Virtual Presentation SRB Virtual Awards 2020

Autofluorescence of endogenous fluorophores: can it detect aneuploidies? (#18)

Cheow Yuen (Tiffany) Tan 1 2 , Saabah Mahbub 3 4 , Carl Campugan 1 2 , Jared Campbell 3 4 , Abbas Habibalahi 3 4 , Sanam Mustafa 1 2 , Jeremy Thompson 1 2 , Ewa Goldys 3 4 , Kylie Dunning 1 2
  1. The University of Adelaide, Adelaide, SA, Australia
  2. Adelaide Medical School, Robinson Research Institute, ARC Centre of Excellence for Nanoscale of BioPhotonics & Institute of Photonics and Advanced Sensing, University of Adelaide, Adelaide, SA, Australia
  3. The Graduate School of Biomedical Engineering, University of New South Wales, Sydney, New South Wales, Australia
  4. ARC Centre of Excellence for Nanoscale of BioPhotonics & Institute of Photonics and Advanced Sensing, University of Adelaide, Adelaide, SA, Australia

Most human embryos are mosaic for chromosome abnormalities: containing cells that are euploid (expected number of chromosomes) and aneuploid (extra or missing chromosomes.). Current diagnosis of aneuploidy in the IVF clinic involves a biopsy of trophectoderm cells followed by sequencing. This is invasive and does not provide an accurate diagnosis of the proportion of aneuploid cells in the fetal or placental cell lineages. Hence, the development of a non-invasive tool to determine the proportion of aneuploid cells would improve IVF success. Aneuploidy is known to lead to altered metabolism in human embryos. We hypothesised that hyperspectral microscopy can discern between euploid and aneuploid cells within the preimplantation embryo using autofluorescent metabolic cofactors reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (flavins). Optical redox ratio (ORR) derived from these molecules was used as a measure of cellular metabolism. We utilised human fibroblast cells with known aneuploidies and a model of embryo mosaicism where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the 4- to 8-cell division, to generate euploid, aneuploid and chimeric embryos at different ratios. Following unsupervised linear unmixing, NAD(P)H was significantly higher in aneuploid cells in both primary human fibroblast cells and individual cells from mouse embryos (P<0.05). Conversely, there were significantly lower levels of flavins in aneuploid compared to euploid cells (P<0.05). This resulted in a significantly reduced ORR in aneuploid cells (P<0.05), indicative of reduced cellular metabolism. Excitingly, mathematical algorithms applied to cellular autofluorescence was able to distinguish between individual euploid and aneuploid cells from mouse embryos (accuracy >98%) and for intact blastocyst embryos with differing ratios of aneuploid cells (accuracy 87%). Collectively, these results demonstrate that hyperspectral imaging can distinguish cells based on their ploidy status making it a potential tool in assessing embryo mosaicism.