Introduction

In medical terminology, spontaneous abortion or miscarriage, is defined as the involuntary termination of pregnancy before 20 weeks’ gestation. Recurrent pregnancy loss (RPL), also known as recurrent miscarriage, is defined as the occurrence of three or more consecutive pregnancy loss with the same partner, and having no more than one live-born child (1).

Miscarriage may occur for many reasons, not all of which can be identified. Thrombophilic disorders are among the few recognized risk factors and potential causes of recurrent miscarriages. During pregnancy, mother's body adapts to the needs of the fetus, since her blood provides all of the nutrients and oxygen the fetus needs throughout the pregnancy (2). The

placenta is the primary site of nutrient and gas exchange between the fetus and the expectant mother. The perfusion of the intervillous space of placenta with maternal blood allows the transfer of nutrients and oxygen from the mother to the fetus and the transfer of waste products and carbon dioxide back from the fetus to the maternal blood supply. In patients with thrombophilia, damage of the placenta happens mostly due to blood clot formation in the maternal as well as the fetal vessels. In addition, since the maternal blood comes out of the maternal vessels and flows very slowly between the branches of the fetal vessels (intervillous space), maternal blood is vulnerable to clot formation. If maternal blood clots in this space, the fetal vessels will be

Table 1. Primer sequence, amplified product, restriction enzyme for detection of C677T polymorphism in MTHFR gen

Note:    * Normal allele         ** Mutant allele

destroyed, since nutrients and oxygen will not be prvided for the vessels; this leads to placental degeneration and fetal vessel damage (3).

Single-nucleotide polymorphisms (SNPs) in metabolic pathways which regulate enzymes such as methylenetetrahydrofolate reductase (MTHFR) are regarded as risk factors for thrombophilia. MTHFR is the key enzyme in folate, methionine and homocysteine metabolism (4). Disturbances in MTHFR activity could be the cause of increased serum level of homocysteine (a potentially toxic amino acid). Hyperhomocysteinemia is considered a risk factor for changes in the coagulation cascade through direct cytotoxic effect on endothelium, atherogenesis, activation of coagulation factor V and VII, increased level of thrombin, platelet aggregation and a tendency toward venous thrombosis (4, 5).

MTHFR irreversibly reduces 5,10-methylene-tetrahydrofolate (substrate) to 5-methyltetra-hydrofolate (product). 5-Methyltetrahydrofolate is the dominant folate form in the circulating human plasma and is used to convert homocysteine to methionine through methionine synthase (5-7). Decreased MTHFR activity hamstrings the methylation cycle, resulting in increasing levels of homocysteine (7). In humans, the gene coding for MTHFR enzyme is located at chromosome 1.p36.3, and is composed of 11 exons (8).

A common polymorphism in the gene for the MTHFR enzyme is known as the C677T MTHFR polymorphism. It leads to an altered amino acid sequence, and is associated with a decreased enzyme activity. A thymine(T)-to-cytosine(C) transition at nucleotide 677 in the exon 4 of MTHFR gene, leads alanine to be substituted by valine residue at position 222 in the catalytic domain of the MTHFR enzyme. The C677T polymorphism of the MTHFR gene encodes a thermolabile variant of the enzyme with reduced activity; reduced activity can lead to elevated levels of homocysteine (also called hyperhomocy-steinemia), especially when folate levels are low.

As a matter of  fact, recent published studies suggest that the C677T polymorphism is likely to modify the enzyme stability rather than enzyme activity. Generally, it was revealed that individuals with homozygous (677TT) genotype have 30% of the MTHFR enzymatic activity of the wild variant, while the heterozygotes (677TT) have 65% of the
enzymatic activity (9-11).

When folate intake is inadequate, individuals who are homozygous (T/T) for the abnormal gene have lower levels of the MTHFR enzyme and thus higher levels of homocysteine in their blood. Improved folate nutritional status appears to stabilize the MTHFR enzyme, resulting in improved enzyme levels and lower homocysteine levels (9, 10).

Considering the effects of MTHFR C677T polymorphism on hyperhomocysteinemia, the increased risk of venous thrombosis and also the association between inherited thrombophilia and recurrent pregnancy loss, the main objective of the present study is to investigate the relationship between the C677T polymorphism of MTHFR gene as a genetic risk factor for idiopathic recurrent miscarriage.

Materials and Methods

Study Population

This prevalence case–control study included 30 women with a history of two or more unexplained consecutive miscarriages, referring to Avicenna Infertility Clinic (AIC) in Tehran. Also, 10 fertile women with at least two live births who were reffering to Baqiyatallah Hospital,Tehran, were included in this study, between August and March, 2009. The study on Avicenna patients was approved by the ethics committee of the clinic and informed consents were obtained from the study participants. Also, the institutional review board approval and informed consents were obtained from all participants in the control group. Women with a previous history of miscarriage, caused by chromosomal abnormality in the embryo, uterine anatomical anomaly, endocrine factors, and infections, were not included in this study. Blood samples were taken for testing.

Mutation analysis

Genomic DNA was extracted from an 80 μl aliquot of whole blood collected in EDTA (ethylene diaminetetraacetic acid) using salting-out method (as the standard method); the extracted DNA was stored at −20°C prior to analysis. To detect the presence or absence of the C677T polymorphism, we selectively amplified a 198-bp fragment of the MTHFR gene by polymerase chain reaction (PCR) (Table 1). PCRs

Figure 1. The electrophoresis image of the PCR product for a 198-bp fragment of the MTHFR gene on agarose gel; the size of PCR product (198 bp) was determined in comparison with 50 bp molecular weight marker in bp on lines 19 and 10 of the image

Figure 2.  Agarose gel electrophoretic analysis of the MTHFR C677T polymorphism; lines 10 and 20 represent 50 bp molecular weight marker; line 1  represents  the 198 bp fragment of PCR product before RFLP; lines  5 and 18 represent the 175 bp fragment corresponding to the genotype TT after digestion of Hinfl as positive control; lines 3, 6, 8, 11, 12, 16, 17, and 19 represent the 198 bp fragment corresponding to the genotype CC after digestion of Hinfl; lines 2, 4, 7, 9, 13, 14, and 15 represent fragments corresponding to the genotype CT after digestion of Hinfl (198, 175 bp)

were performed with 40 ng of genomic DNA, 1 U/μl Taq DNA polymerase, 3.5 µL of 10X PCR buffer [Tris-HCl (pH 8.4), 500 mM KCl], 2.0 mM MgCl₂, 0.25 mM of all four deoxynucleotide triphosphates, and 0.4 µM of each primer, in a volume of 20 µL. After denaturation at 95°C for 5 min, the temperature was cycled 35 times (95°C for 1 min, 61°C for 1 min, and 72°C for 1 min), followed by an extension at 72°C for 7 min to amplify the target DNA.

MTHFR C677T polymorphism was assayed by polymerase chain reaction (PCR) amplification using primers, under the previously described conditions (Figure 1). Ten μl of PCR product was digested overnight at 37 °C with 1 unit of Hinfl restriction enzyme in a final volume of 15 μl, according to the manufacturers instructions (New England Biolabs). This reaction yielded 198 base pair fragments in the presence of allele C, and 175 and 23 base pairs in the presence of allele T. A heterozygous individual will give three fragments—198, 175 and 23 bp in length.

 Digestion results were submitted to electrophoresis in a 3% agarose gel and the bands were visualized using ethidium bromide staining on an ultraviolet transilluminator; the control on each gel included a known homozygous.

Statistical analysis

The genotypes and allele frequencies of MTHFR C677T variations in both groups of patients were compared with the controls, using the χ² test. P

Table 2. The prevalence and allelic frequencies of MTHFR C677T polymorphism among recurrentmiscarriage patients and control group

Note:  In compare with the 2 groups, no significant difference was observed among genotypes.