Factors affecting chromosome copy number and nuclear organisation in human sperm and embryos

Chromosome copy number aberrations are a leading cause of birth defects, stillbirths, pregnancy loss and infertility. Every human male has a proportion of chromosomally abnormal sperm however conditions such as infertility, cancer, cancer treatments, and environmental factors can increase this. Chromosome abnormality is commonplace in human embryos and one reason for the development of the controversial preimplantation genetic screening CPOS). Factors such as embryo quality and maternal age are common correlates. Appropriate nucleus positioning of chromosome territories is also though to be indicative of a "healthy" nucleus with aberrations in such nuclear organization associated with disease. The purpose of this study was to provide insight into the relationship between chromosome copy number, nuclear organization and various aetiological factors in human sperm and early stage embryos. Specifically.

• To investigate the nuclear positioning of telomeric and sub telomeric region in sperm cells and test the hypothesis that such organisation is altered in infertile males.

• To investigate the nuclear positioning of centromeric and locus specific regions of 5 chromosomes in sperm cells from males undergoing chemotherapeutic treatment for testicular cancer and Hodgkin's lymphoma and test the hypothesis that either the cancer, or its treatment significantly alters patterns of nuclear organization.

• To analyse FISH based PGS and "follow up" in 250 treatment cycles to investigate levels of aneuploidy false negative and positive results, also well as effects of different indications such as maternal age.

• To investigate the levels of aneuploidy for all 24 chromosomes using a newly developed multicolour FISH technique. To test hypotheses that factors e.g. maternal age and embryo morphology significantly effect levels, and that day 3 and day 5 results are concordant.

• To assess levels nuclear organisation of human embryos for loci on all 24 chromosomes and their relationship to maternal age, day 3 and day 5 embryo morphology.

Overall, results provide some evidence for differences in nuclear organisation in infertile males compared to controls for telomeric but not sub-telorneric loci. Effects of cancer (testicular cancer and Hodgkin's lymphoma) and chemotherapy were subtle at best with one testicular cancer patient showing a significant difference compared to controls. In embryos, monosomy appeared more common that trisomy and effects of maternal age and embryo quality were apparent when a small subset of chromosomes were analysed. Similar analysis with a 24 FISH assay confirmed monosomy/trisomy ratios however failed to show significant relationship with maternal age and embryo morphology, thereby raising questions about the reliability of the technique. Finally comparison of various parameters and nuclear organization revealed consistent alterations of the position of specific centromeres (e.g. for chromosomes 3 and 4). In conclusion, FISH is now clearly old technology for PGS but has great potential for the analysis of mosaicism and nuclear organisation.