PGT – aneuploidy (PGT-A)

The aim of preimplantation genetic testing of aneuploidy (PGT-A) in embryos by the NGS method is to determine the number of copies of chromosomes, or parts thereof. Determination of the copy number is based on comparison of sequence representation from all regions of the genome, both within the sample and especially in comparison with a reference sample having normal copy number of all chromosomes. 

The input sample is 5–10 trophectoderm cells taken 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 DOP-PCR principle. In the last amplification step, sequences originating from the embryo are added to the amplified sequences to create a library for massively parallel sequencing called NGS. Sequencing data is digitally evaluated through a series of applications to match the obtained sequences to an individual embryo (approximately 5.5 million sequences of 75 nucleotides in length per embryo), map the obtained sequences to a reference genome, and determine their origin position and number for each segment of the genome. The numbers of sequences obtained from each segment of the chromosomes are compared within the sample and against a reference sample of normal chromosome numbers. Mitochondrial DNA sequences are used as an additional layer of quality control in the laboratory and can be used to clearly distinguish embryo groups originating from individual patients. From the obtained data, it is also possible to analyse allelic frequencies in sufficiently covered SNP markers and, in addition to chromosome aneuploidy, to detect abnormally fertilised embryos with a haploid or triploid number of chromosome sets. 

A normal number of chromosomes means two copies of chromosomes 1–22, two copies of the X chromosome in female embryos and one copy of the X and Y chromosomes each in male embryos. Embryos with normal copy numbers are referred to as euploid. Pathological findings – aneuploidy – are trisomy in case of excess copy, or monosomy in case of missing copy of the corresponding chromosome, or part of it. Deviations from the normal number of chromosomes in both directions can be expressed as a percentage and intervals can be set for evaluating the number of copies. Deviations of up to 30% are considered normal. Trisomy, or monosomy, refers to deviations above 70%. Low mosaics refer to deviations in the interval of 30–50% and high mosaics refer to deviations in the range of 50–70%. Deviations involving only part of the chromosome are called segmental aneuploidy. Based on the result of the test, embryos are recommended or not recommended for transfer. For euploid embryos and pathological aneuploid embryos unsuitable for transfer, the diagnostic reliability of the test is greater than 97%. Due to mosaicism naturally occurring in embryos and due to the biological characteristics of the primary sample (5–10 embryo cells), it is not possible to achieve higher reliability of the test. 

Embryos carrying whole chromosome aneuploidy with a deviation from the normal number of chromosomes of more than 70% are considered non-viable in the context of PGT-A by the NGS method. Such aneuploidy is most likely of gamete origin, occurs in all embryo cells and is not compatible with the further development of the foetus into a healthy individual. By excluding these embryos from transfer, it is possible, within the context of IVF, to achieve a higher proportion of implanted embryos after transfer, reduce the risk of foetal abortion due to aneuploidy and shorten the treatment time required for the birth of a healthy child.