title: Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos.
abstract: OBJECTIVE
  To validate a next-generation sequencing (NGS)-based method for 24-chromosome aneuploidy screening and to investigate its applicability to preimplantation genetic screening (PGS).
  
  
  METHODS
  Retrospective blinded study.
  
  
  METHODS
  Reference laboratory.
  
  
  METHODS
  Karyotypically defined chromosomally abnormal single cells and whole-genome amplification (WGA) products, previously analyzed by array comparative genomic hybridization (array-CGH), selected from 68 clinical PGS cycles with embryos biopsied at cleavage stage.
  
  
  METHODS
  None.
  
  
  METHODS
  Consistency of NGS-based diagnosis of aneuploidy compared with either conventional karyotyping of single cells or array-CGH diagnoses of single blastomeres.
  
  
  RESULTS
  Eighteen single cells and 190 WGA products from single blastomeres, were blindly evaluated with the NGS-based protocol. In total, 4,992 chromosomes were assessed, 402 of which carried a copy number imbalance. NGS specificity for aneuploidy call (consistency of chromosome copy number assignment) was 99.98% (95% confidence interval [CI] 99.88%-100%) with a sensitivity of 100% (95% CI 99.08%-100%). NGS specificity for aneuploid embryo call (24-chromosome diagnosis consistency) was 100% (95% CI 94.59%-100%) with a sensitivity of 100% (95% CI 97.39%-100%).
  
  
  CONCLUSIONS
  This is the first study reporting extensive preclinical validation and accuracy assessment of NGS-based comprehensive aneuploidy screening on single cells. Given the high level of consistency with an established methodology, such as array-CGH, NGS has demonstrated a robust high-throughput methodology ready for clinical application in reproductive medicine, with potential advantages of reduced costs and enhanced precision.
authors.affiliation: Genoma Molecular Genetics Laboratory, Rome, Italy. Electronic address: fiorentino@laboratoriogenoma.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Fiorentino
authors.affiliation: Genoma Molecular Genetics Laboratory, Rome, Italy.
authors.forename: Anil
authors.initials: A
authors.lastname: Biricik
authors.affiliation: Genoma Molecular Genetics Laboratory, Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Bono
authors.affiliation: Genoma Molecular Genetics Laboratory, Rome, Italy.
authors.forename: Letizia
authors.initials: L
authors.lastname: Spizzichino
authors.affiliation: Genoma Molecular Genetics Laboratory, Rome, Italy.
authors.forename: Ettore
authors.initials: E
authors.lastname: Cotroneo
authors.affiliation: Genoma Molecular Genetics Laboratory, Rome, Italy.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Cottone
authors.affiliation: Bluegnome, Cambridge, United Kingdom.
authors.forename: Felix
authors.initials: F
authors.lastname: Kokocinski
authors.affiliation: Bluegnome, Cambridge, United Kingdom.
authors.forename: Claude-Edouard
authors.initials: CE
authors.lastname: Michel
mesh_terms: D000328:Adult; D000782:Aneuploidy; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000592:standards / Q000639:trends; D006801:Humans; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques / Q000592:standards / Q000639:trends; D009693:Nucleic Acid Hybridization / Q000379:methods; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000592:standards* / Q000639:trends*; D016032:Randomized Controlled Trials as Topic / Q000592:standards / Q000639:trends; D012189:Retrospective Studies; D016037:Single-Blind Method
keywords: Comprehensive chromosome screening; array comparative genomic hybridization; embryo aneuploidy; next-generation sequencing; preimplantation genetic screening