الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Idiopathic recurrent pregnancy loss (RPL) is a frequent obstetric complication, with approximately 2% of women experience two consecutive pregnancy losses, and 0.4-1% of women experience three or more consecutive losses in the reproductive age. The pathogenesis of RPL is complex, presumably involving the interaction of several genetic and environmental factors. While the exact cause of RPL remains undetermined in most of the cases, genetic predisposition to venous thrombosis, and elevation in total homocysteine (tHcy) levels (hyperhomocysteinaemia) have been described as playing a role in the pathogenesis of RPL. Moreover, anatomic, endocrine, and immune defects as well as infections have been proposed as causes for RPL. After evaluation of all causes, approximately half of all cases will remain unexplained.
Methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) is a key enzyme in the folate dependent remethylation of homocysteine (Hcy). It catalyzes the NADPH-linked reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominating circculating form of folate, and subsequently the vitamin B(12)-dependent methylation of Hcy to methionine. 5-methyltetrahydrofolate participates in single-carbon transfers that occurs as part of the synthesis of nucleotides, the remethylation of Hcy to methionine, and the synthesis of S-adenosylmethionine (SAM) the ultimate methyl donor that is involved in hundreds of biologic transmethylation reactions, including the methylation of DNA, proteins, neurotransmitters and phospholipids. As such, folic acid is indispensable for embryonic development, and deficiencies of folic acid or defects in MTHFR have demonstrated DNA hypomethylation and abnormal biochemical and/or phenotypic changes in model animals, cell culture, and humans.
While several mutations within it were described, the best-characterized MTHFR gene polymorphisms are the alanine-to-valine C677T, and the glutamate-to-alanine A1298C missense mutations. Moreover, while both single-nucleotide polymorphisms (SNPs) induce milder forms of MTHFR deficiency, the A1298C SNP, located in the enzyme regulatory domain, unlike the C677T SNP which is found within the enzyme catalytic domain, does not result in either a thermolabile protein or increased tHcy.
The 677C→T transition (C677T) renders the enzyme thermolabile, and homozygotes (677TT) and heterozygotes (677CT) have about a 70% and 35% reduced MTHFR activity in vitro, respectively. Homozygosity for the 677T allele is associated with elevated homocysteine levels, predominantly in individuals who have a low plasma folate level. Furthermore, the level of plasma homocysteine can be lowered in homozygous individuals by folic acid supplementation.
Through their effect on tHcy levels, MTHFR mutations have been implicated as risk factors in the pathogenesis of RPL. This was highlighted by the findings that the prevalence of homozygous variants of both SNPs was higher among women with more than three idiopathic RPLs. Hyperhomocysteinaemia caused by MTHFR mutations or folate deficiency was associated with placental abruption or infarction, preeclampsia, pregnancy-associated hypertension, and RPL. On the other hand, other investigators failed to demonstrate any association between MTHFR SNP, hyperhomocysteinaemia and RPL, as similar or even lower prevalence rates of MTHFR C677T SNP were seen in patients compared with women with uneventful pregnancies. As such, the relationship between MTHFR C677T gene polymorphism and risk of recurrent pregnancy loss (RPL) is still a matter of debate.
The objective of the current study was to assess the relationship between RPL and MTHFR C677T gene polymorphism in Egyptian women with two or more idiopathic pregnancy losses, and to determine the total homocysteine levels in these RPL cases, to allow proper management and genetic counselling for the high risk carriers.
The population of the present investigation comprised 50 females in the reproductive age, with history of two or more abortions, and a control group consisted of 50 healthy females, with one or more successful pregnancy, and without gestational complications. Moreover, they had no history of pregnancy loss. All females enrolled in this study were of the same ethnic origin.
The two studied groups have undergone full clinical examination with special stress on genetic history including: personal and family history, parental consanguinity, and history of pregnancy and delivery. Both groups were recruited in the molecular study using PCR/RFLP technique for detection of the MTHFR C677T gene polymorphism.
A. DNA extraction
Whole blood samples were collected from all participants in this study by venipuncture, placed into vacutainers with EDTA, kept at 4°C and processed shortly thereafter. Total genomic DNA was isolated by the phenol-chloroform method, as is standard, and was dissolved in nuclease-free water and stored at 4°C.
B. MTHFR genotyping
RPL cases and controls were screened for the C677T gene polymorphism of MTHFR.
1- PCR amplification
The PCR was carried out with specific primers.
2- Detection of PCR product
A part of the PCR amplified product was analyzed on a 2% agarose gel.
3- Restriction fragment length polymorphism (RFLP) analysis
The PCR amplified product was digested with Hinf I restriction enzyme.
4- Analysis of digested DNA product on a 3% agarose gel
Digested DNA fragments were then separated on a 3% agarose gel to determine the genotype.
Furthermore, all RPL cases (homo- and heterozygous variant individuals) were assayed for the measurement of plasma total homocysteine levels by Enzyme Immunoassay (EIA) using a commercial kit according to the manufacturer’s instructions.
Statistical analysis was performed using the IBM SPSS software package version 20.0. Which also computes the odds ratios (ORS) and 95% confidence intervals (95%CI). Pearson’s Chi-square test was used to assess intergroup significance and Student t-test was used to determine differences in means. Statistical significance was set at P ≤ 0.05.
The results of the present work showed the mean age at diagnosis of women with idiopathic recurrent miscarriage was 28.44 years ( range 18.0-40.0). The number of miscarriages ranged from at least 2 consecutive up to 11 times, with the mean number equals 6.5 times. Parental consanguinity was found in 22% (11cases). Seventy percent (35 cases) of women were aborted in the 1st trimester, 6.0% (3 cases) were aborted in the 2nd trimester, while 24.0% (12 cases) were aborted in both the 1st and 2nd trimester of gestation (the combined subgroup). For the control group, the mean age at time of blood sampling was 30.76 years (range 21.0-42.0).
Regarding the point mutation polymorphism located in MTHFR gene-667 two alleles (the C allele and the T allele), and two different genotypes (homozygote T/T and heterozygote C/T) were found, in addition to the wild-type C/C.
The MTHFR genotype distributions in women with RPL were: 4.0% (2/50) T/T; 56.0% (28/50) C/T; and 40.0% (20/50) C/C. In the control group, the homozygous MTHFR-677 T/T was present in 2.0% (1/50); the C/T genotype in 44.0% (22/50); and the C/C genotype in 54.0% (27/50) individuals.
The MTHFR allele frequencies in women with RPL were 32.0% for the T allele (mutant), and 68.0% for the C allele (wild-type). In controls, the T allele was 24.0% whereas the C allele was 76.0%. The most frequent allele in both groups was the C allele (76.0%) in the control group versus (68.0%) in cases with RPL.
Based on Chi-square and odds ratio tests, comparisons of the odds ratios (ORs) among different genotypes showed for RPL cases versus controls, that the individuals carrying the T/T homozygous genotype were at 2.7 fold higher risk when compared to individuals carrying the C/C homozygous genotype. Moreover, the individuals carrying the C/T heterozygous genotype were at about 1.7 fold higher risk when compared to individuals carrying the C/C homozygous genotype. Also, the results showed that the individuals carrying the homozygous T/T genotype were at 1.57 fold higher risk when compared to those carrying the heterozygous C/T genotype. Apparently, these results refer to the T allele as a probable risk factor that could be involved in the induction of RPL, and the individuals carrying the T allele were at about 1.5 fold higher risk for occurrence of RPL when compared to those carrying the C allele. However, for all the above results, there were no statistically significant differences between RPL cases for ORs and genotype distributions or allele frequencies.
For association of trimester of pregnancy loss with MTHFR genotypes:
• Concerning the 1st trimester pregnancy loss individuals, 35cases had: T/T 5.71% (2/35) homozygous for the mutant allele; C/T 54.29% (19/35) heterozygous carriers; C/C 40% (14/35) homozygous for the wild-type allele.
• For the 2nd trimester pregnancy loss individuals, 3 cases had: C/T 66.67% (2/3) heterozygous carriers; C/C 33.33% (1/3) homozygous for the wild-type allele. The homozygous for the mutant allele (T/T) could not be detected (0.0%) in RPL cases experiencing miscarriages in the 2nd trimester.
• For the combined subgroup, 12 cases had C/T 58.33% (7/12) heterozygous carriers; C/C 41.67% (5/12) for the wild-type allele. Again, the homozygous for the mutant allele (T/T) could not be detected (0.0%) in this subgroup.
• Statistical analysis of the results of the current work showed no significant association between the timing of miscarriages (1st trimester; 2nd= trimester or the combined) and the MTHFR genotypes, in comparison with the control group.
• No statistically significant differences for ORs and genotype distributions (for all possible combinations tested) or allele frequencies were observed in RPL cases experiencing miscarriages in the 1st trimester versus controls (32.9% versus 24.0% for the T allele, and 67.1% versus 76.0% for the C allele).
• No statistically significant differences for ORS and genotype distributions(for all possible combinations tested) or allele frequencies were observed in RPL cases experiencing miscarriage in the 2nd trimester versus controls (33.3% versus 24.0% for the T allele, and 66.7% versus 76.0% for the C allele).
• No statistically significant differences for ORs and genotype distributions (for all possible combinations tested) or allele frequencies were observed in the combined subgroup (1st and 2nd trimesters) versus controls (29.2% versus 24.0% for the T allele, and 70.8% versus 76.0% for the C allele).
• Furthrtmore, between women aborted in the 1st trimester and women aborted in the 2nd trimester, no statistically significant differences were observed with respect to genotype distributions or allele frequencies (32.9% versus 33.3% for the T allele, and 67.1% versus 66.7% for the C allele).
• Measurement of total plasma homocysteine levels for all RPL cases showed that the fasting plasma concentrations of homocysteine were comparable of among all C677T genotype carriers in RPL cases. The Mean ± SD was 8.33 ± 2.16 µmol/L for the C/C genotype, 8.02±2.23 µmol/L for the C/T genotype and 8.10±2.40 µmol/L for the T/T genotype. The determined tHcy levels were in the normal range ( <15 µmol/L ). Furthermore, comparison of the total Hcy levels among the different genotypes of the MTHFR gene polymorphism in RPL cases showed no statistically significant difference as P value =0.619.