Optical imaging detects metabolic signatures associated with oocyte and embryo quality

Abstract

Non-invasive assessment of both oocyte and embryo quality is a major focus of research to improve pregnancy and live birth rates following in vitro fertilisation (IVF). The developmental potential of the oocyte and embryo are intimately linked to metabolism. Thus, diagnostic approaches that measure oocyte and embryo metabolism may improve IVF outcomes. Metabolic heterogeneity is known to exist between cells of the cumulus oocyte complex (COC) and the embryo. However, current approaches may fail to accurately predict oocyte and embryo quality as they measure metabolism of the entire COC or embryo, and do not provide spatial information. Label-free optical imaging of NAD(P)H and FAD, provides an overall indicator of metabolism via the optical redox ratio (ORR; FAD / [NAD(P)H + FAD]). Optical imaging of these metabolic co-factors occurs in the complete absence of exogenous tags. In this thesis, I investigated whether label-free optical imaging of cellular autofluorescence could detect metabolic changes associated with oocyte and embryo quality. I confirmed the robustness of label-free optical imaging to measure dynamic metabolic changes in the COC by comparing the ORR with oxygen consumption rate –– the benchmark methodology for the field. Additionally, my work demonstrated the ability of hyperspectral microscopy, a label-free optical imaging modality, to detect metabolic signatures in oocytes with poor developmental potential. I utilised hyperspectral microscopy due to its low power density (energy) requirements for imaging and expected absence of photodamage. This makes this form of microscopy compatible with future clinical implementation. Following demonstration that label-free optical imaging detected metabolic changes associated with oocyte quality, I next investigated whether hyperspectral microscopy could quantify metabolic variance associated with poor embryo quality, specifically, aneuploidy. Current methods for assessing embryo aneuploidy are invasive and do not provide an accurate diagnosis for the presence or absence of aneuploid cells within foetal cell lineage: inner cell mass (ICM). My findings demonstrated that hyperspectral imaging detected significant metabolic differences between euploid and aneuploid cells. Importantly, mathematical algorithms applied to images acquired by hyperspectral microscopy, were able to discriminate between euploid and aneuploid ICM. I also assessed the safety of hyperspectral microscopy by comparing imaged and non-imaged embryos and showed that imaging did not impact embryo development, pregnancy rate or the weight of pups at weaning. Overall, my results demonstrate the potential for label-free optical imaging to be a safe and non-invasive diagnostic for embryo aneuploidy. As I had shown that label-free optical imaging of cellular autofluorescence could discriminate between euploid and aneuploid embryos, I next determined whether preservation of embryos impacted cellular autofluorescence, which could alter determination of ploidy status. Specifically, I investigated the impact of vitrification and fixation on autofluorescence as these techniques are commonly used in the clinic and for research purposes, respectively. My results showed that autofluorescence was impacted by vitrification and fixation. Therefore, caution is warranted when using preserved embryos to measure metabolic state and predict developmental potential. Collectively, these findings demonstrate that label-free optical imaging is a promising non-invasive approach to measure metabolism and predict the developmental potential of oocytes and embryos.