Abstract:
There is a demand for a global shift towards single embryo transfer to ensure safer IVF practice. However, the ability to identify the embryo that is most likely to result in a healthy live birth remains one of the most significant challenges that hinders IVF success. To date, morphological grading has remained the standard method for embryo selection during IVF, however this is poorly predictive of embryo viability. Embryo aneuploidy increases with female age and is the main cause of implantation failure and miscarriage, but morphological grading is unable to diagnose aneuploid embryos. Instead, invasive embryo biopsy coupled with pre-implantation genetic screening (PGS) can identify euploid embryos for transfer, but at best these have an implantation rate of only 65%. This demonstrates that euploidy alone does not completely determine the developmental potential of the embryo. Additional aspects of embryo physiology are surfacing as determinants of viability, and the complex and dynamic relationships that exist between them are beginning to unravel. The mitochondrial genome is emerging as an additional genetic determinant of embryo viability. The first aim of this thesis was to characterise the integrity of the mitochondrial genome in oocytes from a novel bovine model of human IVF, to determine the potential genetic contribution to embryo viability with ageing and ovarian stimulation. Mitochondrial DNA (mtDNA) deletions were more common in oocytes following ageing, however the frequency of heteroplasmic point mutations was universally low. This is the first study to quantify mtDNA heteroplasmy in individual oocytes using comprehensive next-generation sequencing (NGS) technologies. These results warrant further investigation in humans. Novel methods to assess embryo viability are evolving. These methods provide further information about the development, physiology and molecular profile of the embryo. Thus, they offer new paradigms for the way that an embryo is selected for transfer. Accessible biological material that is associated with the embryo can be targeted for the development of biomarkers of embryo viability. One such candidate is the cumulus cells which nurture the oocyte during development. Previous studies have suggested that transcriptomic analysis of cumulus cells may have the potential to provide valuable clues about embryo viability. Beyond the search for biomarkers during traditional embryo culture, time-lapse microscopy has been introduced to provide undisturbed culture. Time-lapse microscopy also allows embryo development to be constantly monitored. With the introduction of this technology, time-lapse parameters have the potential to objectively disclose the developmental time-line of the embryo and predict future viability. Previous studies have investigated biomarkers in isolation, however the novel approach explored in this thesis was to combine two biomarkers within the same cohort of embryos. The second aim of this thesis was to examine the expression of cumulus cell viability genes together with early time-lapse parameters, in order to combine two independent biomarkers for improved embryo selection. The analysis revealed correlations between these two biomarkers and blastocyst quality. The expression of candidate genes involved in energy metabolism, mitochondrial biogenesis, signalling, steroidogenesis and cell stress in cumulus cells correlated to time-lapse parameters of the developing embryo. These results demonstrate that embryo viability is influenced by the ovarian micro-environment in which the oocyte develops. While combining both biomarkers within the model did not increase predictive ability due to small sample size, combining biomarkers for improved embryo selection deserves further investigation in a larger cohort. Recently, spent embryo culture media and blastocoele fluid has been found to contain molecular information that may indicate embryo viability. It has been reported that they contain DNA, but the composition and origin of this genetic material is unknown. The third aim of this thesis was to assess nuclear DNA and mtDNA within both the spent embryo culture media and following artificial blastocoele collapse, in order to determine its potential to be a biomarker of embryo viability. The levels of DNA in spent embryo culture media were found to be extremely low across three types of commercial media, and this was validated using two different genetic platforms. The levels of DNA increased at the blastocyst stage of development, but not following artificial blastocoele collapse. Importantly the background levels of DNA contamination occurring during culture was established, representing a critical analysis for the exploration of this biomarker. While it was found that there was genetic contamination within the culture media, and from maternal cumulus cells, there was preliminary evidence that the DNA did have some embryonic origin. DNA fingerprinting analysis was used to confirm this finding, however the levels of nuclear DNA were too low to produce DNA profiles. Future studies are required to confirm this finding. Further optimisation of methodologies may allow spent embryo media and blastocoele fluid to be used for the genetic viability assessment of embryos non-invasively.