Reliable detection of segmental aneuploidy identified by next generation sequencing (NGS)

O-270 Wednesday, October 19, 2016 12:30 PM RELIABLE DETECTION OF SEGMENTAL ANEUPLOIDY IDENTIFIED BY NEXT GENERATION SEQUENCING (NGS). C. R. Juneau,a K. Scott,b S. Neal,a S. J. Morin,a Y. Zhan,b R. S. Zimmerman,b N. Treff,a J. M. Franasiak,a R. T. Scott.a aRMANJ, BR, NJ; bFEC, BR, NJ. OBJECTIVE: Improved resolution in NGS provides an opportunity to go beyond screening for whole chromosome aneuploidy and also detect segmental aneuploidy. De novo segmental aneuploidy, or dup/dels, represents a new class of findings in PGS. With any novel finding, the first step is to validate the analytical result. Initial reports found the frequency of dup/dels ranges from as low as 3.9% to as high as 70%. Such variability makes the possibility of inaccurate results a major concern. The next step is to characterize the nature of the biologic error identified so clinical implications might be understood. This study seeks to confirm the reliability of the analytical result and to determine the nature of the biologic error involved in embryonic dup/dels. DESIGN: Prospective blinded observational. MATERIALS AND METHODS: All IVF patients who utilized NGS as part of a RCT were reviewed. All blastocysts demonstrating a segmental aneuploidy R5 MB and a concurrent whole chromosome aneuploidy in a separate chromosome were included as these embryos were not suitable for transfer. Each embryo was biopsied at 4 additional sites 3 from the trophectoderm (TE) and 1 from the inner cell mass. Results were classified into 1 of 4 categories: 1) subsequent biopsies all contained the same error as the original confirming the result and indicating an error in meiosis, 2) subsequent biopsies demonstrated reciprocal errors indicating a mitotic error, 3) R1 but not all subsequent biopsies had an error identical to the initial result indicating a mitotic error, or 4) the subsequent biopsies did not demonstrate the original dup/del. RESULTS: 32 embryos from the original RCT met criteria for inclusion. A true dup/del error was confirmed in 28 (88%) of those embryos. 9 of the segmental abnormalities were meiotic and found in all subsequent biopsies, and 19 embryos demonstrated mitotic error. In 4 embryos, the dup/del found in the original biopsy could not be confirmed in subsequent biopsies. The source of this discrepancy cannot be resolved with these data, but likely represents a mix of analytical error

FERTILITY & STERILITYÒ

NGS is able to reliably detect segmental aneuploidy.

Possible Result 1: uniform segmental aneuploidy 2: reciprocal segmental aneuploidy 3: segmental aneuploidy confirmed in R 1 rebiopsy 4: segmental aneuploidy observed in original sample only

Biologic Explanation

N; % of Blastocysts (N¼32)

Meiotic error

9; 28%

Mitotic error

9; 28%

Mitotic error

10; 32%

Mosaicism versus analytical error

4; 12%

and low level mosaics where subsequent biopsies did not contain cells impacted by the dup/del. CONCLUSIONS: A true biologic error was confirmed in 88% of blastocysts which had dup/dels on the embryos’ initial TE biopsy. Given the high prevalence of mitotic errors and limited sampling, this represents a very high level of diagnostic accuracy. Patients should be counseled that detection of segmental aneuploidy in a TE biopsy evaluated by NGS likely represents true biologic error when making decisions about the suitability of embryos for transfer. References: 1. Rodrigo L, Mateu E, Mercader A, Cobo AC, Peinado V, Milan M, et al. New tools for embryo selection: comprehensive chromosome screening by array comparative genomic hybridization. Biomed Res Int 2014;2014:517125. 2. Vanneste E, Voet T, Le Caignec C, Ampe M, Konings P, Melotte C, et al. Chromosome instability is common in human cleavage-stage embryos. Nat Med 2009;15:577-83. Supported by: Foundation for Embryonic Competence

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