Euploid/aneuploid mosaicism affects up to 17.3 % of human blastocyst embryos with trophectoderm biopsy or spent media currently utilised to diagnose aneuploidy and mosaicism in clinical in vitro fertilisation. Based on their design, these approaches will fail to diagnose the presence of aneuploid cells within the fetal lineage (inner cell mass (ICM)) of some blastocyst embryos. In this study we determined whether non-invasive hyperspectral autofluorescence microscopy can discern between euploid and aneuploid cells within the ICM of mouse preimplantation embryos.
Mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the 4- to 8-cell division. Individual blastomeres were dissociated from control and reversine-treated 8-cell embryos and imaged directly or used to generate chimeric blastocysts with differing ratios of control: reversine-treated cells. Individual blastomeres and embryos were interrogated by hyperspectral imaging. Changes in metabolism were determined by quantification of metabolic co-factors: reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavins, with subsequent calculation of optical redox ratio (ORR: Flavins/[NAD(P)H + Flavins]). Separately, autofluorescence signals obtained from hyperspectral imaging were examined mathematically to extract features. This was used to discriminate between different cell populations.
An increase in the abundance of NAD(P)H and decrease in flavins led to a significant reduction in the ORR for reversine-treated mouse blastomeres (P < 0.05). Mathematical analysis of cell autofluorescence achieved separation between (i) control and reversine-treated mouse blastomeres cells, (ii) control and reversine-treated chimeric blastocysts, (iii) 1:1 and 1:3 chimeric blastocysts and (iv) confirmed euploid and aneuploid ICM from mouse blastocysts. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve of 0.99, 0.87, 0.88 and 0.93, respectively.
Hyperspectral autofluorescence imaging was able to discriminate between euploid and aneuploid ICM in mouse blastocysts. This approach may lead to an accurate and non-invasive diagnostic for embryo analysis.