51. Extracellular embryo genomic DNA and its potential for genotyping applications
- Author
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Aurora Diotallevi, Mauro Magnani, Luca Galluzzi, Francesca Andreoni, Simone Palini, Mariangela Primiterra, Silvia De Stefani, and Carlo Bulletti
- Subjects
culture medium ,Short Communication ,Embryo ,Embryo culture ,Biology ,Preimplantation genetic diagnosis ,Molecular biology ,blastocoele fluid ,genomic DNA ,chemistry.chemical_compound ,human embryos ,chemistry ,Genotype ,MTHFR ,gDNA ,Allele ,Genotyping ,DNA ,Biotechnology - Abstract
Background: Preimplantation genetic diagnosis (PGD) currently relies on biopsy of one or few embryo cells. Our aim was to evaluate the embryo extracellular matrices (spent medium and blastocoele fluid) as source of DNA for embryo genotyping. Results/methodology: We first evaluated the amplifiability and the amount of genomic DNA in spent embryo culture media from day 3 (n = 32) and day 5/6 (n = 54). Secondly, we evaluated the possibility to genotype the MTHFR polymorphism C677T from media at day 5/6 (n = 8) and blastocoele fluids (n = 9) by direct sequencing. The C677T polymorphism detection rate was 62.5 and 44.4% in medium and fluid, respectively. Conclusion: A noninvasive approach for embryo genotyping was possible, but still with limitations due to low detection rate and possible allele dropout., PGD currently relies on biopsy of embryo cells, which could imply some risk of embryo damage. Since embryo DNA was retrieved both in blastocoele cavity and culture medium, we evaluated if this extracellular DNA could be useful to obtain medical-related genetic information from the embryo. First, we used multicopy genes to verify amplifiability and amount of DNA in medium, then we amplified and sequenced one gene fragment containing a polymorphism of medical importance in a subset of samples. The polymorphism detection rate was not yet high enough to warrant clinical application but we demonstrated that this approach was possible.
- Published
- 2015