Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo

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 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.

Non-invasive assessment of oocyte developmental competence

“…In the field of reproductive biology, hyperspectral microscopy demonstrated the capacity to discern between good and poor quality bovine embryos (Sutton-McDowall et al 2017;Santos Monteiro et al 2021) and oocytes denuded of cumulus cells from young and aged mice (Bertoldo et al 2020). Additionally, mathematical algorithms applied to autofluorescence signals obtained from hyperspectral imaging were able to discriminate between different cell populations (Gosnell et al 2016a;Sutton-McDowall et al 2017;Habibalahi et al 2019;Mahbub et al 2021;Tan et al 2022b). Our recent work successfully demonstrated the ability of hyperspectral imaging to separately measure metabolism in the oocyte and cumulus cell compartments of the intact COC, when compared with conventional laser scanning confocal microscopy (Fig.…”

Reproductive biology research down under: highlights from the Australian and New Zealand Annual Meeting of the Society for Reproductive Biology, 2021

“…Such changes alter the autofluorescence signal of cells and can be quantified and correlated using mathematical approaches, thus enabling discrimination between different cell populations (Gosnell et al 2016). Dr. Dunning's team has recently demonstrated that hyperspectral imaging can be used to successfully distinguish healthy euploid from aneuploid cells within the ICM of mouse preimplantation embryos (Tan et al 2021). Future studies will determine the effectiveness of hyperspectral imaging at detecting aneuploidy in human embryos, which would ultimately provide an affordable and practical clinical tool for determining embryo health non-invasively.…”

The effect of discrete wavelengths of visible light on the developing murine embryo

“…As label-free optical imaging to determine embryo developmental potential increase in popularity [ 21 , 22 ], it is imperative that the impact of different wavelengths of light on the preimplantation embryo is carefully characterized. Mapping the stress tolerance embryos show for each wavelength may be advantageous in identifying how damage can be mitigated in clinical manipulation and modern imaging techniques.…”