Next-generation sequencing based preimplantation genetic testing of 24-chromosome aneuploidy and monogenic disorders. N. R. Treff1,2,3, X. Tao1, A. Fedick1,2, D. Taylor1,2,3, K. H. Hong1,2, E. J. Forman1,2, R. T. Scott Jr.1,2 1) Reproductive Medicine Associates of New Jersey, Basking Ridge, NJ., USA; 2) Dept. of Obstertics, Gynecology, and Reproductive Sciences, UMDNJ-Robert Wood Johnson Medical School, Basking Ridge, NJ, USA; 3) Dept. of Genetics, Rutgers-The State University of New Jersey, Piscataway, NJ, USA.

   Comprehensive chromosome screening (CCS) of preimplantation embryo aneuploidy has recently been shown to improve IVF outcomes in multiple randomized controlled trials. Next-generation sequencing (NGS) may provide a unique opportunity to perform CCS and monogenic disorder testing in parallel with high throughput and reduced costs. However, the unique and major challenge of applying NGS to preimplantation genetic testing (PGT) is maintaining accuracy on the limited quantity of starting material from an embryo, while also controlling costs per case (since multiple embryos are evaluated). This study develops and validates a new cost-effective methodology for NGS-based detection of monogenic disorders and 24-chromosome aneuploidy in the human blastocyst. Phase I of development involved the characterization of 5-cell samples from positive control cell lines in order to mimic the number of cells obtained in a typical trophectoderm biopsy. These samples included a variety of cell lines with known aneuploidy, triploidy, or mutation carrier status. NGS was performed using semiconductor sequencing on either the Ion PGM or Proton with molecular barcoding for analysis of multiple samples per chip. Specific disease related mutations, highly polymorphic SNPs, and repetitive sequences with chromosome specific alignment capability were targeted during PCR based capture. Sample processing and data analysis criteria were established to provide 100% consistency with the known genotypes and karyotypes of each control sample. Phase II involved blinded analysis of trophectoderm biopsies from previously characterized human blastocysts and additional cell line samples. Up to 1010 bases were sequenced with up to 96 samples on an individual P1 chip (4,608 chromosomes in a single run). 80 embryo and 240 cell line samples were evaluated. NGS resulted in 100% consistency with known karyotypes and genotypes. Targeted sequencing operating costs were 1/3rd lower than the most efficient of the currently used methodologies. In conclusion, targeted NGS provides an accurate and high-throughput methodology for preimplantation 24-chromosome aneuploidy screening and monogenic disorder diagnosis. This NGS method also represents a more cost-effective solution for PGT and may soon become a standard in reproductive medicine.

You may contact the first author (during and after the meeting) at