers generates a 198-bp PCR product. Subsequent digestion with HinfI will generate two fragments of 175 and 23 bp in length in the presence of an affected allele and in a homozygous individual. A heterozygous individual will give three fragments—198, 175 and 23 bp in length.
Prospective pregnancy study
Study population
The outcome of the next pregnancy of those participating in the prevalence study was determined. No woman took any medication during pregnancy apart from folic acid (400 µg/day) as prophylaxis against neural tube defects. Women with antiphospholipid syndrome were excluded, as were those who took aspirin (Figure 1). Both the patients and clinicians were blinded to the results of thrombophilic mutation status.
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Women were followed from the time of a positive urine pregnancy test and had fortnightly serial pelvic ultrasound scans at our clinic until 12 weeks’ gestation. After this time, the remainder of their antenatal care was provided at St Mary’s Hospital.
Statistical analysis
Statistical analysis was performed using SPSS version 12 (SPSS, Chicago, IL, USA). Discrete variables were analysed using Fisher’s exact test or the c2 test, and continuous variables were analysed using the Mann–Whitney U-test. P-values less than 0.05 were taken as statistically significant. Relative Risk and 95% confidence intervals were calculated where appropriate.
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Results |
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The frequencies of FVL, PTG and MTHFR C677T alleles were similar between both individuals and couples with RM compared with controls (Table II). The allele frequencies for all three mutations were similar to those previously reported (Holmes et al., 1999
; Pickering et al., 2001; Rai et al., 2001; Alonso et al., 2002; Carp et al., 2002b; Pauer et al., 2003). The prevalence of multiple thrombophilic mutations (greater than one mutation in the same individual) was also similar among (i) individuals with RM and controls and (ii) couples with RM and controls (Table III).
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The live birth rate in the next pregnancy in which no thromboprophylaxis was administered was significantly lower amongst couples in whom either partner carried multiple (greater than one) mutations [2 of 12 (17%)] compared with couples in whom neither partner carried a mutation [28 of 50 (56%); P < 0.03] (Table IV). A breakdown of thrombophilic genotypes for each partner with multiple mutations, together with gestational ages at which the next pregnancy ended, is summarized in Table V. Women in the group with multiple mutations were of similar age (median, 34 years; range, 25–46) and had a similar number of previous miscarriages (median, 3; range, 3–5) compared with women in the group with no mutations (median age, 33 years; range, 25–46; P = 0.15; median number of previous miscarriages, 3; range 3–5; P = 0.73). Of note, 50% of miscarriages occurred at 
8 weeks’ gestation. This is higher than the 2% that has been previously reported (Brigham et al., 1999).
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The live birth rate amongst women (i) with no thrombophilic mutations was 56% (28 of 50) and (ii) with one thrombophilic mutation (FVL, PTG or homozygous MTHFR) 55% (11 of 20) and (iii) amongst those with greater than one mutation 25% (1 of 4).
The relative contribution of the paternal as compared with the maternal genotype in determining pregnancy is summarized in Tables IV and V. Of note, the miscarriage rate amongst couples in whom the male partner carried greater than 1 thrombophilic mutation was 87.5% (7 of 8) versus 75% (3 of 4) when the female partner carried greater than one thrombophilic mutation.
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Discussion |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionAcknowledgementsReferences |
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This is the first study to report the prevalence of three thrombophilic mutations (FVL, PTG and MTHFR) in both the female and male partners of couples with RM. The allele frequencies of the three thrombophilic mutations examined were similar in couples with RM compared with parous controls with no history of miscarriage. However, the prospective pregnancy study reported that pregnancies amongst couples in whom either partner carried more than one thrombophilic mutation were at significantly increased risk of future miscarriage compared with couples in whom neither partner carried a mutation.
The discordance between the prevalence data and the pregnancy outcome data clearly suggests that there is an additional factor other than parental thrombophilic mutation status that determines pregnancy outcome. In this respect, we are currently investigating the role of the fetal thrombophilia genotype.
This study has certain potential limitations. Whilst it should be acknowledged that heterozygosity for the MTHFR mutation is not thought to be an independent risk factor for systemic venous thrombosis, we have included those heterozygous for this mutation as we are examining (i) the allele frequencies of the three thrombophilic mutations and (ii) the contribution of the MTHFR allele in combination with either the FVL allele or the PTG allele to pregnancy outcome. Folic acid supplementation could also lower plasma homocysteine levels, which are elevated amongst homozygous carriers of MTHFR, which would potentially reduce the likelihood of this being an important risk factor for miscarriage. However, on direct questioning, all women in this study took only a low dose of folic acid (400 µg) as prophylaxis against neural tube defects.
The placenta has a dual blood supply—one maternal and one fetal. The maternal blood, originating from uterine spiral arteries, circulates in the intervillous space, coming into contact with the syncytiotrophoblast and subsequently draining back through decidual veins. The fetal blood supply within the placenta takes origin from the umbilical arteries. Blood circulates in the fetal villi within stem vessels and subsequently drains back via the umbilical vein. A hypercoagulable state within the fetal circulation could lead to fetal stem vessel thrombosis, placental infarction in the distribution of fetal vessels and subsequent miscarriage. In support of this, fetal carriage of FVL has been reported in association with histologically proven placental infarction (Dizon-Townson et al., 1997).
This large prevalence study addresses many of the deficiencies in the existing data on the prevalence of genetic thrombophilias amongst women with RM. Published studies have been plagued by (i) the small size of individual studies leading to a lack of power (Wramsby et al., 2000; Younis et al., 2000; Carp et al., 2002b; Finan et al., 2002; Rey et al., 2003), (ii) stratification bias in which there is an underestimation of the prevalence amongst controls and a spurious increase in prevalence amongst cases and (iii) admixture bias in which there has been poor matching of cases and controls because of racial heterogeneity (Carp et al., 2002b).
Data reporting the prospective outcome of untreated pregnancies amongst women with RM and a genetic thrombophilia are sparse. There are two prospective studies of the pregnancy outcome of women with RM in whom a genetic thrombophilic defect has been identified (Carp et al., 2002a; Rai et al., 2002). The study from our unit (Rai et al., 2002) reported a significantly lower live birth rate (38%) amongst 16 women who carried the FVL mutation compared with those who have a normal factor V genotype (69%). The second study reported the outcome of one patient with FVL (miscarried), three with the prothrombin gene mutation (all live births) and 16 homozygous for the MTHFR mutation (25% live birth rate).
Despite the lack of prospective randomized placebo-controlled studies, women with RM who carry a thrombophilic defect are being offered thromboprophylaxis with heparin during pregnancy. Heparin does not cross the placenta and would therefore not have an anticoagulant effect on the fetal side of the placental vasculature. As this study demonstrates that the paternal thrombophilic genotype and, by inference, the fetal genotype contribute to determining pregnancy outcome, the role of thromboprophylactic agents that cross the placenta needs to be investigated. Of these agents, warfarin is thought to be teratogenic in the first trimester (Ageno et al., 2004), but aspirin is not associated with fetal development abnormalities (Kozer et al., 2003).
In conclusion, our study demonstrates that the prevalence of multiple thrombophilic mutations is similar in couples with recurrent miscarriage and parous controls. In couples with recurrent early miscarriage, multiple genetic thrombophilic mutations in either partner significantly increase the risk of miscarriage in a subsequent pregnancy.
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Acknowledgements |
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Save the Baby Charity (UK) funded this study. We thank all patients and staff of the Recurrent Miscarriage Clinic, Antenatal Clinic, Labour Ward and the Lindo Wing at St Mary’s Hospital, London. We thank Mrs Pilar Dariba for her help in collecting blood samples, Mrs Sarah Choy for her help with laboratory work and Dr Abdul Shlebak and Mr Kevin Marriott for providing laboratory training on genotyping of samples.
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References |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionAcknowledgementsReferences |
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Ageno W, Crotti S and Turpie AG (2004) The safety of antithrombotic therapy during pregnancy. Expert Opin Drug Saf 3,113–118.[CrossRef][Medline]
Alonso A, Soto I, Urgelles MF, Corte JR, Rodriguez MJ and Pinto CR (2002) Acquired and inherited thrombophilia in women with unexplained fetal losses. Am J Obstet Gynecol 187,1337–1342.[CrossRef][Web of Science][Medline]
Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA and Reitsma PH (1994) Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 369,64–67.[CrossRef][Medline]
Brigham SA, Conlon C and Farquharson RG (1999) A longitudinal study of pregnancy outcome following idiopathic recurrent miscarriage. Hum Reprod 14,2868–2871.
Carp H, Dolitzky M, Tur-Kaspa I and Inbal A (2002a) Hereditary thrombophilias are not associated with a decreased live birth rate in women with recurrent miscarriage. Fertil Steril 78,58–62.[CrossRef][Web of Science][Medline]
Carp H, Salomon O, Seidman D, Dardik R, Rosenberg N and Inbal A (2002b) Prevalence of genetic markers for thrombophilia in recurrent pregnancy loss. Hum Reprod 17,1633–1637.
Clark P, Brennand J, Conkie JA, McCall F, Greer IA and Walker ID (1998) Activated protein C sensitivity, protein C, protein S and coagulation in normal pregnancy. Thromb Haemost 79,1166–1170.[Web of Science][Medline]
Dizon-Townson D, Meline L, Nelson L, Varner M and Ward K (1997) Fetal carriers of the factor V Leiden mutation are prone to miscarriage and placental infarction. Am J Obstet Gynecol 177,402–405.[CrossRef][Web of Science][Medline]
Finan RR, Tamim H, Ameen G, Sharida HE, Rashid M and Almawi WY (2002) Prevalence of factor V G1691A (factor V-Leiden) and prothrombin G20210A gene mutations in a recurrent miscarriage population. Am J Hematol 71,300–305.
Frosst P, Blom HJ et al. (1995) A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 10,111–113.[CrossRef][Web of Science][Medline]
Holmes ZR, Regan L, Chilcott I and Cohen H (1999) The C677T MTHFR gene mutation is not predictive of risk for recurrent fetal loss. Br J Haematol 105,98–101.[CrossRef][Web of Science][Medline]
Kozer E, Costei AM, Boskovic R, Nulman I, Nikfar S and Koren G (2003) Effects of aspirin consumption during pregnancy on pregnancy outcomes: meta-analysis. Birth Defects Res B Dev Reprod Toxicol 68,70–84.[CrossRef][Web of Science][Medline]
Kutteh WH (1996) Antiphospholipid antibody-associated recurrent pregnancy loss: treatment with heparin and low-dose aspirin is superior to low-dose aspirin alone. Am J Obstet Gynecol 174,1584–1589.[Web of Science][Medline]
Out HJ, Kooijman CD, Bruinse HW and Derksen RH (1991) Histopathological findings in placentae from patients with intra-uterine fetal death and anti-phospholipid antibodies. Eur J Obstet Gynecol Reprod Biol 41,179–186.[CrossRef][Web of Science][Medline]
Pauer HU, Burfeind P, Kostering H, Emons G and Hinney B (2003) Factor XII deficiency is strongly associated with primary recurrent abortions. Fertil Steril 80,590–594.[CrossRef][Web of Science][Medline]
Pickering W, Marriott K and Regan L (2001) G20210A prothrombin gene mutation: prevalence in a recurrent miscarriage population. Clin Appl Thromb Hemost 7,25–28.
Poort SR, Rosendaal FR, Reitsma PH and Bertina RM (1996) A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 88,3698–3703.
Rai R, Backos M, Elgaddal S, Shlebak A and Regan L (2002) Factor V Leiden and recurrent miscarriage—prospective outcome of untreated pregnancies. Hum Reprod 17,442–445.
Rai R, Cohen H, Dave M and Regan L (1997) Randomised controlled trial of aspirin and aspirin plus heparin in pregnant women with recurrent miscarriage associated with phospholipid antibodies (or antiphospholipid antibodies). BMJ 314,253–257.
Rai R, Regan L, Hadley E, Dave M and Cohen H (1996) Second-trimester pregnancy loss is associated with activated C resistance. Br J Haematol 92,489–490.[Web of Science][Medline]
Rai R, Shlebak A, Cohen H, Backos M, Holmes Z, Marriott K and Regan L (2001) Factor V Leiden and acquired activated protein C resistance among 1000 women with recurrent miscarriage. Hum Reprod 16,961–965.
Rey E, Kahn SR, David M and Shrier I (2003) Thrombophilic disorders and fetal loss: a meta-analysis. Lancet 361,901–908.[CrossRef][Web of Science][Medline]
Rushton DI (1988) Placental pathology in spontaneous miscarriage. In Beard RW and Sharp F (eds) Early Pregnancy Loss: Mechanisms and Treatment. RCOG, London, pp. 149–158.
Stirling Y, Woolf L, North WR, Seghatchian MJ and Meade TW (1984) Haemostasis in normal pregnancy. Thromb Haemost 52,176–182.[Web of Science][Medline]
Wramsby ML, Sten-Linder M and Bremme K (2000) Primary habitual abortions are associated with high frequency of factor V Leiden mutation. Fertil Steril 74,987–991.[CrossRef][Web of Science][Medline]
Younis JS, Brenner B, Ohel G, Tal J, Lanir N and Ben-Ami M (2000) Activated protein C resistance and factor V Leiden mutation can be associated with first—as well as second—trimester recurrent pregnancy loss. Am J Reprod Immunol 43,31–35.
Submitted on May 20, 2005; resubmitted on October 6, 2005; accepted on November 15, 2005.
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