Optimization and application of molecular preimplantation genetic diagnosis (PGD) with study of C677T polymorphism of the MTHFR gene involved in recurrent miscarriage

Background: PGD is screening method for couples who wish to have a healthy child and can determine genotype of embryos before implantation to prevent from transfer and implantation of affected embryos. The aim of study was to optimization and application of molecular preimplantation genetic diagnosis (PGD) with examination of methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism and sex determination in human preimplantation embryos.Methods: A number of 50 cleavage stage embryos (not usable for IVF) for MTHFR C677T polymorphism and sex determination study were tested. After cell lysis, whole genome amplification (WGA) was performed with primer extension preamplification (PEP-PCR) method. To determine this polymorphism in the blastomeres, fluorescent PCR was used with labeled primers and subsequently cut with HinfI enzyme and finally, the exact size of PCR productes were determined using capillary electrophoresis.Result: Only 37 samples out of 50 blastomeres amplified successfully that among them 23 male and 14 female embryos were observed and 25 normal genotype, 9 heterozygous and 3 mutant genotype were detected. The frequencies of 677C and 677T allele were 80% and 20% respectively.Conclusion: In this study, MTHFR c.677C˃T genotype frequency in human preimplantatin embryos was evaluated. Studies trying to establish an association between folic acid metabolism and preimplantation embryo development have shown that MTHFR is expressed in both human oocytes and embryos .Given the relative ease with which embryonic MTHFR genotype can be determined, this polymorphisms may represent valuable new biomarker for the assessment of embryo competence and improve in vitro fertilization (IVF) process.

کلیدواژه ها:

C677T polymorphism ، MTHFR gene ، PEP-PCR ، Recurrent miscarriage ، PGD