Molecular and cytogenetic approaches to the analysis of chromosomes in human preimplantation embryos

The original focus of the research for this thesis was concentrated on establishing strategies to detect chromosome imbalance in embryos derived from chromosomal rearrangement carrier parents. Optimising conditions and investigating the possibility of different probe combinations was an important part of this study. The research carried out involved the gathering of data regarding segregation patterns during meiosis in gametogenesis, as well as acquiring information as to the mechanisms involved in the formation of mosaic and chaotically dividing embryos. Dual and triple probe combinations were developed, and evaluated in 69 surplus IVF embryos, using FISH. In the normally developing group 77% were uniformly normal, while in the abnormally developing group, 24% were normal. PGD strategies were designed for forty-four referrals, 20% of which reached the embryo biopsy stage of PGD. Over 13 PGD cycles, a total of 166 oocytes were retrieved and 113 of the resulting embryos were biopsied. FISH analysis showed that 29% of the embryos were normal for the chromosomes tested, whilst 71% were chromosomally unbalanced. The three live births and one biochemical pregnancy achieved, despite the poor history in almost all cases, is proof that a policy of biopsying two cells from embryos consisting of 6 or more cells and a single cell from 4 or 5 cell embryos is compatible with a positive outcome and reduces the risk of misdiagnosis in cases of balanced/aneuploid mosaics. The use of Comparative Genomic Hybridisation (CGH) was investigated as an alternative, global strategy for PGD. The final part of the research for this thesis involved assessing the efficiency of CGH, improving the protocol for optimised use on single cells, and its application to human embryonic material. Results suggested that CGH is a laborious and technically demanding technique not standard enough for routine clinical application. It therefore proved not to be the best approach to PGD for sub fertile patients carrying translocations since deletions and small duplications were not readily detected in single cells. Delineating the involvement of the WHS for a referred patient by examining the status of selected polymorphic markers, and performing molecular cytogenetic analysis of embryonic DNA samples from fetal tissues obtained from social terminations, were projects performed as part of the training required for CGH analysis on single cells