Preimplantation Genetic Diagnosis for Aneuploidy and Translocations Using Array Comparative Genomic Hybridization

Abstract

At least 50% of human embryos are abnormal, and that increases to 80% in women 40 years or older. These abnormalities result in low implantation rates in embryos transferred during in vitro fertilization procedures, from 30% in women <35 years to 6% in women 40 years or older. Thus selecting normal embryos for transfer should improve pregnancy results. The genetic analysis of embryos is called Preimplantation Genetic Diagnosis (PGD) and for chromosome analysis it was first performed using FISH with up to 12 probes analyzed simultaneously on single cells. However, suboptimal utilization of the technique and the complexity of fixing single cells produced conflicting results. PGD has been invigorated by the introduction of microarray testing which allows for the analysis of all 24 chromosome types in one test, without the need of cell fixation, and with staggering redundancy, making the test much more robust and reliable. Recent data published and presented at scientific meetings has been suggestive of increased implantation rates and pregnancy rates following microarray testing, improvements in outcome that have been predicted for quite some time. By using markers that cover most of the genome, not only aneuploidy can be detected in single cells but also translocations. Our validation results indicate that array CGH has a 6Mb resolution in single cells, and thus the majority of translocations can be analyzed since this is also the limit of karyotyping. Even for translocations with smaller exchanged fragments, provided that three out of the four fragments are above 6Mb, the translocation can be detected.

Clinical application of next-generation sequencing in preimplantation genetic diagnosis cycles for Robertsonian and reciprocal translocations

“…Firstly, over half of the embryos may be abnormal due to unbalanced segregation of the two involved chromosomes [2,14]. Further, in those embryos that are diagnosed as balanced, there is the possibility of additional incidental aneuploidies, including whole or partial chromosomal gains and losses, mosaicism, and segmental imbalances in other chromosomes that are known to commonly arise by either non-disjunction errors [15] or breakage-fusion-bridge cycles [16].…”

Current methods for preimplantation genetic diagnosis

“…Although it is not possible to use aCGH for haploid or polyploid embryo, low-grade mosaicism and single mutations, research has proved that a significant majority of embryos with such dysfunctions also have additional disorders that can be detected with aCGH (such as aneuploidy), and which are not present in approximately 0.2% of cases [19,20].…”

Selecting a Single Embryo for Transfer after In-vitro Fertilization: A Translational Medicine Perspective

“…Over 50% of IVF embryos have abnormal chromosomes increasing to up to 80% in women over 40 years of age [33]. Despite the presence of these abnormalities, few embryos arrest between day 3 and 5.…”

“…Despite the presence of these abnormalities, few embryos arrest between day 3 and 5. Most progressed into the blastocyst stage [34]. The implication of this is that genetically incompetent embryos that are likely going to miscarriage or failed to implant cannot be detected by the rate of embryo development alone observed morphologically.…”

“…This is because FISH screening does not make it possible to test for all 23 chromosome pairs. At best only 12 of the 23 chromosome pairs of an embryo can be analyzed using FISH thereby allowing some chromosomal abnormalities to be missed [33]. This limitation in FISH technology indicates a need for newer technologies like metaphase comparative genomic hybridization (mCGH), array Comparative Genomic Hybridization (aCGH), Qualitative Polymerase Chain Reaction(qPCR), Single Nucleotide Polymorphism (SNP) array and more recently NextGeneration Sequencing (NGS).…”