STUDY QUESTION Can label-free, non-invasive optical imaging by hyperspectral autofluorescence microscopy discern between euploid and aneuploid cells within the inner cell mass (ICM) of the mouse preimplantation embryo? SUMMARY ANSWER Hyperspectral autofluorescence microscopy enables discrimination between euploid and aneuploid ICM in mouse embryos. WHAT IS KNOWN ALREADY Euploid/aneuploid mosaicism affects up to 17.3% of human blastocyst embryos with trophectoderm biopsy or spent media currently utilized to diagnose aneuploidy and mosaicism in clinical in vitro fertilization. Based on their design, these approaches will fail to diagnose the presence or proportion of aneuploid cells within the foetal lineage ICM of some blastocyst embryos. STUDY DESIGN, SIZE, DURATION The impact of aneuploidy on cellular autofluorescence and metabolism of primary human fibroblast cells and mouse embryos was assessed using a fluorescence microscope adapted for imaging with multiple spectral channels (hyperspectral imaging). Primary human fibroblast cells with known ploidy were subjected to hyperspectral imaging to record native cell fluorescence (4–6 independent replicates, euploid n = 467; aneuploid n = 969). For mouse embryos, blastomeres from the eight-cell stage (five independent replicates: control n = 39; reversine n = 44) and chimeric blastocysts (eight independent replicates: control n = 34; reversine n = 34; 1:1 (control:reversine) n = 30 and 1:3 (control:reversine) n = 37) were utilized for hyperspectral imaging. The ICM from control and reversine-treated embryos were mechanically dissected and their karyotype confirmed by whole genome sequencing (n = 13 euploid and n = 9 aneuploid). PARTICIPANTS/MATERIALS, SETTING, METHODS Two models were employed: (i) primary human fibroblasts with known karyotype and (ii) a mouse model of embryo aneuploidy where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the four- to eight-cell division. Individual blastomeres were dissociated from control and reversine-treated eight-cell embryos and either 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 cellular metabolism were determined by quantification of metabolic co-factors (inferred from their autofluorescence signature): NAD(P)H and flavins with the subsequent calculation of the optical redox ratio (ORR: flavins/[NAD(P)H + flavins]). Autofluorescence signals obtained from hyperspectral imaging were examined mathematically to extract features from each cell/blastomere/ICM. This was used to discriminate between different cell populations. MAIN RESULTS AND THE ROLE OF CHANCE An increase in the relative abundance of NAD(P)H and decrease in flavins led to a significant reduction in the ORR for aneuploid cells in primary human fibroblasts and reversine-treated mouse blastomeres (P < 0.05). Mathematical analysis of endogenous cell autofluorescence achieved separation between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres cells, (iii) control and reversine-treated chimeric blastocysts, (iv) 1:1 and 1:3 chimeric blastocysts and (v) 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 0.97, 0.99, 0.87, 0.88 and 0.93, respectively. We believe that the role of chance is low as mathematical features separated euploid from aneuploid in both human fibroblasts and ICM of mouse blastocysts. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Although we were able to discriminate between euploid and aneuploid ICM in mouse blastocysts, confirmation of this approach in human embryos is required. While we show this approach is safe in mouse, further validation is required in large animal species prior to implementation in a clinical setting. WIDER IMPLICATIONS OF THE FINDINGS We have developed an original, accurate and non-invasive optical approach to assess aneuploidy within the ICM of mouse embryos in the absence of fluorescent tags. Hyperspectral autofluorescence imaging was able to discriminate between euploid and aneuploid human fibroblast and mouse blastocysts (ICM). This approach may potentially lead to a new diagnostic for embryo analysis. STUDY FUNDING/COMPETING INTEREST(S) K.R.D. is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58-2019). This study was funded by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CE140100003) and the National Health and Medical Research Council (APP2003786). The authors declare that there is no conflict of interest.

Study question: Can label-free, non-invasive optical imaging by hyperspectral microscopy discern between euploid and aneuploid cells within the inner cell mass of the mouse preimplantation embryo? Summary answer: Hyperspectral microscopy shows a variance in metabolic activity which enables discrimination between euploid and aneuploid cells. What is known already: 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 or proportion of aneuploid cells within the fetal lineage (inner cell mass (ICM)) of some blastocyst embryos. Study design, size, duration: The impact of aneuploidy on cellular metabolism of primary human fibroblast cells and mouse embryos was assessed by a fluorescence microscope adapted for imaging with multiple spectral channels (hyperspectral imaging). Primary human fibroblast cells with known ploidy were subjected to hyperspectral imaging to record native cell fluorescence (euploid n= 467; aneuploid n= 969). For mouse embryos, 50-70 individual euploid and aneuploid blastomeres (8-cell stage embryo) and chimeric blastocysts (40-50 per group: euploid; aneuploid; or 1:1 and 1:3 ratio of euploid:aneuploid) were utilised for hyperspectral imaging. Participants/materials, setting, methods: Two models were employed: (i) Primary human fibroblasts with known karyotype and (ii) a mouse model of embryo aneuploidy where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the 4- to 8-cell division. Individual blastomeres were dissociated from reversine treated (aneuploid) and control (euploid) 8-cell embryos and either imaged directly or used to generate chimeric blastocysts with differing ratios of euploid:aneuploid cells. Individual blastomeres and embryos were subjected to hyperspectral imaging. Changes in cellular metabolism were determined by quantification of metabolic cofactors (inferred from their autofluorescence signature): reduced nicotinamide adenine dinucleotide (NAD(P)H), flavins with the subsequent calculation of the optical redox ratio (ORR: Flavins/[NAD(P)H + Flavins]). Mathematical algorithms were applied to extract features from the autofluorescence signals of each cell/blastomere/inner cell mass to discriminate between euploid and aneuploid. Main results and the role of chance: An increase in the relative abundance of NAD(P)H with a decrease in flavins led to a significant reduction in the ORR for aneuploid cells in both primary human fibroblasts and individual mouse blastomeres (P < 0.05). Mathematical algorithms were able to achieve good separation between (i) euploid and aneuploid primary human fibroblast cells, (ii) euploid and aneuploid mouse blastomeres cells and (iii) euploid and aneuploid chimeric blastocysts and (iv) 1:1 and 1:3 chimeric blastocysts. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve of 0.85, 0.99, 0.87 and 0.88, respectively. We believe that the role of chance is low as multiple cellular models (human somatic cells and mouse embryos) demonstrated a consistent shift in cellular metabolism in response to aneuploidy as well as the robust capacity of mathematical features to separate euploid and aneuploid cells in a statistically significant manner. Limitations, reasons for caution: There would be added value in determining the degree of embryo mosaicism by sequencing the inner cell mass (ICM) of individual blastocysts to correlate with metabolic profile and level of discrimination achieved using the mathematical features approach. Wider implications of the findings: Hyperspectral imaging was able to discriminate between euploid and aneuploid human fibroblasts and mouse embryos. This may lead to the development of an accurate and non-invasive optical approach to assess mosaicism within the ICM of human embryos in the absence of fluorescent tags. Study funding/competing interest(s): K.R.D. is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58-2019). This study was funded by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CEI40100003). The authors declare that there is no conflict of interest. Key words: hyperspectral microscopy/ pre-implantation/ mosaicism/ aneuploidy/ autofluorescence/ NAD(P)H/ Flavins/ embryo assessment/ non-invasive/ cellular metabolism/ optical microscopy

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