PGT – monogenic disease (PGT‐M)

Preimplantation genetic testing of monogenic diseases in embryos by karyomapping (SNP array) to determine their genotype and to test aneuploidy. Embryos carrying a genotype associated with the development of a genetic disorder or a variation in the number of chromosomes are not recommended for transfer.

PGT testing by karyomapping is performed in several stages. The input sample is 5–10 trophectoderm cells collected on days 5–6 of embryo development at the blastocyst stage. The cells are first lysed and the released DNA then amplified by genome-wide amplification based on the MDA principle. The next step is DNA analysis of the patient and relatives together with amplified embryo DNA using SNP array technology. By detecting fluorescence on a special chip, it is possible to determine the nucleotide composition and therefore the genotype of individual tags (SNPs) at precisely defined positions in the human genome. Approximately 700,000 SNPs covering all chromosomes and the entire genome are used for the analysis. By evaluating the fluorescence intensity in two channels for all SNPs on the chip, information about the genotype and allele ratio between the SNPs and the whole genome is obtained. SNP alleles are transmitted from parents to offspring on the principle of Mendelian inheritance. In the last phase, the obtained SNP data are digitally evaluated by a series of applications in order to determine the genotype of the embryo in the gene tested and to determine the number of copies of all chromosomes, or parts thereof, in the embryo. 

Embryo genotyping is performed using the method of indirect genetic diagnosis, where instead of the mutation itself, SNP markers that are bound to it (on one strand of DNA) are tested. The set of all SNP markers in association with a mutant or normal allele is called a haplotype. An embryonic genotype is a combination of haplotypes that represent and bind to a mutant or normal allele of the gene being tested. The length of the haplotype and the number of SNP markers in association with a particular allele vary for each embryo depending on the positions of the crossing overs. The diagnostic reliability of embryo genotyping within PGT-M in this way is very high (>99.5%) and compensates for the risk of ADO (allelic drop-out) when testing very small amounts of DNA from several cells taken from the embryo. 

Aneuploidy testing in embryos is performed simultaneously with genotyping using whole-genome SNP analysis. Using DNA samples from the mother and father, in case of chromosome aneuploidy, or parts thereof, it is possible to determine whether the aneuploidia in the embryo is meiotic (originating from an ovum or sperm) or is of mitotic origin and occurred only after the formation of the zygote during early embryonic development. Aneuploidy of meiotic origin is considered non-viable because it is most likely to occur in all cells and embryos and is not compatible with the further development of the foetus into a healthy individual. 

Examined embryos are recommended or not recommended for transfer based on the combination of the result of embryo genotyping with aneuploidy testing, in accordance with a genetic indication for testing that reflects the type of inheritance, severity of manifestations and risk of developing hereditary disease associated with the embryonic genotype.