Authors: Anderson RE, JB Whitney, CM Rios, A Jones, Harutunian A, MC Schiewe

Study question: Are human blastocysts homogeneous for chromosomal copy number between multiple samples of the trophectoderm and does trophectoderm ploidy accurately predict ICM ploidy?

Summary answer: Upon re-biopsy, trophectoderm homogeneity for full gain or loss was 97%, while ICM concordance was 100%. Human blastocyst ploidy proved consistent between cell samples.

What is known already: Blastocyst biopsy and preimplantation genetic screening (PGS) allows for ploidy status determination before implantation without adversely impacting embryo competence or baby health and well-being. The efficacy and invasive nature of the trophectoderm biopsy, as well as resultant NGS reports of mosaicism, have been scrutinized. The importance of mosaic profiles on PGS outcomes is not well defined and the effect to the offspring not fully understood. Importantly, the uniformity of aneuploidy within the trophectoderm and ICM remains unclear. It has been shown that increased mosaicism decreases implantation, while low-level mosaics can result in viable pregnancies, questioning the ICM concordance with trophectoderm.

Study design, size, duration: Nine research consented embryos were thawed and a minimum of 3 additional trophectoderm samples were taken. Samples were tubed according to standard NGS amplification protocols and sent blinded to two separate reference labs, including the original testing lab. Furthermore, both labs were unaware of the experimental plan or cell types being tested (i.e, double-blinded design). The geneticists performed NGS and profile examination to produce standard euploid/aneuploid results in accordance with their own established lab guidelines.

Participants/materials, setting, methods: Research consented embryos possessing single/dual chromosomal aneuploidies were selected and blindly sampled. MicroSecure vitrified blastocysts were warmed (100% intact) and cultured in LifeGlobal medium (37°C, tri-gas humidified conditions) for a minimum of 4 hours. Each blastocyst was biopsied 3-5 times according to standard protocols. A final biopsy per embryo attempted to isolate only ICM cells as a separate sample. A total of 28 re-biopsies were performed for a total of 37 samples.

Main results and the role of chance: A full gain or loss was determined if the NGS profile reached >95% gain/loss for each chromosome. This cut-off is contrary to standard calls of ~30%. Of the original samples,7 of the 9 had a single full gain or loss, 1 embryo had a full loss and a mosaic loss of >50%, and another sample had only a mosaic gain of 65%. A total of 36 samples, or 97% were concordant for the same chromosomes called. A total of 7 ICM samples achieved 100% concordance with the original trophectoderm sample result. The one sample not producing concordance was the original 65% mosaic embryo with all re-biopsies resulting in euploidy. Unfortunately, this embryo did not have an ICM tubed to determine relevant mosaicism. Additional low-level mosaics occurred on 3 samples (8%). It is incredibly encouraging that so many of the profiles exhibited mirror images proving the efficacy of NGS repeatability. Our laboratory has previously reported the importance of biopsy technique, so every attempt was made to sample healthy cellular masses free of degeneration. Of note, all mosaic profiles had a documented fair to poor cellular mass result from the biopsy, reinforcing the need for proper technique.

Limitations, reasons for caution: Trophectoderm biopsy with NGS is heavily reliant on proper technique and geneticist interpretations. Genetic calling policies and definitions of euploidy/aneuploidy and mosaic embryos can differ from lab to lab, adding to the growing uncertainty of PGS. Understanding result interpretation is imperative to improving patient education on the risks of mosaicism.

Wider implications of the findings: This study supports that full gain/losses are extremely reproducible among all cells/types of the human blastocyst. Mosaic profiles are potentially detrimental or possibly just artifact. Further research is needed help alleviate the ambiguity of mosaic profile risks, but this study has proven NGS efficacy when full aneuploidy is present.