Study question What is the effect of exposing the embryo to discrete wavelengths of light on preimplantation development and resultant offspring health? Summary answer Exposure of embryos to red or yellow wavelengths negatively impacted embryo health, pregnancy rate and resulted in offspring that were heavier at weaning. What is known already Previous studies have indicated a potential negative impact of shorter wavelengths of light on embryo health. Red and yellow wavelengths are widely considered benign and utilised clinically in time-lapse equipped incubators within IVF clinics. However previous studies had not uniformly and correctly irradiated embryos to enable a fair comparison between different wavelengths. Study design, size, duration A current aim of the field is to use optical imaging to predict embryo developmental potential. Such approaches use varying wavelengths of light. The impact of irradiating the embryo with discrete wavelengths of light is not fully understood. Here, we assess the impact of various wavelengths on the developing embryo and for the first time, ensured that the energy dose applied was consistent between wavelengths, thus mimicking fluorescence and time-lapse imaging (470 – 620 nm). Participants/materials, setting, methods Preimplantation mouse embryos were exposed daily to blue (470 nm), green (520 nm), yellow (590 nm) or red (620 nm) wavelengths and compared to embryos that were not exposed. We assessed embryo development, DNA damage, and postnatal outcomes following transfer to pseudopregnant recipients. Main results and the role of chance We found exposure to the yellow wavelength significantly impaired embryo development to the blastocyst stage (P < 0.05). While exposure to blue, green and red wavelengths resulted in significantly higher levels of DNA damage when compared to unexposed embryos (P < 0.05). The pregnancy rate was significantly lower when embryos were exposed to the red wavelength (P < 0.05). Interestingly, resultant offspring were significantly heavier when derived from red or yellow light exposed embryos compared to those derived from unexposed embryos (P < 0.01). Towards understanding the effect on offspring weight we assessed intracellular lipid abundance in the embryo. We found lipid abundance to be significantly elevated following exposure to yellow wavelength (1.8-fold, P < 0.0001) but not red. We believe that the role of chance is low as results were collected from multiple independent experimental replicates that were tested using appropriate statistical analyses. Limitations, reasons for caution While we demonstrate the distinct impacts of discrete wavelengths of light on the developing mouse embryos including post-natal effects, confirmation of these results in human embryos is required. Wider implications of the findings Red and yellow wavelengths are utilised clinically in time-lapse equipped incubators within IVF clinics. Our results demonstrate the potential need to re-evaluate these assumptions. Mapping the stress tolerance embryos show for each wavelength may be advantageous in identifying how damage can be mitigated in clinical manipulation and imaging techniques. Trial registration number not applicable
Study question Can we separate between control and reversine-treated cells within the inner cell mass (ICM) of the mouse preimplantation embryo by using label-free and non-invasive hyperspectral microscopy? Summary answer Hyperspectral microscopy is able to discern between control and reversine-treated cells using cellular autofluorescence in the complete absence of fluorescence tags. What is known already Embryo mosaicism (containing cells that are euploid (46 chromosomes) and aneuploid (deviation from the expected number of chromosomes)) affects up to 17.3% of human blastocyst embryos. Current diagnosis of aneuploidy in the IVF clinic involves a biopsy of trophectoderm (TE) cells or spent media followed by sequencing. In some blastocyst embryos these approaches will fail to diagnose of the proportion of aneuploid cells within the fetal lineage (ICM). Study design, size, duration The impact of aneuploidy on cellular metabolism was assessed by using cellular autofluoresence and hyperspectral microscopy (broad spectral profile). Two models were employed: (i) Primary human fibroblast cells with known karyotypes (4-6 independent replicates, euploid n = 467; aneuploid n = 969) and reversine induced aneuploidy in mouse embryos (5-8 independent replicates, 30-44 cells per group). Both models were subjected to hyperspectral imaging to quantify native cell fluorescence. Participants/materials, setting, methods The human model is comprised of euploid (male and female) and aneuploid (triploid and trisomies: 13, 18, 21, XXX, and XXY) primary human fibroblast cells. For the mouse model, we treated embryos 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. Blastomeres were either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Main results and the role of chance Following unsupervised linear unmixing, the relative abundance of metabolic cofactors was quantified: reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavins with the subsequent calculation of the optical redox ratio (ORR: Flavins/[NAD(P)H + Flavins]). Primary human fibroblast cells displayed an increase in the relative abundance of NAD(P)H with a decrease in flavins, leading to a significant reduction in the ORR for aneuploid cells (P < 0.05). The mouse embryos displayed an identical trend as the human model between control and reversine-treated embryos. Mathematical algorithms were applied and able to distinguish between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres and (iii) chimeric blastocysts with differing ratios of control and reversine-treated cells. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve. We also showed that hyperspectral imaging of the preimplantation embryo does not impact on embryo developmental competence, pregnancy outcome and offspring health in a mouse model. We believe the role of chance is low as both human somatic cells and mouse embryos showed a consistent shift in cellular metabolism in response to human fibroblast cells that are aneuploid and reversine treated mouse embryos. Limitations, reasons for caution Further validation of our approach could include sequencing of the ICM of individual blastocysts to determine the proportion of aneuploid cells in ICM and correlate this with the metabolic profile obtained through hyperspectral imaging. Wider implications of the findings With hyperspectral imaging able to discriminate between (i) euploid and aneuploid human fibroblast cells and (ii) control and reversine-treated mouse embryos, this could be an accurate, non-invasive and label-free optical imaging approach to assess mosaicism within the ICM of mouse embryos, potentially leading to a new diagnostic tool for embryos. Trial registration number Not applicable
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