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 as well as exploring the phenomenon of mosaicism and its underlying mechanisms in human preimplantation embryos. High levels of chromosomal mosaicism have been detected in human preimplantation embryos mostly by fluorescent in situ hybridisation (FISH) but also by comparative genomic hybridisation (CGH) and karyotyping. Mosaicism could arise through several mechanisms including abnormal cell divisions (mitotic non-disjunction or anaphase lag), failure of cytokinesis or endoreduplication. The FISH procedure has been criticised, as it is prone to failure. Two separate studies were developed and carried out in order to detect the level of mosaicism in embryos. In the first study a FISH protocol for the use of two different probes per chromosome was developed. The aim was to gain information on mechanisms leading to aneuploidy mosaicism and its true incidence. Three colour FISH was performed in three sequential rounds. In the first and second round different probes were used for chromosomes 1, 11, 18. In the third round probes were used for chromosomes X, Y and 18. Each FISH procedure included a control slide to assess FISH efficiency in all rounds of FISH. Two groups of embryos were spread on day 5 of development embryos grown in cleavage media throughout and embryos transferred to blastocyst media after day 3. A total of 21 embryos were analysed in each Group. The FISH results revealed one uniformly diploid and 20 mosaic embryos for Group I and 2 uniformly diploid and 19 mosaic embryos for Group II. Use of 2 different probes per chromosome was able to detect FISH artefacts and failure of hybridisation. Post- zygotic chromosome loss was the predominant mechanism leading to aneuploidy mosaicism for both groups, followed by chromosome gain, with only a few examples of mitotic non-disjunction. The relatively high percentage of tetraploidy in the blastocyst medium group was considered to reflect normal embryonic development. The use of CGH was investigated as an alternative strategy to detect the true level of mosaicism in the whole genome. The second 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 however, can provide extra information when used as a research tool. CGH was combined with FISH in order to assess chromosomal abnormalities in day 3 and day 5 embryos respectively. CGH was employed in 1-2 biopsied cells from a day 3 embryo, which was grown up to day 5 and further analysed by multi-colour FISH. The aim of this study was to observe the full chromosomal status of 1 -2 blastomeres biopsied at the cleavage stage (day 3) of development followed by FISH analysis of the rest of the embryo on day 5. This would allow the assessment of abnormalities in day 3 embryos by a full karyotype and then confirm whether the abnormality persists until day 5 using FISH for the chromosome(s) involved. In summary 30 embryos were fully analysed and only 3 (10%) were uniformly normal, while the rest were mosaic or chaotic. CGH was able to provide results in 83.3% of the embryos subjected to analysis. FISH and CGH showed either agreeing or complimentary results for all embryos analysed. The predominant mechanism of aneuploidy mosaicism was whole chromosome loss. Furthermore, partial aneuploidy was also detected, with partial chromosome loss being the principal mechanism. In the final part of the thesis the development of PGD protocols for a single gene disorder, namely DM, were devised using polymerase chain reaction (PCR) techniques. Two PGD protocols were devised and employed clinically in two patients undergoing PGD for DM using fluorescent PCR. Due to the extensive workup needed to develop the specific PCR protocols for each patient, a universal-like protocol was researched. Such a protocol would involve production of a sufficient amount of DNA through whole genome amplification techniques i.e. DOP-PCR from a single cell to carry out subsequent analysis with F-PCR markers as well whole chromosome analysis using CGH. DOP-PCR amplified DNA was subjected to amplification of five markers that would have been used during a PGD workup for DM and also subjected to CGH analysis. Initially genomic DNA was tested which produced high fidelity of amplification. Single cell DNA was then utilised in order to assess the amplification rate, allele dropout (ADO) and contamination levels.