1 Department of Reproductive Science and Medicine, Imperial College School of Medicine at St Mary's, 2 Department of Haematology, St Mary's Hospital NHS Trust and 3 Department of Haematology, University College London Hospitals Trust, London, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: activated protein C resistance/factor V Leiden/prevalence/recurrent miscarriage
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A key component in the anticoagulant pathway is protein C, which when activated inhibits the actions of coagulation factors V and VIII. Resistance to the anticoagulant properties of activated protein CAPC resistancewas first reported in 1993 (Dahlback et al., 1993). It was later demonstrated that APC resistance may either be congenital or acquired. Congenital APC resistance is almost exclusively due to a single point mutation (G
A) at nucleotide position 1691 in the factor V gene, which results in a mutated form of factor V, known as factor V Leiden (Bertina et al., 1994
). Mutated factor V is resistant to inactivation by APC, resulting in increased thrombin generation and a hypercoaguable state. Acquired APC resistance is associated with lupus anticoagulant and high concentrations of coagulation factor VIII (Laffan and Manning, 1996
). Both factor V Leiden and acquired APC resistance are risk factors for systemic venous thrombosis (de Visser et al., 1999
) and factor V Leiden has been reported in association with placental thrombosis (Rai et al., 1996; Dizon et al., 1997
).
The prevalence of factor V Leiden and acquired APC resistance among women with recurrent miscarriage has been variably reported to be either similar to or increased compared to parous controls (Balasch et al., 1997; Brenner et al., 1997
; Grandone et al., 1997
; Dizon-Townson et al., 1997
; Metz et al., 1997
; Ridker et al., 1998
; Kutteh et al., 1998
; Souza et al., 1999
; Tal et al., 1999
). Previous studies have included only small numbers of women, have been prone to selection bias and some have not divided women into those with recurrent first trimester miscarriage and those with later pregnancy loss.
The aim of this study was to address the limitations of existing data by determining the prevalence of both factor V Leiden and acquired APC resistance among more than 1000 consecutive women with recurrent miscarriage.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
A variety of causes for recurrent miscarriage has been suggested over the decades. However, despite intensive investigation, only antiphospholipid antibodies (aPL)lupus anticoagulant (LA) and anticardiolipin antibodies (aCL)and parental karyotype abnormalities are established causes for recurrent miscarriage (Royal College of Obstetricians and Gynaecologists, 1998). The prevalence of aPL in the study cohort was 20.7% (230/1111).
Protocol for the investigation of recurrent miscarriage
All women attending the Recurrent Miscarriage Clinic are screened for the factor V Leiden mutation, APC resistance and for both LA and aCL prior to pregnancy. In addition, peripheral blood karyotyping is carried out on both partners.
Factor V Leiden
Genomic DNA was extracted from EDTA whole blood using standard techniques. The polymerase chain reaction (PCR) using known primers was used to amplify exon 10 of the factor V gene which contains the G A mutation at nucleotide position 1691 (Bertina et al., 1994
). Following amplification, a 20 µl aliquot of the product was digested overnight with 5 IU of the enzyme Mnl I (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 ethium bromide. The undigested PCR product measures 223 base-pairs (bp) in size. Following cleavage with Mnl I, 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 Mnl I cleavage site. Controls on each gel included a known heterozygote, a normal control known not to possess the factor V Leiden mutation and a water blank containing no input DNA.
Activated protein C resistance
Venous blood samples were collected with minimal stasis using a 19-gauge butterfly needle into 0.109 mol/l trisodium citrate, nine parts blood to one part anticoagulant. Platelet poor plasma was prepared by double centrifugation of samples at 2700 g at room temperature for 20 min and stored at 70°C. APC resistance was assessed by measuring the anti-coagulant response in plasma on the addition of APC (Coatest®; Chromogenix, Epsom, Surrey, UK). A ratio of <2.26 for the clotting time in the presence of APC/clotting time in the absence of APC was taken to represent APC resistance (Rosen et al., 1994).
Definitions of congenital and acquired activated protein C resistance
Congenital APC resistance was defined as an APC ratio in the Coatest® of <2.26 in an individual who carried the factor V Leiden mutation and acquired APC resistance as a ratio of <2.26 in an individual with a normal factor V genotype.
Antiphospholipid antibodies
All women were screened for aPL on at least two occasions more than 6 weeks apart prior to pregnancy. Lupus anticoagulant 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% or more in the ratio was considered to be positive for lupus anticoagulant (Lupus Anticoagulant Working Party on behalf of the BCSH Haemostasis and Thrombosis Taskforce, 1991
). Anticardiolipin antibodies were identified using a standardized enzyme linked immunosorbent assay (ELISA). An immunoglobulin (Ig)G anticardiolipin value
5 GPL units and an IgM anticardiolipin value
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 8 weeks after the initial sample. Only women with persistently positive tests for either LA or aCL were considered to have the antiphospholipid syndrome.
Statistical analysis
Discrete variables were analysed using the 2 test and continuous variables analysed using the MannWhitney U-test. P values of < 0.05 were taken as statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Women with acquired APC resistance were significantly (P < 0.01) less likely to have had a previous live birth compared to those with a normal APC ratio (Table III). There was no association between APC resistance, congenital or acquired, and aPL positivity (Table III
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In a previous much smaller study, in which APC resistance was assessed among women with recurrent miscarriage but no differentiation was made between congenital and acquired causes, it was reported that the frequency of APC resistance was similar among women with early miscarriage compared with controls (Rai et al., 1996a). This is also the case in the present study, which emphasizes the importance of discriminating between congenital and acquired causes of APC resistance (Table II
).
Previous studies reporting an increased frequency of the factor V Leiden allele among women with recurrent miscarriage have been based on small numbers of women, and may have been prone to acquisition bias. For example, the study reporting the highest frequency of the factor V Leiden allele among women with recurrent miscarriage (15%) is based on those referred for investigation to a specialist haemostasis unit (Brenner et al., 1997). Similarly, prevalence studies of acquired APC resistance among women with recurrent miscarriage have also been based on small numbers of patients and may also have been prone to acquisition bias (Balasch et al., 1997
; Brenner et al., 1997
; Tal et al., 1999
).
Although both factor V Cambridge (Williamson et al., 1998) and the HR2 haplotype (Bernardi et al., 1997
) have been reported to be associated with APC resistance, these are both rare and no common genetic cause for APC resistance apart from the factor V Leiden mutation has been reported.
In contrast, high concentrations of coagulation factor VIII (Laffan and Manning, 1996), antiphospholipid antibodies (Aznar et al., 1997
), pregnancy (Cumming et al., 1995
) and the combined oral contraceptive pill (Olivieri et al., 1995
) are associated with acquired APC resistance. In this study, no woman was taking the contraceptive pill and all were tested at least 12 weeks after their last pregnancy. No association was found between aPL and APC resistance among this miscarriage population. This is not surprising as aPL are a heterogeneous family of auto-antibodies. Indeed, attention is shifting away from the concept of aPL associated pregnancy loss being purely thrombotic in aetiology towards emphasizing the adverse non-thrombotic effects of aPL on the trophoblast and the decidua (Rai, 2000
).
As women were tested after their miscarriages, an important caveat when interpreting the coagulation data is that it does not establish a temporal relationship between the development of APC resistance and pregnancy loss. This caveat, of course, applies to all non-genetic epidemiological studies of miscarriage. Despite this limitation, these data suggest that APC resistance may be an important cause of pregnancy loss, the mechanism of which is likely to be thrombosis of the placental vasculature. APC resistance has been demonstrated to develop during normal pregnancy (Cumming et al., 1995; Clark et al., 1998
) and it is possible that among women who are APC resistant prior to pregnancy, this effect is amplified. The placental vasculature would be particularly prone to thrombosis as during pregnancy there is a relative increase in intravascular coagulation in the placental bed compared with haemostatic changes in the systemic circulation (Bonnar et al., 1970
).
An interesting finding in the current study is that the prevalence of acquired APC resistance was significantly higher among those with recurrent first-trimester miscarriage compared with controls. If maternal thrombophilic defects are a cause of recurrent first-trimester miscarriage and the mechanism of pregnancy loss is placental vasculature thrombosis, it is important to determine at what gestational age a placental intervillous circulation is established. Recent histological data suggest that while before 8 weeks gestation, the maternal arterial connections with the intervillous space are restricted to tortuous networks, after this time direct channels of communication are established. While these channels are initially of small calibre they become well delineated by the end of the first trimester (Burton et al., 1999).
We are currently examining the prospective outcome of untreated pregnancies among women with recurrent miscarriage and acquired APC resistance in order to determine the significance of our findings.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balasch, J., Reverter, J.C., Fabregues, F. et al. (1997) First-trimester repeated abortion is not associated with activated protein C resistance. Hum. Reprod., 12, 10941097.[ISI][Medline]
Beauchamp, N.J., Daly, M.E., Hampton, K.K. et al. (1994) High prevalence of a mutation in the factor V gene within the U.K. population: relationship to activated protein C resistance and familial thrombosis. Br. J. Haematol., 88, 219222.[ISI][Medline]
Bernardi, F., Arcieri, P., Bertina, R.M. et al. (1997) Contribution of factor VII genotype to activated FVII levels. Differences in genotype frequencies between northern and southern European populations. Arterioscler. Thromb. Vasc. Biol., 17, 25482553.
Bertina, R.M., Koeleman, B.P., Koster, T. et al. (1994) Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature, 369, 6467.[ISI][Medline]
Bonnar, J., Prentice, C.R.M., McNichol, G.P. and Douglas, A.S. (1970) Haemostatic mechanism in the uterine circulation during placental separation. Brit. Med. J., 2, 564567.[ISI][Medline]
Brenner, B., Mandel, H., Lanir, N. et al. (1997) Activated protein C resistance can be associated with recurrent fetal loss. Brit. J. Haematol., 97, 551554.[ISI][Medline]
Brenner, B., Sarig, G., Weiner, Z. et al. (1999) Thrombophilic polymorphisms are common in women with fetal loss without apparent cause. Thromb. Haemost., 82, 69.[ISI][Medline]
Burton, G.J., Jauniaux, E. and Watson, A.L. (1999) Maternal arterial connections to the placental intervillous space during the first trimester of human pregnancy: the Boyd collection revisited. Am. J. Obstet. Gynecol., 181, 718724.[ISI][Medline]
Clark, P., Brennand, J., Conkie, J.A. et al. (1998) Activated protein C sensitivity, protein C, protein S and coagulation in normal pregnancy. Thromb. Haemost., 79, 11661170.[ISI][Medline]
Cumming, A.M., Tait, R.C., Fildes, S. et al. (1995) Development of resistance to activated protein C during pregnancy. Brit. J. Haematol., 90, 725727.[ISI][Medline]
Dahlback, B., Carlsson, M. and Svensson, P.J. (1993) Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc. Natl Acad. Sci. USA, 90, 10041008.[Abstract]
de Visser, M.C., Rosendaal, F.R. and Bertina, R.M. (1999) A reduced sensitivity for activated protein C in the absence of factor V Leiden increases the risk of venous thrombosis. Blood, 93, 12711276.
Dizon-Townson, D.S., Kinney, S., Branch, D.W. and Ward, K. (1997) The factor V Leiden mutation is not a common cause of recurrent miscarriage. J. Reprod. Immunol., 34, 217223.[ISI][Medline]
Dizon, T.D., Meline, L., Nelson, L.M. et al. (1997) Fetal carriers of the factor V Leiden mutation are prone to miscarriage and placental infarction. Am. J. Obstet. Gynecol., 177, 402405.[ISI][Medline]
Grandone, E., Margaglione, M., Colaizzo, D. et al. (1997) Factor V Leiden is associated with repeated and recurrent unexplained fetal losses. Thromb. Haemost., 77, 822824.[ISI][Medline]
Khamashta, M.A. and Hughes, G.R. (1993) ACP broadsheet no. 136: February 1993. Detection and importance of anticardiolipin antibodies. J. Clin. Pathol., 46, 104107.[ISI][Medline]
Kupferminc, M.J., Eldor, A., Steinman, N. et al. (1999) Increased frequency of genetic thrombophilia in women with complications of pregnancy. N. Engl. J. Med., 340, 913.
Kutteh, W.H., Park, V.M. and Deitcher, S.R. (1998) Hypercoagulable state mutation analysis in white patients with early first-trimester recurrent pregnancy loss. Fertil. Steril., 71, 10481053.[ISI]
Laffan, M.A. and Manning, R. (1996) The influence of factor VIII on measurement of activated protein C resistance. Blood Coagul. Fibrinolysis, 7, 761765.[ISI][Medline]
Lupus Anticoagulant Working Party on behalf of the BCSH Haemostasis and Thrombosis Taskforce (1991) Guidelines on testing for the lupus anticoagulant. J. Clin. Path., 44, 885889.[ISI][Medline]
Metz J., Kloss, M., O'Malley C.J. et al. (1997) Prevalence of factor V Leiden is not increased in women with recurrent miscarriage. Clin. Appl. Thrombosis/Hemostasis, 3, 137140.[ISI]
Olivieri, O., Friso, S., Manzato, F. et al. (1995) Resistance to activated protein C in healthy women taking oral contraceptives. Br. J. Haematol., 91, 465470.[ISI][Medline]
Preston, F.E., Rosendaal, F.R., Walker, I.D. et al. (1996) Increased fetal loss in women with heritable thrombophilia. Lancet, 348, 913916.[ISI][Medline]
Rai, R. (2000) Obstetric management of antiphospholipid syndrome. J. Autoimmun., 15, 203207.[ISI][Medline]
Rai, R., Regan, L., Hadley, E. et al. (1996a) Second-trimester pregnancy loss is associated with activated C resistance. Br. J Haematol., 92, 489490.[ISI][Medline]
Rai, R.S., Regan, L., Chitolie, A. et al. (1996b) Placental thrombosis and second trimester miscarriage in association with activated protein C resistance. Brit. J. Obstet. Gynaecol., 103, 842844.[ISI][Medline]
Rees, D.C. (1996) The population genetics of factor V Leiden (Arg506Gln). Br. J. Haematol., 95, 579586.[ISI][Medline]
Ridker, P.M., Miletich, J.P., Buring, J.E. et al. (1998) Factor V Leiden mutation as a risk factor for recurrent pregnancy loss. Ann. Intern. Med., 128, 10001003.
Royal College of Obstetricians and Gynaecologists (1998) The management of recurrent miscarriage. RCOG, London.
Rosen, S., Johansson K., Lindberg, K. and Dahlback B. (1994). Multicentre evaluation of a kit for activated protein C resistance on various coagulation instruments using plasmas from healthy individuals. Thromb. Haemost., 72, 255260.[ISI][Medline]
Rushton, D.I. (1988) Placental pathology in spontaneous miscarriage. In: Beard, R.W. and Sharp, F. (eds) Early pregnancy loss: mechanisms and treatment. RCOG, London, pp. 149158.
Souza, S.S., Ferriani, R.A., Pontes, A.G. et al. (1999) Factor V Leiden and factor II G20210A mutations in patients with recurrent abortion. Hum. Reprod., 14, 24482450.
Stirling, Y., Woolf, L., North, W.R. et al. (1984) Haemostasis in normal pregnancy. Thromb. Haemost., 52, 176182.[ISI][Medline]
Tal, J., Schliamser, L.M., Leibovitz, Z. et al. (1999) A possible role for activated protein C resistance in patients with first and second trimester pregnancy failure. Hum. Reprod., 14, 16241627.
Williamson, D., Brown, K., Luddington, R. et al. (1998) Factor V Cambridge: a new mutation (Arg306>Thr) associated with resistance to activated protein C. Blood, 91, 11401144.
Younis, J.S., Ohel, G., Brenner, B. and Ben-Ami, M. (1997) Familial thrombophilia the scientific rationale for thrombophylaxis in recurrent pregnancy loss? Hum. Reprod., 12, 13891390.
Submitted on October 23, 2000; accepted on February 6, 2001.