Webinar: Advances in Simultaneous Analysis to Determine Embryo PGT-A and PGT-M Status with NGS

Webinar summary

This presentation examines recent developments in preimplantation genetic testing (PGT), with emphasis on next-generation sequencing (NGS)-based methodologies for preimplantation genetic testing for aneuploidy (PGT-A) and monogenic disorders (PGT-M). The presentation reviews research findings related to analytical performance, methodological considerations, and emerging challenges related to embryo sample mosaicism characterization using the Ion ReproSeq PGS kits on the Ion GeneStudio System next-generation sequencing platform.

Study overview

The presentation contextualizes the transition from legacy cytogenetic and array-based methodologies to NGS-based platforms for embryonic genomic analysis. NGS-based workflows enable multiplexed sequencing through barcoding strategies and provide enhanced analytical resolution for chromosomal copy number assessment across embryonic samples.

Analytical validation was performed using characterized cell lines to assess detection capabilities across:

• Whole chromosome aneuploidies
• Segmental chromosomal alterations
• Mosaic genomic profiles generated through controlled admixture experiments

In parallel, the presentation reviews linkage-based and SNP-based approaches for PGT-M, comparing traditional STR methodologies with NGS-based SNP analysis incorporating expanded marker density across evaluated sample sets.

Key findings

Analytical performance of NGS in PGT-A

• NGS-based workflows enable high-throughput parallel sequencing across multiplexed sample cohorts via barcoding strategies
• Increased dynamic range facilitates detection of both whole chromosome copy number changes and sub-chromosomal segmental events within embryonic samples
• Analytical verification studies demonstrated accurate identification of euploid, aneuploid, and segmental aberrations using reference samples and control materials

Mosaicism detection

• NGS-based analysis enables identification of intermediate chromosomal states consistent with mosaicism in embryonic samples
• Reported mosaicism frequencies vary substantially (approximately 5–20%) depending on analytical thresholds and laboratory-specific parameters

Biological and technical considerations in mosaicism

• Mosaicism reflects the presence of multiple chromosomal populations within a single embryo, arising from mitotic errors during embryonic development
• Concordance between repeated biopsies of mosaic embryo samples is low (~35%), supporting evidence of spatial heterogeneity rather than analytical variability alone
• Concordance between trophectoderm and inner cell mass sample analyses remains high (~96%), though discordance persists in a subset of evaluated samples

Workflow and platform characteristics

• The ReproSeq workflow requires approximately 16 hours of total processing time with ~3 hours hands-on input for sample preparation and processing
• Automated liquid handling systems reduce operator-dependent variability and improve reproducibility across processed sample batches
• Integrated bioinformatics pipelines support automated data processing and result reporting

NGS-based approaches for PGT-M

• Traditional STR-based linkage analysis is limited by marker density and amplification variability across analyzed samples
• SNP-based NGS approaches enable analysis of a larger number of markers (~200), increasing analytical robustness and genomic coverage
• Concurrent analysis of PGT-A and PGT-M from the same amplified sample is feasible without additional biopsy samples

NGS-based methodologies provide a high-resolution, scalable framework for preimplantation genetic analysis, enabling detection of chromosomal abnormalities, segmental alterations, and mosaic genomic states. Analytical validation demonstrates robust performance across multiple variant classes, while expanded SNP-based approaches enhance the resolution of monogenic disorder testing.

Despite these advances, variability in mosaicism and interpretation underscores the need for standardization of analytical thresholds, expanded multi-site feasibility studies, and continued investigation into the significance of mosaic findings.

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PMR-004833