In the field of in vitro fertilisation (IVF), there is a need to understand how cell properties of the cumulus oocyte complex may be used to predict successful pregnancy and live birth rates post-IVF. Here we used optical tweezers for the first time to measure the viscosity of the cumulus cell matrix surrounding the oocyte (egg). This study aimed to determine whether the viscosity of the cumulus cell matrix – prior to fertilisation – is reflective of subsequent embryo developmental potential and indicative of pregnancy success.
Measurements were performed using a 1µm diameter silica probe particle trapped by a focused 1064nm laser. We benchmarked the accuracy of the system by measuring the viscosity of glycerol with varying mass fractions. Viscosity measurements of the cumulus cell matrix were performed in isolation from both the cumulus cells and the oocyte. This showed that the viscosity of cumulus matrix was significantly higher when sampled from oocytes with a higher developmental potential (in vivo matured) compared to those of lower quality (in vitro matured).
Embryo quality is a crucial factor affecting live birth outcomes. However, an accurate diagnostic for embryo quality remains elusive in the in vitro fertilization clinic. Determining physical parameters of the embryo may offer key information for this purpose. Here, we demonstrate that digital holographic microscopy (DHM) can rapidly and non-invasively assess the refractive index of mouse embryos. Murine embryos were cultured in either low- or high-lipid containing media and digital holograms recorded at various stages of development. We showed that DHM can detect spatio-temporal changes in refractive index during embryo development that are reflective of its lipid content. As accumulation of intracellular lipid is known to compromise embryo health, DHM may prove beneficial in developing an accurate, non-invasive, multimodal diagnostic.
Our approach to multispectral fluorescence microscopy can non-invasively identify the biomolecular composition of cells and capture complex biological heterogeneity which is fundamentally important for biological research and medical diagnostics. We have applied this technology to demonstrate the embryo quality for chromosomal abnormalities (containing euploid and aneuploidy cells) and understanding the biochemical signatures of polycystic ovarian syndrome (PCOS) oocytes. We then explored oocyte quality following treatment with the NAD+ precursor NMN. These findings demonstrate the utility of our approach to the multispectral assessment of autofluorescence for the non-destructive, label-free assessment of clinically relevant problems.
Automated, unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biomedical research. Label-free imaging provides information about endogenous autofluorescent metabolites, enzymes and cofactors. Our multispectral fluorescence imaging technique allows precise quantification of native fluorophores in cells and tissues. This study uses label-free multispectral analysis to extract different fluorophores and redox ratio from single cells (oocytes, cultured cancer cells) as well as blastocyst embryos. Additionally, we characterise the molecular composition, structure and functional status of ex vivo healthy bovine and osteoarthritic human knee articular cartilage to assess 2 types of experimental treatments.
Despite its wide-spread use, the success rate of assisted reproductive technologies including in vitro fertilization is less than 20%. Most human embryos are mosaic for chromosome abnormalities: containing cells that are euploid (normal) and aneuploid (incorrect number of chromosomes). Currently, a cell biopsy is used in IVF clinics to diagnose aneuploidy in the embryo but this does not provide a diagnosis of how many cells are aneuploid in the entire embryo. Hence, the development of a non-invasive tool to determine the proportion of aneuploid cells would likely improve IVF success. Aneuploidy in human embryos leads to altered metabolism. The co-factors utilized in cellular metabolism are autofluorescent and can be used to predict the metabolic state of cells. Here we used hyperspectral imaging to noninvasively assess intracellular fluorophores and thus metabolism. In this study, we utilized a powerful model of embryo aneuploidy where we generate mouse embryos with differing ratios of euploid:aneuploid cells. We also used primary human fibroblast cells with known aneuploidies to make comparison with euploid cells. Hyperspectral imaging of 1:3 chimeric embryos showed a distinct spectral profile compare to the control/normal embryos. The abundance of FAD in the inner cell mass (cells that form the fetus) of euploid and aneuploid blastocysts was strikingly different. For human cell lines, we were able to clearly distinguish between euploid and aneuploid with different karyotypes. These data show hyperspectral imaging is able to distinguish cells based on their ploidy status making it a promising tool in assessing embryo mosaicism.
Despite its wide-spread use, the success rate of assisted reproductive technologies including IVF is less than 20% in Australia/New Zealand. Most early human embryos are mosaic for chromosome abnormalities, containing a proportion of normal and abnormal cells. The most common chromosomal abnormality is aneuploidy: incorrect number of chromosomes. This form of mosaicism is thought account for early pregnancy loss in IVF. Current single cell biopsies of embryos are not diagnostic for the proportion of cells that are aneuploid (degree of heterogeneity/mosaicism). Thus, development of a non-invasive tool to determine the proportion of aneuploid cells facilitating segregation of embryos with a low percentage of aneuploid cells would likely improve IVF success rates. In other cells, including cancer cells, aneuploidy results in altered cellular metabolism. In this study we utilised hyperspectral imaging as a means of non-invasively measuring cellular metabolism in the early embryo. We utilised a mouse model where we manipulated the ratio of aneuploid:normal cells. Aneuploid embryos were generated by treatment during division from 4 to 8 cells using a reversible spindle assembly check point inhibitor, reversine. Eight-cell aneuploid embryos were dissociated and joined with control/normal cells to generate 1:1 aneuploid:normal chimeras. Hyperspectral imaging of 1:1 chimeric embryos had a distinct spectral profile that varied dramatically from the control/normal embryos. Interestingly, entirely aneuploid embryos showed a spectral profile dissimilar from both normal and chimeric embryos. These data show hyperspectral imaging is capable of distinguishing between embryos with varying degrees of aneuploidy making it a promising tool in assessing embryo health.
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