ANALYTICAL APPROACHES AND EFFECT ON PLOIDY IN PGT-A

Abstract

Preimplantation genetic testing for aneuploidy (PGT-A) is routinely offered to patients undergoing in vitro fertilization (IVF). Commonly performed via next generation sequencing (NGS) on trophectoderm biopsy samples, it provides a higher resolution than previous technologies and the ability to detect mosaicism. Embryos with corresponding samples that tested negative for aneuploidy can be selected for transfer, as they have the greatest probability of leading to a successful pregnancy. However, as laboratories use different platforms for PGT-A and often have differing approaches, a single embryo biopsy sample may yield seemingly different results, which (combined with clinic policies) may determine whether or not embryo transfers will occur and affect transfer outcomes. Results can be affected by the laboratory’s chosen mosaicism threshold or sensitivity settings, among other factors. Laboratories should be transparent about this information and work with clinics to develop policies that are in the best interest of the patients. The goal of this study was to compare our laboratory’s reported PGT-A euploid, mosaic, and aneuploid rates when samples’ NGS data is re-analyzed with different settings, including mosaicism threshold and sensitivity setting changes. Samples received from mid-October 2019 through mid-November 2019 were included in our analysis. PGT-SR (structural rearrangements) samples or samples from cases with suspected rearrangements were excluded. Whole-genome amplification and NGS analysis was performed using the Ion ReproSeqTM PGS Kit and data analysis was performed utilizing the Ion ReporterTM software (Thermo Fisher Scientific). Data analysis was conducted under 3 different approaches: A) our standard settings (which include a mosaicism threshold of 30-70% and a copy number variant [CNV] penalty setting of -3), B) a smaller mosaicism threshold of 40-60% with our standard CNV penalty of -3, and c) our standard mosaicism threshold (30-70%) with a lower CNV penalty of -5. Results were reported as 1) no abnormal cells detected, 2) abnormal cells detected (with mosaicism percentage, if identified), or 3) did not pass quality control (QC) metrics, leading to the following embryo categories: euploid, full mosaic, partial mosaic, partial aneuploid, full aneuploid, and QC failure. Results were tallied for each of the analytical approaches and chi square analyses were performed to compare the data. A total of 1,512 samples from 286 patient cycles were included in the analysis. Table 1 displays the ploidy rates for each of the analytical approaches as well as the corresponding chi square p-values. Changes to mosaicism thresholds and sensitivity settings both affected ploidy rates, though only changes to the former led to statistically significant differences in the euploid and mosaic rates. Given the potential impact of PGT-A results on embryo transfer decisions, it is important that laboratories are transparent about their mosaicism thresholds and other key analytical factors that affect results. This will allow for proper pre- and post-test counseling for patients opting for PGT-A and maximize the benefit of the testing as technology continues to change.