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

Capsule:
Study reporting extensive preclinical validation and accuracy assessment of next-generation sequencing (NGS)-based comprehensive aneuploidy screening on single cells. NGS has demonstrated a robust methodology, ready for clinical application in reproductive medicine.

Authors:
Francesco Fiorentino, Ph.D., Anil Biricik, M.Sc., Sara Bono, B.Sc., Letizia Spizzichino, B.Sc., Ettore Cotroneo, B.Sc., Giuliano Cottone, B.Sc., Felix Kokocinski, Ph.D., Claude-Edouard Michel, Ph.D.

Volume 101, Issue 5, Pages 1375–1382.e2

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).

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.

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