A single trophectoderm biopsy at blastocyst stage is mathematically unable to determine embryo ploidy accurately enough for clinical use

Abstract

Background: It has become increasingly apparent that the trophectoderm (TE) at blastocyst stage is much more mosaic than has been appreciated. Whether preimplantation genetic screening (PGS), utilizing a single TE biopsy (TEB), can reliably determine embryo ploidy has, therefore, increasingly been questioned in parallel.

Methods: We for that reason here established 2 mathematical models to assess probabilities of false-negative and false-positive results of an on average 6-cell biopsy from an approximately 300-cell TE. This study was a collaborative effort between investigators at The Center for Human Reproduction in New York City and the Center for Studies in Physics and Biology and the Brivanlou Laboratory of Stem Cell Biology and Molecular Embryology, the latter two both at Rockefeller University in New York City.

Results: Both models revealed that even under best case scenario, assuming even distribution of mosaicism in TE (since mosaicism is usually clonal, a highly unlikely scenario), a biopsy of at least 27 TE cells would be required to reach minimal diagnostic predictability from a single TEB.

Conclusions: As currently performed, a single TEB is, therefore, mathematically incapable of reliably determining whether an embryo can be transferred or should be discarded. Since a single TEB, as currently performed, apparently is not representative of the complete TE, this study, thus, raises additional concern about the clinical utilization of PGS.

Keywords: In vitro fertilization; Mathematical model; Premplantation genetic screening; Trophectoderm biopsy.

Figures

Fig. 1

Schematics of PGS. The currently in use PGS procedure (PGS 2.0) is based on a single TEB of on average approximately 6 cells, which is alleged to accurately reflect the chromosomal status of the developing embryo/fetus. The procedure has entered worldwide clinical use without prior clinical validation. Three crucial questions (unresolved issues in the figure) still require resolution. This manuscript attempts to answer the first of these questions, whether a single TEB accurately enough reflects the whole TE to discriminate between embryos that can undergo transfer and those that should be discarded

Fig. 2

P-values for observing no mosaicism, given different hypotheses r and a threshold of 0,05 (dotted line). With the curve crossing the significance line (P = 0.05) at 0.6, the figure demonstrates that any value of r larger than 60% euploid cells leads to a P-value that is larger than the usual significance level of 5% (indicated as a dashed line in the figure), meaning that even the hypothesis that there are 40% aneuploid cells in the embryo cannot be rejected when not observing any aneuploid cells in the biopsy

Fig. 3

P-values for observed mosaicism, given different hypotheses r, and varying numbers of abnormal-aneuploid cells in biopsy. The curves demonstrate that, even when obtaining a mosaic TEB, with decreasing aneuploidy cell numbers in the TEB (from 6 to 1), any explanation with increasing r from r < 0.4 to r < 0.99 could be a reasonable explanation of the observed data since it leads to observing mosaicism in >5% of cases. The threshold at which r crosses the significance level increases with decreasing aneuploidy. This means that a given r is more likely to explain the obtained biopsy result the lower the measured aneuploidy is. For example, if one (or more) cells are aneuploid, the threshold will be approximately r = 0.99, meaning that even a hypothesis of r = 0.99, basically an entirely euploid embryo, is compatible with the data. On the other hand, if three (or more) cells are aneuploid, a hypothesis with an r as high 0.99 is too unlikely to give the observed data, and only an r < 0.85 hypothesis is compatible with this biopsy outcome