Objectives: To assess the extent to which exposure to Intracytoplasmic Sperm Injection (ICSI) is associated with significant health, developmental and psychosocial adjustment outcomes. To investigate the incidence of assisted conception in children with Beckwith-Wiedemann syndrome.;Methods: A population case-control study of 510 school age children (and their families) born after ICSI (n=189), conventional in-vitro fertilisation (IVF) (n=158) or natural conception (n=163). Outcome measures included: A full physical examination of the child which included enumeration of physical abnormalities and an assessment of general health. An assessment of childhood IQ using the Weschler Preschool and Primary Scale of Intelligence (WPPSI). An assessment of gross and fine motor skills using the WPPSI and McCarthy Motor Skills tests. Questionnaires to assess of parent-child relationships. A survey of parental attitudes towards disclosing the method of conception to their IVF children. A survey of 160 members of the Beckwith-Wiedemann Syndrome (BWS) support group enquiring about conception.;Results: There was no difference between conception groups for overall physical health or fine/gross motor difficulties. There was evidence of an increase in congenital abnormalities in the assisted reproduction groups. Parent-child relationships were similar between groups. The majority of ICSI / IVF parents wish to disclose the method of conception to their child. There is an increased likelihood of children with BWS being conceived after IVF compared to the general population.;Conclusion: The studies in this thesis are reassuring, in terms of physical and neurodevelopmental health of ICSI children aged 4-5 years and their family relationships. The increase in congenital abnormalities after IVF/ICSI requires further study. Families of assisted conception children wish to disclose conception method to their child but require more support. There is evidence of an increased risk of BWS in children conceived after assisted reproduction.

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.

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No description available.

Success rates of in vitro fertilisation (lVF) are unsatisfactory and consequently, multiple embryo transfers are performed despite the risk of multiple pregnancies. A major goal in assisted conception is to perform elective single embryo transfer (eSET) without compromising a patient's chances of pregnancy. In order to achieve this goal, methods are required for identifying oocytes with the greatest potential Follicular fluid (FF), the fluid which surrounds an oocyte during its development in the ovary, may be a suitable medium in which to test for biomarkers of oocyte quality. Nuclear magnetic resonance (NMR)-based metabolomics is ideal for the global analysis of biological samples to identify trends and biomarkers in groups of individuals, but yet is still in its infancy in the field of female fertility. With this in mind, the aim of this work was to use these techniques to achieve a greater understanding of several aspects of infertility, with the ultimate goal of aiding eSET and success rates in IVF. FF and blood plasma from NF patients were analysed for biomarkers of oocyte quality; patients who had clinical pregnancies could be identified by high rates of anaerobic glycolysis and oocyte energy sources. Supervised analysis revealed that plasma may be predictive of FF composition, offering an alternative test medium which can be collected far less invasively than FF. The change in composition of FF and plasma during three phases of the menstrual cycle was investigated, aiding understanding of the changing environment of the oocyte during its development. This study also revealed the metabolic effects of the IVF-drug, human chorionic gonadotrophin (hCG). FF composition was also investigated for changes with the degree of vascularisation to the oocyte; lH-NMR- based metabolomics did not identify any difference in FF composition with vascularity, but 31P-NMR identified • that lower levels of phospholipids were associated with poorer vascularisation. Finally, serum samples from NF patients with three reproductive diseases implicated in fertility were compared to each other and to those of IVF patients without any diseases. No differences were found, suggesting that the NF drug regime may counteract any metabolic effects of these diseases. The studies performed here were on small-scales; however, some very interesting preliminary results have been produced and the potential of NMR-based metabolomics in the field of human female infertility has been demonstrated.

Human reproduction has been aptly split into three distinct eras. The first of these is when sexual intercourse results in conception, followed by pregnancy and childbirth. The second era of human reproduction occurs via in vitro fertilisation (IVF), whereby the foetus is fertilised outside the woman, but is later implanted into a female host where it is gestated until birth. In the third phase, the foetus is fertilised and gestated entirely in vitro, outside the female host and in an artificial womb/incubator (ectogenesis). Reproductive technologies are marked by the rapidity in which they develop, and as the reproductive revolution shows no sign of relenting, science may be about to propel us into this third wave of reproduction.

Differentiation of human endometrial stromal cells (HESCs) into specialized decidual cells is critical for embryo implantation and maintenance of a successful pregnancy. Initiation of this differentiation process, termed decidualization, is strictly dependent on elevated cAMP levels, whereas its maintenance requires continuous progesterone signalling. Here I show that NADPH oxidase-dependent reactive oxygen species (ROS) play a critical role in initiating and maintaining the decidual process. I first show that cAMP-dependent induction of decidual marker genes can be attenuated or enhanced upon inhibition or activation of the NADPH oxidase complex, respectively. Time-course analysis demonstrated that cAMP enhances endogenous ROS production, apparent after 12 hours of stimulation, which coincides with a marked induction of differentiation markers. By a process of elimination, I identified NOX4 as the main catalytic subunit involved in decidualization. Silencing of NOX4, or its cofactor p22PHOX, impaired the decidual process. I then show that the NOX4/p22PHOX complex regulates the transcriptional activity of CCAAT/enhancer binding protein β, a key regulator of HESC differentiation. Furthermore, microarray analysis revealed that the NOX4/p22PHOX complex functions downstream FOXO1, a multifaceted transcription factor involved in antioxidant defences, DNA repair and cell cycle regulation. In agreement, knockdown of NOX4/p22PHOX complex disrupted endogenous ROS production and resulted in a paradoxical prooxidant stress response further characterized by activation of the DNA repair pathway in the absence of primary DNA damage or cell death. Finally, I provide preliminary data that p22PHOX is downregulated at the transcript and protein level in the eutopic endometrium of patients with endometriosis. In summary, endogenous ROS signalling is critical for the differentiation and redox homeostasis of HESCs. Furthermore, deregulation of endometrial NADPH oxidase-dependent ROS production and signalling may be a hallmark of endometriosis, a prevalent and debilitating reproductive disorder.

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No description available.