1 Department of Obstetrics and Gynecology, Case Western Reserve University at Metro Health Medical Center, 2500 Metro Health Drive, Cleveland, OH 441091998, USA, 2 Department of Obstetrics and Gynecology, Faculty of Medicine, Osaka University 22, Yamadaoka, Suita, Osaka, 565-0871, 3 Department of Obstetrics and Gynecology, Osaka Police Hospital, 1031 Kitayama-cho, Tennouji-ku, Osaka 543-8502 and 4 Shimizu Women's Clinic, 224 Minamiguchi, Takarazuka, Hyogo, 665-0011, Japan
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Abstract |
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Key words: factor V Leiden mutation/recurrent spontaneous abortion
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Introduction |
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The FVL mutation is detectable in ~35% of the Caucasian population in Europe and the United States (Rees et al., 1995). In contrast, its prevalence is <1% in southeast Asia, the Middle East, and Africa (Rees et al., 1995
). In accordance with the Asian statistics which include Indonesia, Taiwan, Mongolia, and Hong Kong Chinese (Rees et al., 1995
), the prevalence is also extremely low in the Japanese (Takamiya et al., 1995
; Ozawa et al., 1996
; Odawara and Yamashita, 1997
). However, the prevalence of the FVL mutation has not yet been examined in Japanese women with a history of RSA or other obstetric complications.
The aim of the present study is to clarify the association between the FVL mutation and recurrent pregnancy loss in the Japanese population, in part since the controversial results in the previous studies may be due to the racial differences in study populations. It is also possible that the frequency of the mutation may be concentrated in a population with potential thrombotic diseases, even though the basal prevalence in the general population is low. Another question is that if the FVL mutation is linked to fetal loss, which factor is responsible for the situation; maternal carriers, fetal carriers, or both? In fact, one study suggested that fetal carriers of the FVL mutation are at high risk during prenatal life (Dizon-Townson et al., 1997). In this study, we examined both parental peripheral blood samples instead of using fetal tissues.
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Materials and methods |
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All of the materials were collected after obtaining informed consent. From each subject, ~1 ml of peripheral blood sample was obtained and stored at 80°C in a heparinized tube until use. Peripheral blood DNA was prepared from 75 µl with a rapid method described elsewhere (Gelhaus et al., 1995). A 267 base pair (bp) fragment in exon 10 of the factor V gene was amplified by the polymerase chain reaction (PCR) method with the forward primer 5'-TGCCCAGTGCTTAACAAGACCA-3' and the reverse primer 5'-TGTTATCACACTGGTGCTAA-3' (Bertina et al., 1994
). Amplification was carried out in 10 µl of reaction mixture which contained 10 mM TrisHCl (pH 8.3), 50 nM KCl, 1.5 mM MgCl2, 0.01% gelatin, 50 ng of template DNA, 200 µM of each dNTP, 200 nM of each primer, and 0.2 IU of Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany). The PCR was performed in a thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT, USA) under the following conditions: denaturation for 1 min at 94°C; then 35 cycles of 40 s at 94°C, 40 s at 55°C and 40 s at 72°C. A portion of the PCR products was digested with Mnl I and electrophoresed on 4% agarose gels (Nu Sieve GTG, FMC, Rockland, ME, USA) in Tris-acetate-EDTA buffer. The fragments were stained with ethidium bromide and visualized using ultraviolet light. Normal alleles with a G at nucleotide 1691 yielded 163, 67 and 37 bp fragments, whereas FVL alleles (G to A substitution) yielded 200 and 67 bp fragments.
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Results and discussion |
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The possibility remains that our conclusion is not warranted, based on the relatively small sample size. However, our sample size and negative results are comparable to those of the previous studies; the prevalence of FVL mutation in women with RSA and in a control group were 0/40 and 0/25 (Dizon-Townson et al., 1997) and 10.7% (9/84) and 9.2% (8/87) (Pauer et al., 1998
) respectively. To the best of our knowledge, our report is the first to describe the prevalence of the FVL mutation in women with RSA in an Asian population. Our data contribute to the clarification of the world wide distribution of the mutation in women with RSA. It is of interest that negative results with regard to the association between recurrent fetal loss and the FVL mutation have been reported in both high-risk (Dizon-Townson et al., 1997
; Pauer et al., 1998
) and low-risk populations, as in this study. This may indicate that there is little, if any, relationship between the FVL mutation and RSA. Only a large prospective study would enable a definite conclusion regarding this relation.
It is also a matter of controversy whether routine screening for the FVL mutation should be done to diagnose and manage patients with recurrent pregnancy losses (Balasch et al., 1997; Pauer et al., 1998
). Considering the extremely low prevalence of FVL alleles in our present population in general and in patients with RSA, we feel that it is not necessarily advisable to screen for this mutation in Japanese patients with RSA at present. The screening policy in a given country should be based on that country's own population data.
In addition to early fetal miscarriage, the FVL mutation may be a factor of predisposition to other pregnancy complications (Dizon-Townson et al., 1996; Grandone et al., 1997a
). The FVL mutation was more than twice as common in pregnant women with severe pre-eclampsia (8.9%) compared to those with normal blood pressure (4.2%) (Dizon-Townson et al., 1996
). A case report suggested that the FVL mutation, together with another mutation in 5,10-methylene-tetrahydrofolate reductase, was associated with pregnancy complications such as recurrent fetal loss, intrauterine growth restriction, and coagulopathies during pregnancy (Grandone et al., 1997c
). Further studies may resolve the question of whether the FVL mutation is a cause of such obstetrical complications in the Japanese population.
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Acknowledgments |
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Notes |
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References |
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Submitted on October 7, 1998; accepted on March 10, 1999.