Although selection of chromosomally normal embryos has the potential to improve outcomes for patients undergoing IVF, the clinical impact of aneuploidy screening by fluorescence in situ hybridization (FISH) has been controversial. There are many putative explanations including sampling error due to mosaicism, negative impact of biopsy, a lack of comprehensive chromosome screening, the possibility of embryo self-correction and poor predictive value of the technology itself. Direct analysis of the negative predictive value of FISH-based aneuploidy screening for an embryo's reproductive potential has not been performed. Although previous studies have found that cleavage-stage FISH is poorly predictive of aneuploidy in morphologically normal blastocysts, putative explanations have not been investigated. The present study used a single nucleotide polymorphism (SNP) microarray-based 24 chromosome aneuploidy screening technology to re-evaluate morphologically normal blastocysts that were diagnosed as aneuploid by FISH at the cleavage stage. Mosaicism and preferential segregation of aneuploidy to the trophectoderm (TE) were evaluated by characterization of multiple sections of the blastocyst. SNP microarray technology also provided the first opportunity to evaluate self-correction mechanisms involving extrusion or duplication of aneuploid chromosomes resulting in uniparental disomy (UPD). Of all blastocysts evaluated (n = 50), 58% were euploid in all sections despite an aneuploid FISH result. Aneuploid blastocysts displayed no evidence of preferential segregation of abnormalities to the TE. In addition, extrusion or duplication of aneuploid chromosomes resulting in UPD did not occur. These findings support the conclusion that cleavage-stage FISH technology is poorly predictive of aneuploidy in morphologically normal blastocysts.

Many studies estimate that chromosomal mosaicism within the cleavage-stage human embryo is high. However, comparison of two unique methods of aneuploidy screening of blastomeres within the same embryo has not been conducted and may indicate whether mosaicism has been overestimated due to technical inconsistency rather than the biological phenomena. The present study investigates the prevalence of chromosomal abnormality and mosaicism found with two different single cell aneuploidy screening techniques. Thirteen arrested cleavage-stage embryos were studied. Each was biopsied into individual cells (n = 160). The cells from each embryo were randomized into two groups. Those destined for FISH-based aneuploidy screening (n = 75) were fixed, one cell per slide. Cells for SNP microarray-based aneuploidy screening (n = 85) were put into individual tubes. Microarray was significantly more reliable (96%) than FISH (83%) for providing an interpretable result (P = 0.004). Markedly different results were obtained when comparing microarray and FISH results from individual embryos. Mosaicism was significantly less commonly observed by microarray (31%) than by FISH (100%) (P = 0.0005). Although FISH evaluated fewer chromosomes per cell and fewer cells per embryo, FISH still displayed significantly more unique genetic diagnoses per embryo (3.2 ± 0.2) than microarray (1.3 ± 0.2) (P < 0.0001). This is the first prospective, randomized, blinded and paired comparison between microarray and FISH-based aneuploidy screening. SNP microarray-based 24 chromosome aneuploidy screening provides more complete and consistent results than FISH. These results also suggest that FISH technology may overestimate the contribution of mitotic error to the origin of aneuploidy at the cleavage stage of human embryogenesis.

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Methods of comprehensive microarray-based aneuploidy screening in single cells are rapidly emerging. Whole-genome amplification (WGA) remains a critical component for these methods to be successful. A number of commercially available WGA kits have been independently utilized in previous single-cell microarray studies. However, direct comparison of their performance on single cells has not been conducted. The present study demonstrates that among previously published methods, a single-cell GenomePlex WGA protocol provides the best combination of speed and accuracy for single nucleotide polymorphism microarray-based copy number (CN) analysis when compared with a REPLI-g- or GenomiPhi-based protocol. Alternatively, for applications that do not have constraints on turnaround time and that are directed at accurate genotyping rather than CN assignments, a REPLI-g-based protocol may provide the best solution.