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Accurate 23 chromosome aneuploidy screening in human blastomeres using single nucleotide polymorphism (SNP) microarrays

2007; Elsevier BV; Volume: 88; Linguagem: Inglês

10.1016/j.fertnstert.2007.07.025

1556-5653

Nathan R. Treff, Jing Su, J. Mavrianos, Paul A. Bergh, Kathleen A. Miller, Richard T. Scott,

Chromosomal and Genetic Variations

ObjectiveThe combination of whole-genome amplification (WGA) and microarray technologies may provide an attractive solution to the many limitations of fluorescence in situ hybridization (FISH) based screening for PGD. This study seeks to validate a WGA- and single nucleotide polymorphism (SNP)-based microarray paradigm, and provide an accurate single cell 23 chromosome aneuploidy screening technology.DesignProspective, randomized, and blinded.Materials and methodsPhase I: Three single cells from each of 9 stable cell lines with various previously established karyotypes were studied. These included lines which were trisomic (8, 9, 13, 15, 16 and 21, 18, and X), one that had monosomy 21, and one 46,XX cell line. Single cells were loaded into individual tubes and were randomized and blinded. WGA was performed using a modification of the GenomePlex system (Sigma-Aldrich). Microarray analysis was performed on a genome-wide 250K SNP genotyping microarray (Affymetrix). Copy number analysis was performed using the copy number analysis tool (CNAT) 4.0.1 (Affymetrix). Each cell was analyzed and a final diagnosis resulted for each chromosome prior to unblinding. Phase II: Eighty-two blastomeres obtained by biopsy of 19 discarded embryos were similarly analyzed.ResultsPhase I: Two of the 27 single cells resulted in indeterminate diagnosis (93% overall reliability), the remaining 25 were diagnosed accurately. Although two cells did not produce an evaluable result, there were no misdiagnoses amongst those with assigned karyotypes (100% accuracy). This included the characterization of the ploidy status of 575 individual chromosomes without a single misdiagnosis. Phase II: Interpretable results were obtained on all 82 blastomeres obtained from 19 embryos. Six of the discarded embryos were euploid for all 23 chromosomes. The remaining embryos demonstrated aneuploidy consistent with meiotic and mitotic errors, and mosaicism. Most striking was the consistency in abnormalities throughout individual embryos. While some mosaicism was present, a missing chromosome in one blastomere was typically accompanied by an extra copy of the same chromosome in other cells.ConclusionsMicroarray based aneuploidy screening has excellent reliability and accuracy, and holds enormous promise in clinical PGD of aneuploidy. This study represents the first validated method of single-cell whole-genome SNP microarray-based aneuploidy assessment. ObjectiveThe combination of whole-genome amplification (WGA) and microarray technologies may provide an attractive solution to the many limitations of fluorescence in situ hybridization (FISH) based screening for PGD. This study seeks to validate a WGA- and single nucleotide polymorphism (SNP)-based microarray paradigm, and provide an accurate single cell 23 chromosome aneuploidy screening technology. The combination of whole-genome amplification (WGA) and microarray technologies may provide an attractive solution to the many limitations of fluorescence in situ hybridization (FISH) based screening for PGD. This study seeks to validate a WGA- and single nucleotide polymorphism (SNP)-based microarray paradigm, and provide an accurate single cell 23 chromosome aneuploidy screening technology. DesignProspective, randomized, and blinded. Prospective, randomized, and blinded. Materials and methodsPhase I: Three single cells from each of 9 stable cell lines with various previously established karyotypes were studied. These included lines which were trisomic (8, 9, 13, 15, 16 and 21, 18, and X), one that had monosomy 21, and one 46,XX cell line. Single cells were loaded into individual tubes and were randomized and blinded. WGA was performed using a modification of the GenomePlex system (Sigma-Aldrich). Microarray analysis was performed on a genome-wide 250K SNP genotyping microarray (Affymetrix). Copy number analysis was performed using the copy number analysis tool (CNAT) 4.0.1 (Affymetrix). Each cell was analyzed and a final diagnosis resulted for each chromosome prior to unblinding. Phase II: Eighty-two blastomeres obtained by biopsy of 19 discarded embryos were similarly analyzed. Phase I: Three single cells from each of 9 stable cell lines with various previously established karyotypes were studied. These included lines which were trisomic (8, 9, 13, 15, 16 and 21, 18, and X), one that had monosomy 21, and one 46,XX cell line. Single cells were loaded into individual tubes and were randomized and blinded. WGA was performed using a modification of the GenomePlex system (Sigma-Aldrich). Microarray analysis was performed on a genome-wide 250K SNP genotyping microarray (Affymetrix). Copy number analysis was performed using the copy number analysis tool (CNAT) 4.0.1 (Affymetrix). Each cell was analyzed and a final diagnosis resulted for each chromosome prior to unblinding. Phase II: Eighty-two blastomeres obtained by biopsy of 19 discarded embryos were similarly analyzed. ResultsPhase I: Two of the 27 single cells resulted in indeterminate diagnosis (93% overall reliability), the remaining 25 were diagnosed accurately. Although two cells did not produce an evaluable result, there were no misdiagnoses amongst those with assigned karyotypes (100% accuracy). This included the characterization of the ploidy status of 575 individual chromosomes without a single misdiagnosis. Phase II: Interpretable results were obtained on all 82 blastomeres obtained from 19 embryos. Six of the discarded embryos were euploid for all 23 chromosomes. The remaining embryos demonstrated aneuploidy consistent with meiotic and mitotic errors, and mosaicism. Most striking was the consistency in abnormalities throughout individual embryos. While some mosaicism was present, a missing chromosome in one blastomere was typically accompanied by an extra copy of the same chromosome in other cells. Phase I: Two of the 27 single cells resulted in indeterminate diagnosis (93% overall reliability), the remaining 25 were diagnosed accurately. Although two cells did not produce an evaluable result, there were no misdiagnoses amongst those with assigned karyotypes (100% accuracy). This included the characterization of the ploidy status of 575 individual chromosomes without a single misdiagnosis. Phase II: Interpretable results were obtained on all 82 blastomeres obtained from 19 embryos. Six of the discarded embryos were euploid for all 23 chromosomes. The remaining embryos demonstrated aneuploidy consistent with meiotic and mitotic errors, and mosaicism. Most striking was the consistency in abnormalities throughout individual embryos. While some mosaicism was present, a missing chromosome in one blastomere was typically accompanied by an extra copy of the same chromosome in other cells. ConclusionsMicroarray based aneuploidy screening has excellent reliability and accuracy, and holds enormous promise in clinical PGD of aneuploidy. This study represents the first validated method of single-cell whole-genome SNP microarray-based aneuploidy assessment. Microarray based aneuploidy screening has excellent reliability and accuracy, and holds enormous promise in clinical PGD of aneuploidy. This study represents the first validated method of single-cell whole-genome SNP microarray-based aneuploidy assessment.