Label-free Optical Approaches for the Non-invasive Assessment of Embryo Quality

In assisted reproductive technologies, embryo quality is a key contributor to IVF success. Accurate assessment of embryo quality remains a challenge, which if overcome, would improve the ~30% success rate of IVF, which has remained stagnant or more than a decade. Current evaluations of embryo quality rely on subjective visual inspection or invasive cell biopsy, which currently, fail to improve life birth rate. Optical imaging may address this gap providing an accurate and non-invasive means of assessing embryo quality. In recent years, optical imaging has gained prominence due to its innate capacity to unravel the intricacies of biological processes with remarkable spatio-temporal resolution. Optical imaging has historically been used in conjunction with exogenous fluorescent tags or stains. However, there has been a recent shift towards label-free optical imaging. This approach can capture information on how light changes as it passes through cells, or it can capture the natural fluorescence emitted by endogenous molecules, termed autofluorescence. Thus, label-free optical imaging holds great promise for the evaluation of preimplantation embryo quality. In this thesis, I investigated whether optical imaging approaches can non-invasively detect changes in embryos during preimplantation development that are associated with embryo quality. I confirmed that label-free optical imaging can be used to characterise the bio-physical properties of embryos during preimplantation development. Specifically, my work demonstrated the ability of digital holographic microscopy to detect spatio-temporal changes in refractive index that were associated with increased intracellular lipid abundance, in the absence of exogenous tags. This underscores the potential for refractive index measurement as a label-free indicator of embryo quality, a feature compatible with clinical implementation. Further, I investigated the capacity of light sheet microscopy to capture cellular autofluorescence from the preimplantation embryo as a non-invasive means of measuring cellular metabolism. This is based on the knowledge that essential metabolic co-factors involved in ATP production are naturally fluorescent. As embryo quality is linked to metabolic activity, the use of light sheet microscopy for metabolic imaging is an exciting approach as it is known to be gentle, inflicting minimal or no photodamage in other cell types. I confirmed this to be true when imaging the preimplantation embryo: light sheet microscopy did not impact DNA integrity with imaged embryos having comparable levels of DNA damage to embryos that were not imaged. Conversely, optical imaging using confocal microscopy resulted in significantly higher levels of DNA damage compared to non-imaged and light sheet imaged embryos. Excitingly, I showed that light sheet microscopy can detect autofluorescence signals from blastocyst-stage embryos with comparable signal intensity to confocal microscopy. Overall, my results demonstrate the capacity of light sheet microscopy to capture cellular autofluorescence from embryos and in a safe manner. As I had shown that light sheet microscopy can detect autofluorescence signals with no impact on embryo DNA integrity, I next determined whether an advanced form of light sheet microscopy could be used for metabolic imaging. Specifically, I investigated the capacity for light sheet microscopy with a phasor-based hyperspectral detection method to capture embryo metabolism non-invasively and in situ. My results showed that the phasor-based hyperspectral detection method was more sensitive at detecting subtle metabolic changes in the embryo compared to the conventional bandpass filter method. Importantly, I showed that this metabolic imaging did not impact embryo developmental potential. This highlights the potential of this form of microscopy to be further developed as an accurate and non-invasive diagnostic tool for embryo quality. Collectively, these findings demonstrate that label-free optical imaging shows great promise as an accurate, non-invasive approach to assess embryo quality and ultimately improve patient outcomes for those undergoing IVF.

Author dc:creator

• Chow, Darren Jin Xiang

Advisors dc:contributor.advisor

• Dunning, Kylie
• Dholakia, Kishan

• embryo assessment
• optical imaging
• autofluorescence
• label-free imaging
• metabolic imaging
• non-invasive
• hyperspectral imaging
• digital holographic microscopy
• light sheet microscopy

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/2440/142633

OAI identifier oai:identifier

oai:digital.library.adelaide.edu.au:2440/142633