Chromosome segregation and recombination in human meiosis: Clinical applications and insight into disjunction errors

Chromosome copy number errors (or aneuploidy) of gametes and embryos occurs in humans more frequently than in any other studied species, with a spectrum of manifestations from implantation failure to affected live births. It is predominantly problem arising in maternal meiosis with at least 20% of oocytes being aneuploid, a proportion that increases dramatically with advancing maternal age. Currently the only intervention to reduce the chances of transmitting aneuploidy is by invasive embryo biopsy procedures in high-risk groups (mainly patients with advanced maternal age) undergoing in-vitro fertilisation. Despite the severity of this problem, aneuploidy of the human preimplantation embryo is relatively poorly understood. With this in mind the purpose of this thesis is to explore the premise underpinning the use of preimplantation genetic screening (PGS) in human embryos and investigate its clinical applications and current methodologies. A series of published works demonstrate what I believe to be a significant contribution to the development of applications for studying human preimplantation aneuploidy, also providing insight into its origins and mechanisms at the earliest stages of human development.

Specifically, I present a novel standard set of protocols as a general reference work from practitioners in the fields of embryo biopsy and array comparative genomic hybridisation (CGH - the current ‘gold standard’ for preimplantation aneuploidy screening). I present a summary of work encapsulated in three published clinical papers using a linkage based analysis of Single Nucleotide Polymorphism (SNP) karyotypes (Karyomapping). Karyomapping was designed as a near-universal approach for the simultaneous detection of chromosomal and monogenic disorders in a PGS setting and these results demonstrate the utility of the technique in three separate scenarios.

In order to study the underlying mechanisms of female meiosis I present my findings on the use of a calcium ionophore to activate human oocytes artificially. An algorithm based on Karyomapping (termed MeioMapping) is demonstrated for the first time specifically to investigate human female meiosis. By recovering all three products of human female meiosis (oocyte, and both polar biopsies – herein termed “Trios”) using calcium ionophore, I present a novel protocol (commissioned by Nature Protocols) to allow exploration of the full extent of meiotic chromosome recombination and segregation that occurs in the female germline. Finally I present a published set of experiments using this protocol to provide new insight into meiotic segregation patterns and recombination in human oocytes. This work uncovers a previously undescribed pattern of meiotic segregation (termed Reverse Segregation), providing an association between recombination rates and chromosome mis-segregation (aneuploidy). This work demonstrates that there is selection for higher recombination rates in the female germline and that there is a role for meiotic drive for recombinant chromatids at meiosis II in human female meiosis.

The work presented in this thesis provides deeper understanding of meiotically derived maternal aneuploidy and recombination. More importantly it provides a vehicle within an ethical framework to continue to expand our knowledge and uncover new insights into the basis of meiotic errors that may aid future reproductive therapies.