24sure microarray

PGS analysis was performed by array comparative genomic hybridization (aCGH). In particular the 24sure microarray (BlueGnome Ltd, Illumina) was used, which is a bacterial artificial chromosome (BAC) array with >5000 DNA clones not in disease or known copy number variant (CNV) regions. From 2009 to the end of 2013, samples were sent to Genesis Genetics (Plymouth, MI, USA) for analysis using BlueGnome technology and protocols followed by BlueFuse Multi™ software (Illumina Inc., San Diego, CA, USA) for data analysis. Since January 2014, samples have been analysed by the laboratory at the CReATe Fertility Centre using the same technology, protocols and software.

Samples were lysed and the DNA released was fragmented and subjected to whole-genome amplification with the Repli-g® single cell kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Two reference DNA samples (genomic male and female DNA) and one negative control followed the same amplification protocol as the embryo samples. Electrophoresis was performed to confirm that all samples and the positive controls amplified appropriately. The negative control did not produce any amplified DNA, indicating that there was no contamination. Amplified embryo DNA samples and references were labelled with Cy3 and Cy5 using the Illumina fluorescent labelling system, according to the manufacturer's instructions. Labelled samples and reference DNA were combined and applied to a 24Sure microarray (Illumina, USA) and co-hybridized overnight. After hybridization the slides were washed as described by Jaroudi and Wells (2013) , dried by centrifugation and scanned with a microarray scanner (InnoScan 710AL, Innopsys, Carbonne, France). Scanned images were analysed and quantified, and whole chromosomal copy number ratios were reported using the CytoChip algorithm fixed settings in BlueFuse Multi Software, version 3.3 (Illumina).