Current quantitative methodologies for preimplantation genetic testing frequently misclassify meiotic aneuploidies as mosaic

Abstract

Objective: To study the biological origin of chromosomal abnormalities in embryos reported as mosaic after next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) and to assess the accuracy of current NGS-based methods in distinguishing meiotic from mitotic errors.

Design: Retrospective cohort study utilizing single-nucleotide polymorphism genotyping and karyomapping to identify meiotic aneuploidy in clinically reported mosaic embryos.

Subjects: A total of 146 embryos from 87 patients who underwent in vitro fertilization cycles with NGS-based PGT-A between 2018 and 2020 at The Evewell, London, United Kingdom.

Exposure: Embryos underwent clinical NGS-based PGT-A to identify chromosomal abnormalities. Haplotype-based methods such as single-nucleotide polymorphism-based genotyping and karyomapping were performed on the same amplified material used for NGS-based PGT-A to determine the origin of the chromosomal errors.

Main outcome measures: The proportion of embryos reported as mosaic by NGS that contained meiotic errors, and the distribution of meiotic vs. mitotic origin among chromosomal abnormalities identified in the mosaic range.

Results: Of the 141 embryos identified as mosaic after NGS-based PGT-A, 32.6% (n = 46/141) contained an error of meiotic origin, challenging their classification as "mosaic embryos." In total, 191 individual chromosomal errors were reported in the mosaic range by NGS-based PGT-A. Of those, 29.3% (56/191) errors (both below and above the 50% copy number threshold) were found to be of meiotic origin. The majority (94.6%) of meiotic errors were maternal in origin. Embryos with multiple chromosomal abnormalities were significantly more likely to have at least one meiotic error. Higher intermediate copy number values correlated with an increased probability of an error being of meiotic origin.

Conclusion: This study presents the first direct evidence that a third of embryos reported as mosaic (both low- and high-level mosaic) by NGS-based PGT-A contain meiotic errors, highlighting the potential misclassification of aneuploid embryos as mosaic by current NGS-based PGT-A methods that cannot accurately distinguishing between meiotic and mitotic errors. Single-nucleotide polymorphism genotyping provides essential information for accurately determining the origin of chromosomal abnormalities and should be integrated with NGS-based copy number analysis to enhance diagnostic accuracy. Further studies are needed to refine mosaicism classification and to better understand its true implications in in vitro fertilization treatment.

Keywords: karyomapping; mosaicism; next-generation sequencing (NGS); pre-implantation genetic testing for aneuploidy (PGT-A); single-nucleotide polymorphism (SNP).