|The Study of MTHFR gene C677T Polymorphism in Human Embryonic Blastomers by Fluorescence PCR Method|
|Saeedeh Saeedi1, Mohammad Reza Mashayekhi1, Masoud Maleki1|
|1Department of Biology, Tabriz Branch, Islamic Azad University, Tabriz, Iran|
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Keywords : PGD, PEP-PCR, MTHFR Gene, C677T Polymorphism, Recurrent Miscarriage
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Objective: The aim of the present study was to evaluate Methylene tetrahydrofolatereductase (MTHFR)gene C677T polymorphismin human pre-implantation embryos.
Materials and Methods: 50 embryos after biopsy at cleavage stage (which were inappropriate for assisted reproductive techniques) were analyzed in order to study of MTHFR C677T polymorphism and sex determination. After cell lysis, in the first step, whole genome amplification (WGA) was performed by primer extension pre-amplification( PEP) PCR method. In the next step, fluorescence PCR with labeled primers was used to examine this polymorphism in the blastomers. Finally, the HinfI enzyme was employed to cutting the fragments and the capillary electrophoresis was used for determination the PCR products size.
Results: only 37 blastomeres among 50 samples were successfully amplified that 23 male and 14 female embryos were observed and the frequencies of CC,CT and TT genotypes in embryos were 67/56%,24.32%,8/10%,respectively. Additionally, the frequencies of 677C and 677T allele were 80% and 20% , respectively.
Conclusion: In this study, MTHFR c.677C˃T genotype frequency was evaluated in human embryonic blastomeres before implantation.So this polymorphism may provide a new and valuable biomarker for evaluating the embryo usefulness and improving the IVF process by choosing the best embryo.Our findings revealed that the C677T polymorphism detection rate was %74 in the samples.