11. HIGH CONSISTENCY IN EMBRYO ANEUPLOIDY TESTING OF UNIFORM, MOSAIC AND SEGMENTAL ANEUPLOIDIES WITH THE APPLICATION OF A VALIDATED ALGORITHM

Abstract

Introduction Detection of mosaic embryos and degree of mosaicism is a hot topic in preimplantation genetic testing of aneuploidy (PGT-A). Several groups have reported healthy life births from mosaic embryos. A key point is the degree of mosaicism that can be detected in a trophectoderm biopsy and the impact of next generation sequencing (NGS) protocol and interpretation of sequencing data. The aim of this study was to determine the robustness of the diagnosis using a validated algorithm to establish the thresholds for mosaicism degree and the minimal resolution for de novo segmental aneuploidies. Material & methods NGS protocol combined automatic library preparation (IonChefTM system, ThermoFisher Scientific), and multiplexing for up to 96 samples (S5-XL sequencer; ThermoFisher). Pools of 4-6 cells with uniform and segmental aneuploidies (10-23 Mb) from cell lines with known karyotypes were employed for platform validation. For mosaicism validation, different proportions of gDNA (0-100%) from cell lines with whole chromosome aneuploidies were sequenced. Quality parameters were evaluated to determine the conditions for accurate detection. Biopsies were diagnosed as mosaic, if only one or two chromosomes fitted the mosaicism thresholds, without uniform aneuploidy for any other chromosome. After validation, results of 34,147 biopsies from 9 diagnostic laboratories using the same diagnostic algorithm were analysed (October 2018-December 2018). Results The minimal resolution for de novo segmental aneuploidies was established at 10 Mb, and the cut-off values for mosaicism detection at 30% and 70%. Biopsies were classified as: euploid ( Finally, we wanted to assess the incidence of mosaicism for chromosomes considered suitable for transfer after genetic and psychological counselling according to Gratti et al (2018). The percentage of low degree mosaicism for low risk chromosomes (1, 3, 4, 5, 10, 12, 19) was 1.1% and for chromosomes with moderate risk (2, 6, 7, 9, 11, 15, 17, 20, 22) was 1.5%. Conclusions High informativity rates were achieved using a high-throughout platform. The application of an internally validated algorithm for PGT-A showed high consistency among different laboratories, avoiding subjectivity and interindividual differences in the diagnosis, and minimizing the risks for overdiagnosis of mosaicism. Low mosaicism rates were identified and even lower if we consider only low mosaics for chromosomes suitable for transfer in the absence of euploid embryos and after proper genetic and psychological counselling. Reference Grati FR, Gallazzi G, Branca L, Maggi F, Simoni G, Yaron Y. An evidence-based scoring system for prioritizing mosaic aneuploid embryos following preimplantation genetic screening. Reprod Biomed Online. 2018 Apr;36(4):442-449.