Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos

Fertil Steril. 2014 May;101(5):1375-82. doi: 10.1016/j.fertnstert.2014.01.051. Epub 2014 Mar 6.


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).

Design: Retrospective blinded study.

Setting: Reference laboratory.

Patient(s): 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.

Intervention(s): None.

Main outcome measure(s): Consistency of NGS-based diagnosis of aneuploidy compared with either conventional karyotyping of single cells or array-CGH diagnoses of single blastomeres.

Result(s): 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%).

Conclusion(s): 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.

Keywords: Comprehensive chromosome screening; array comparative genomic hybridization; embryo aneuploidy; next-generation sequencing; preimplantation genetic screening.

Publication types

  • Validation Study

MeSH terms

  • Adult
  • Aneuploidy*
  • Female
  • High-Throughput Nucleotide Sequencing / standards
  • High-Throughput Nucleotide Sequencing / trends
  • Humans
  • Middle Aged
  • Nucleic Acid Amplification Techniques / standards
  • Nucleic Acid Amplification Techniques / trends
  • Nucleic Acid Hybridization* / methods
  • Pregnancy
  • Prenatal Diagnosis / standards*
  • Prenatal Diagnosis / trends*
  • Randomized Controlled Trials as Topic / standards
  • Randomized Controlled Trials as Topic / trends
  • Retrospective Studies
  • Single-Blind Method