FISH Reanalysis on Embryos Diagnosed as Abnormal on Day 3: Does Day 3 Single Cell Biopsy Accurately Represent the Numerical Chromosomal Status of the Embryo?

Authors: Catherine Marin DeUgarte MD2 Man Li MD PhD*1, Alan DeCherney MD2, Hal Danzer MD1, Mark Surrey MD1 David L. Hill PhD1. 1ART Reproductive Center, 450 N. Roxbury Dr. Beverly Hills, Ca 90210 USA 2Division of OBGYN, UCLA, CA, USA

Objective: The purpose of this study is to determine the chromosomal make-up of those embryos diagnosed as abnormal by PGD (Preimplantation Genetic Diagnosis).

Design: Prospective

Materials and Methods: All embryos determined to be abnormal by PGD, that would have otherwise been discarded, were included in this study. These abnormal embryos were fixed one or two days after PGD and FISH analysis was performed on all nuclei that were fixed. Fifty-eight embryos were included in the study from 27 IVF/ICSI patients that had PGD. Single blastomeres were biopsied on day 3, 4-12 cell stage embryos.

Chromosomes 13, 18, 21, X and Y were analyzed by Fluorescence in Situ Hybridization (FISH). Euploid embryos (on average 2.5/patient) were transferred back on day 4-6 post-retrieval or cryopreserved. The abnormal embryos, that would have otherwise been discarded, were used for this study after patient consent was obtained.

Results: Out of the 58 abnormal embryos, on FISH confirmation, the day 3 diagnosis was confirmed in 28, and 12 embryos were deemed as normal on reconfirmation (graph 1). The remaining 18 embryos had a different abnormality or a combination of abnormalities (eg a monosomic embryo on day 3 may have trisomic result on day 4).

Of the embryos that had false positive results (diagnosed as abnormal by PGD when they were in fact normal), the most common abnormalities encountered were monosomies and trisomies and they were also the most frequently diagnoses that were confirmed Graph 2 and table 1). 6 chromosomally normal embryos were also analyzed for this study. These embryos would have otherwise been discarded and all 6 were confirmed as normal when FISH analysis was done day 4 or 5.

Conclusion: This study shows that in embryos diagnosed as abnormal, the exact diagnosis was confirmed in 48 %, whereas 21 % were falsely diagnosed as abnormal when they were in fact normal. The remaining 31 % of the embryos were diagnosed as abnormal on reanalysis. However, in these embryos, other abnormalities than the one originally assigned were discovered.

Technical considerations such as overlapped or split FISH probe signals, hybridization failure, loss of nuclear materials during processing may account for some of the results. The majority of discordant results may also be due to mosaicism in Day 3 embryos. From this preliminary data we can see that PGD technologies may at times diagnose normal embryos as abnormal.

Therefore we propose that embryos that are diagnosed as abnormal be evaluated day 4 and if appropriate, repeat biopsy should be considered as well as freezing those embryos that are deemed to be normal on rebiopsy.

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