Parental genomes segregate into different blastomeres during multipolar zygotic divisions leading to mixoploid and chimeric blastocysts

The zygotic division enables two haploid genomes to segregate into two biparental diploid blastomeres. This fundamental tenet was challenged by the observation that blastomeres with different genome ploidy or parental genotypes can coexist within individual embryos. We hypothesized that whole parental genomes can segregate into distinct blastomere lineages during the first division through "heterogoneic division". Here, we map the genomic landscape of 82 blastomeres from 25 embryos that underwent multipolar zygotic division. The coexistence of androgenetic and diploid or polyploid blastomeres with or without anuclear blastomeres, and androgenetic and gynogenetic blastomeres within the same embryo proofs the existence of heterogoneic division. We deduced distinct segregation mechanisms and demonstrate these genome-wide segregation errors to persist to the blastocyst stage in both human and cattle. Genome-wide zygotic segregation errors contribute to the high incidence of embryonic arrest and provide an overarching paradigm for the development of mixoploid and chimeric individuals and moles.

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

Study questionCan 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 answerHyperspectral microscopy shows a variance in metabolic activity which enables discrimination between euploid and aneuploid cells.What is known alreadyEuploid/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, durationThe 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, methodsTwo 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 chanceAn 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 cautionThere 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 findingsHyperspectral 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.

Production of rabbit chimeric embryos by aggregation of zona-free nuclear transfer blastomeres

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16 h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p<0.05), but significantly lower than in TR<>N chimeric blastocysts (p<0.001). Similarly, a higher proportion (p<0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.