recurrent spontaneous abortion ( rsa ) represents a significant clinical problem , which an estimated 1% to 5% of all women of reproductive age experience [ 1 , 2 ] . however , the pathogenesis of rsa is complicated , and the cause in 40%50% of the cases is not well understood . genetic , anatomic , endocrine , immunologic , infectious , and environmental factors have been proposed as causes of rsa [ 3 , 4 ] . various reports have postulated thrombophilia as a risk for rsa [ 5 , 6 ] . deficiency in the homocysteine metabolism pathway resulting in an elevation of homocyteine level in plasma ( hyperhomocysteinemia ) has been regarded as a cause of thrombophilia . methylenetetrahydrofolate reductase ( mthfr ) is one of the main regulatory enzymes in the metabolism of homocysteine that catalyses the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate . mutations in mthfr gene lead to decreased activity of enzyme and hyperhomocysteinemia , which induces platelet aggregation through promotion of endothelial oxidative damage . although several mutations within the mthfr gene were described , c677 t and a1298c mutations are the two most common mutations . c677 t transition is a missense mutation in the exon 4 of this gene , which converts an alanine to a valine codon ( at codon 222 ) in the n - terminal catalytic domain of the protein leading to a thermolabile protein , with decreased enzymatic activity . the second mutation is mthfr a1298c , that is , associated with decreased activity of enzyme , but not with thermolability . a1298c transversion is a point mutation in exon 7 , characterized by a glutamate to alanine substitution ( at codon 429 ) within the c - terminal regulatory domain of the protein . numerous investigations have been performed on the incidence of mthfr c677 t and a1298c mutations in women with rsa [ 9 , 1217 ] . some of these studies have demonstrated a relationship between these mutations and rsa [ 9 , 10 , 12 , 13 , 18 ] , whereas others have been unable to confirm these results [ 14 , 19 ] . thus , the role of mthfr c677 t and a1298c mutations in rsa is still controversial . here , we evaluated the prevalence of mthfr c677 t and a1298c mutations in rsa patients in the northwest of iran and compared it with healthy controls using two methods , pcr - restriction fragment length polymorphism ( pcr - rflp ) and amplification refractory mutation system - pcr ( arms - pcr ) . we enrolled 89 patients with a history of first trimester rsa from many different regions of northwest of iran that were all referred by obstetricians . the patients with chromosomal abnormality , uterine anomalies , genital infections , and endocrinological disorder were excluded from the study . fifty healthy women with at least two live births from the same region were selected as a control group . the mean age of the case and control groups were 30.17 and 31.54 years , respectively . the average abortion in the cases was 2.94 ( ranged from 2 to 7 ) , and the average successful pregnancy in the controls was 2.2 ( ranged from 2 to 4 ) . peripheral blood samples ( 5  l ) were obtained from all cases and controls in tubes containing edta . total genomic dna was extracted from leukocytes by a standard salting - out protocol as previously published . the dna concentration was firstly measured by spectrophotometer and adjusted to 100  ng/l by adding double distillation h2o and then stored in 20c . we applied two methods , pcr - rflp and arms - pcr , for identification of mthfr c677 t and a1298c gene mutations . to perform pcr - rflp , each dna sample was amplified by polymerase chain reaction using primers that are shown in table 1 . pcr was carried out on 25  l volume , in an eppendorf thermal cycler ( eppendorf , germany ) . initial denaturation at 94c for 5  min was followed by 35 cycles of denaturation at 95c for 40  s ; annealing at 58c ( mthfr c677 t ) and 62c ( mthfr a1298c ) and extension at 72c for 40  s and final extension at 72c for 7  min were performed . pcr amplifications were then confirmed by electrophoresis on 2% agarose gel , and after staining with ethidium bromide , viewed with ultraviolet light . finally , the amplified products were digested by restriction enzymes that are shown in table 1   and the digested fragments were separated by electrophoresis on 8% polyacrylamide gels that stained with silver nitrate . arms - pcr was performed using three primers for each mutation , one forward primer and two reverse primers specific for the wild type and mutant alleles that are shown in table 2 [ 12 , 22 ] . pcr was carried out on 20  l volume , in an eppendorf thermal cycler ( eppendorf , germany ) . first denaturation step ( 96c , 2  min ) was followed by 10 cycles of denaturation ( 96c , 15  s ) and annealing / extension ( 65c , 60  s ) , and a final 20 cycles of denaturation ( 96c , 10  s ) , annealing ( 61c , 50  s ) , and extension ( 72c , 30  s ) . the genotype distributions of each mutation , the frequency of heterozygous and homozygous were compared between patients and controls with pearson 's chi - square test . a p value of < 0.05 was regarded as significant . the homozygote and heterozygote genotypes of each group were unified as a new group and then odds ratios and 95% confidence intervals were calculated . analysing mthfr gene mutations among the rsa and control samples by using pcr - rflp and arms - pcr methods clarified that there was no difference between these two methods . the genotype distribution of each mthfr mutations in patients and controls are shown in ( figure 1 ) . the frequency of 677c / t genotype mthfr gene was 33.70% in patients and 44% in controls while the frequency of 677t / t genotype was 6.74% in patients and 2% in controls ( figure 2 ) . the frequency of 677 t allele was 23.59% in patients and 24% in controls ( figure 3 ) . there is no significant difference in the prevalence of 677t / t genotype among women with rsa and healthy controls ( p = 0.285 ) . the frequency of 1298a / c genotype mthfr gene was 51.68% in patients and 68% in controls . in contrast with the heterozygote genotype , the frequency of homozygote genotype was higher in rsa patients compared with the control group . no statistically significant difference in the frequency of a1298c mthfr gene mutation was detected between the two groups ( p = 0.17 ) . the frequencies of mthfr 677 t and mthfr 1298c alleles were ( 23.4% , 34.8% ) in patients and ( 24% , 40% ) in controls , respectively ( figure 4 ) . the total mutant allele frequencies were 29.21% in women experiencing rsa and 32% in fertile controls . all possible mthfr c677t / a1298c genotype combinations were represented in both groups ( table 3 ) . the frequencies of 677ct/1298ac combined heterozygosity in patients were 15.73% and 32% in the control group . the odds ratios ( ors ) of the mthfr 677c / t ( or = 0.69 ; 95% confidence interval ( ci ) = 0.331.42 ) and the mthfr 1298a / c ( or = 0.50 ; 95% ci = 0.231.09 ) . although most researchers generally use the pcr - rflp method for detection of gene mutations , here we have focused on studying the concordance between the pcr - rflp and arms - pcr methods . we applied the two methods of pcr - rflp and arms - pcr for detection of genotypes . the results from pcr - rflp method were the same as arms - pcr method . therefore , due to the fact that it is relatively easy to perform low costs , and the possibility of detecting a larger number of samples , we proposed arms - pcr as a useful method that could be used as an alternative for identification of these mutations . many pieces of literature have discussed the matter that mthfr gene mutations might be a risk factor for recurrent spontaneous abortions [ 9 , 10 , 12 , 13 , 18 ] ; hence , we investigated the prevalence of c677 t and a1298c , two common mthfr gene mutations in northwestern iran , to determine whether these mutations related with rsa . the genotypes distribution of c677 t mthfr gene mutation was compared in the two studied groups . it is clear from table 3 and figure 2 that the heterozygosity in nucleotide 677th of the mthfr gene has no statistically significant difference among the two groups . however , homozygosity for 677 t allele of the mthfr gene in women with rsa was higher than healthy controls , which were concordant with previous reports [ 9 , 10 , 18 ] . the total frequency of 677 t alleles for mthfr gene 677 t was also compared between women experiencing rsa with fertile control women ( 23.59% , 24% ) , respectively . on the whole , our data has indicated no statistically significant difference in the prevalence of c677 t mutation between the two groups . these observations are in contrast with a previous report on the literature [ 14 , 19 ] . this difference may be explained by differences in the populations or by using low numbers of samples . on the other hand , our findings suggest that c677 t mutation probably has no significant role in the etiology of first - trimester rsa in patients in the northwest of iran or that other hyper coagulant gene mutations may have a role in rsa . furthermore , the frequency of a1298c mthfr gene mutation was also compared in patients and healthy women . as shown in table 3 and figure 2 , the frequency of 1298a / c genotype mthfr gene in patients was less than the control group . the frequency of 1298c / c genotype was higher in patients with rsa compared with the control group , in contrast with the heterozygote genotype . the total frequency of 1298 t alleles for mthfr gene 677 t were also compared between women experiencing rsa compared with the fertile women in the control group ( 34.83% , 40% ) , respectively ( figure 2 ) . this was in accordance with earlier investigations [ 13 , 15 ] , which reported no association between mthfr 1298a / c and rsa . findings of previous studies have shown that the presence of combined c677t / a1298c genotypes highly increased the risk of rsa . therefore , prevalence comparison of 677ct/1298ac compound heterozygosity in patients and healthy controls showed no significant difference . in conclusion , our results showed no significant variations in mthfr c677 t and a1298c genotype distribution among patients who suffered from rsa and controls . further studies on larger population may be needed . to better understand the pathobiology of rsa disease , we need to identify novel genetic variants and the interactive effects of these variants with each other and the environment . due to the fact that , it is possibile to detect a large number of samples and low costs , we proposed arms - pcr as a useful method that could be used for identification of these mutations .
introduction . 
 recurrent spontaneous abortion ( rsa ) is a significant obstetrical complication that may occur during pregnancy . 
 various studies in recent years have indicated that two common mutations ( c677 t and a1298c ) of the methylenetetrahydrofolate reductase ( mthfr ) gene are risk factor for rsa . 
 this study was carried out to determine the influence of ( c677 t and a1298c ) of the methylenetetrahydrofolate reductase ( mthfr ) gene mutations with rsa . materials and methods . 
 a total of 139 women were included in this study : 89 women with two or more consecutive miscarriages and 50 healthy controls . 
 total genomic dna was isolated from blood leukocytes . to determine the frequency of the two common c677 t and a1298c mthfr gene mutations in the patients and controls , we used two methods , amplification refractory mutation system - pcr and pcr - restriction fragment length polymorphism . 
 results . 
 there is no significant difference in the prevalence of 677t / t genotype among women with rsa and healthy controls ( p = 0.285 ) . 
 also no statistically significant difference in the frequency of a1298c mthfr gene mutation was detected between the two groups ( p = 0.175 
 ) . conclusion . in conclusion , 
 the results indicate that the amplification refractory mutation system - pcr method was in complete concordance with the results obtained by standard pcr - restriction fragment length polymorphism method . 
 the results also show no significant difference in mthfr c677t 
 / a1298c genotype distribution among the two groups ; therefore , further studies on larger population and other genetic variants to better understand the pathobiology of rsa are needed .