The development of low-cost and non-invasive methods of preimplantation genetic testing

Over the past thirty years, numerous methods have been developed to assess genetic abnormalities in preimplantation embryos during IVF relevant to embryo health and viability, or that cause severe inherited disease (preimplantation genetic testing of aneuploidy; PGT-A or monogenic disease; PGT-M, respectively); however, these are often expensive, restricting patient access. Accordingly, this thesis aimed to develop low-cost methods of PGT. By applying long-read nanopore sequencing to parental gDNA samples, a novel low-cost means of identifying and phasing informative SNPs during the pre-clinical work-up phase of PGT-M was developed. Approaches based on long-range PCR amplicon sequencing and CRISPR- Cas9 enrichment are validated, with the potential to enhance patient access and improve test accuracy. Strategies based on the analysis of DNA within spent embryo culture media (SCM) during IVF (non-invasive PGT; niPGT) were also explored, with the potential to reduce costs and improve safety compared to traditional expensive and invasive embryo biopsy techniques. Specifically, a multiplex PCR-NGS protocol was developed for the non- invasive diagnosis of β-thalassaemia and sickle-cell anaemia, providing promising results during the optimisations. Moreover, niPGT-M assays for recessive X-linked disorders and sickle-cell anaemia were optimised, adopting an inexpensive recombinase polymerase amplification and lateral flow assay approach: although initial validations were promising, further optimisations are necessary to avoid misdiagnosis. To assess the potential for niPGT-A, a novel pipeline was developed utilising a whole genome amplification and next-generation sequencing approach. To date, the optimal method of amplifying DNA in SCM, which is of low quantity and likely somewhat degraded, has been unclear. After applying multiple methods to SCM, SurePlex achieved the highest levels of amplification and diagnostic concordance with paired trophectoderm biopsy specimens; however, rates were too low for clinical application. Therefore, analysis of the cumulus cell transcriptome was investigated as an alternative minimally-invasive and low-cost means of identifying oocyte-derived chromosomal abnormalities in the embryo. Transcriptome sequencing and qPCR analysis identified multiple novel candidate biomarkers, and most convincingly cytoskeletal protein PTSPIP1, with altered expression in cumulus cells associated with aneuploid embryos.