Evaluating the Status of Complex Aneuploidy to Locate Additional Transferrable Embryos

Recently, the term "chaotic" has come into the PGT-A reporting lexicon, defined (perhaps arbitrarily) as 6 or more aneuploidies detected. Complex aneuploidy refers to embryos with multiple chromosomal aneuploidies. In one large study, complex aneuploidy at the cleavage stage was found in 41% of embryos overall, 29% of embryos in women 27-37 years old, and 45% of embryos in women 38-45 years old. By the blastocyst stage, only 10% of all embryos were complex aneuploid, with 13.7% of embryos in women aged 38-47 years compared to 4.5% in women aged 27-37 years demonstrating complex aneuploidy. Multiple aneuploidies were also detected in a retrospective study of 1502 spontaneous abortions, with 4.6% of cases showing double aneuploidy and only 0.4% showing 3 or more aneuploidies (2).

The high rate of complex aneuploidy in cleavage and blastocyst stage embryos even among young patients suggests multiple mitotic errors in the first few cell divisions, which would likely lead to developmental arrest (2). Therefore, if transferred, such embryos would be expected not to implant or to miscarry. Historically, embryos with multiple chromosomal abnormalities have therefore been de-selected for transfer or even discarded. However, recently Lin et al. reported a healthy live birth following chaotic embryo transfer (3), calling into question the assumed non-viability of chaotic embryos. Might some of these embryos correct multiple aneuploidies and become euploid? It is plausible that any euploid population of cells could have a higher survival rate and a higher mitotic rate, and if abnormal cells undergo apoptosis, a euploid embryo could result. This could explain the low rate of complex aneuploidy seen in miscarriages. An additional explanation is that imperfections in the biopsy, amplification, and/or NGS PGT-A process occasionally lead to noisy reads, that are interpreted as multiple chromosomal abnormalities, but actually reflect artifact. For such embryos, re- testing may result in euploid results.

Small studies (published in abstract form only) have evaluated the reproducibility of chaotic results from NGS-based PGT-A. A retrospective cohort study evaluated 58 'chaotic' embryos via PGT-A of re-biopsy samples. Thirty-eight percent (n=22) of embryos had euploid re-biopsy results (4). Our group reported re-biopsy of 64 embryos initially classified as chaotic and identified 17% (n=11) of embryos in the cohort as euploid and only 67% concordance between initial and re-biopsy sample results (5). Four blastocysts with euploid results on re- biopsy were transferred, resulting in two ongoing pregnancies. Furthermore, we have pooled data from multiple IVF clinics within our network that have conducted re-biopsy of embryos with multiple aneuploidies and found that overall, 22% had euploid PGT-A results on re-biopsy (Background Table, unpublished data).

In anticipation of conducting a prospective observational study to characterize the nature of PGT-A results indicating 3 or more aneuploidies, we have begun to catalog and hold these embryos in storage. To date, 112 patients with have explicitly expressed interest in contributing 128 embryos resulted as 'chaotic' or 'complex aneuploid' of 3-5 chromosomes, for the purposes of this research, and we anticipate identifying multiples of this number upon a

retrospective database search and with prospective accumulation of additional PGT-A results. Following a retrospective database search, we have identified a total of 274 'chaotic' and 5251 'complex aneuploid' embryos having been reported using NGS based PGT-A. Limited data exist on the reproductive potential of embryos classified as having multiple chromosomal abnormalities, and preliminary data, including from our center, suggest a high false positive rate. The large volume of ART performed within our network represents a unique opportunity to characterize the clinical significance of these results, in the hopes of minimizing embryo wastage and maximizing live birth from IVF cycles using PGT-A.

The aim of this study is to evaluate the reproductive potential of embryos classified as "complex aneuploid" of three or more chromosomes or "chaotic" on initial trophectoderm biopsy sample analyzed by next generation sequencing based preimplantation genetic testing for aneuploidy. We plan to do this by prospectively enrolling patients with embryos classified as such, re-biopsy of the embryos, and analysis of repeat biopsy samples. We also plan to have an independent, blinded geneticist make diagnoses, as possible, based on the NGS profiles.