Validation of arrayCGH on day-4 single blastomeres from day-3 embryos diagnosed as abnormal by FISH


Fluorescence in situ Hybridisation (FISH) is the technique most commonly applied to Preimplantation Genetic Screening (PGS). Array Comparative Genomic Hybridisation (aCGH) has emerged in the field of PGS as a new technique to analyse aneuploidies from all the human chromosomes. Biopsy can be performed on oocytes (polar bodies), cleavage stage (blastomere) or blastocyst stage (trophoectoderm). Whole Genome Amplification (WGA) is necessary to obtain good quality DNA to perform aCGH. The aim of this study was to evaluate the potential of single cell aCGH by comparing the results

Material and Methods:
This study included 28 blastomeres from embryos diagnosed as abnormal by FISH. FISH analysis was performed on single day-3 biopsied blastomeres for 9 chromosomes in two consecutive hybridisations: 1) Multivysion™ PB panel for chromosomes 13, 16, 18, 21 and 22; 2) MultiVysion™ 4 Colour Custom panel for chromosomes 15, 17, X and Y (Vysis®, Inc. Downers Grove, IL, USA). Additionally, re-hybridisation rounds were conducted to rescue non-informative or monosomic results using subtelomeric probes. The 28 abnormal embryos were re-biopsied on day 4. A single cell from each embryo underwent WGA using Sureplex® (BlueGnome Ltd., Cambridge, UK). WGA products and genomic DNA used as control were labelled in Cy3 and Cy5 fluorophores respectively. Both labelled DNAs were co-hybridised in a 24sure® array (BlueGnome Ltd., Cambridge, UK). After washing, slides were scanned using Innoscan® 710 (Innopsys, Carbonne, France). Data were analysed by BlueFuse Multi® software (BlueGnome Ltd., Cambridge, UK). The aCGH protocol spends arround 24 hours from single cell amplification to the interpretation of the results.

P. Mir, L. Rodrigo, A. Cervero, A. Mercader, A. Delgado, P. Buendía, A. Pellicer, C. Rubio and J. Martín
Institut Universitari IVI, PGD, Valencia, Spain


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