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|Title:||Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo|
|Citation:||Human Reproduction, 2022; 37(1):14-29|
|Publisher:||Oxford University Press (OUP)|
|Tiffany C.Y. Tan, Saabah B. Mahbub, Jared M. Campbell, Abbas Habibalahi, Carl A. Campugan, Ryan D. Rose, Darren J.X. Chow, Sanam Mustafa, Ewa M. Goldys, and Kylie R. Dunning|
|Abstract:||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 n¼467; aneuploid n¼969). For mouse embryos, blastomeres from the eight-cell stage (five independent replicates: control n¼39; reversine n¼44) and chimeric blastocysts (eight independent replicates: control n¼34; reversine n¼34; 1:1 (control:reversine) n¼30 and 1:3 (control:reversine) n¼37) were utilized for hyperspectral imaging. The ICM from control and reversine-treated embryos were mechanically dissected and their karyotype confirmed by whole genome sequencing (n¼13 euploid and n¼9 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 þ 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.|
|Keywords:||hyperspectral microscopy; preimplantation; mosaicism; aneuploidy; autofluorescence; NAD(P)H; flavins; embryo assessment; non-invasive; cellular metabolism|
|Description:||Advance Access Publication on November 6, 2021|
|Rights:||© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved.|
|Appears in Collections:||Paediatrics publications|
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