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In this thesis I have studied the possible receptors on decidual leukocytes which will bind to the HLA class I ligands on trophoblast. Of particular importance is the identity and function of the receptor for HLA-G as this molecule is only found on invading trophoblast cells. I have studied the expression, binding characteristics and functions of two possible HLA-G receptors, KIR2DL4 and ILT2. cDNA for these receptors was transfected into NK cell line, several functional assays were performed including cytotoxicity and cytokine production. The results showed the recognition of HLA-G by ILT2 and the functional consequences in these in vitro studies. There was no report on KIR2DL4 antigen expression in decidual NK cells. A rabbit polyclonal antibody against KIR2DL4 was made and purified. KIR2DL4 protein was detected from NK cell lysate in a Western blotting assay with this rabbit antibody. To further characterise the possible outcome of this maternal interaction in pregnancy, first I studied the cytokine profiles of decidual leukocytes, NK cells and trophoblast. In addition to previously published cytokines (GM-CSF and TNF-a), IL-6 and IFN-g have been found to be produced by decidual NK cells. These cytokines could have some major functions in the roles NK cells play in pregnancy. To dissect the change of cytokine profile after decidual leukocytes' recognition of placental cells, an in vitro culture system was developed using primary trophoblast cells and isolated decidual leukocytes. We found two cytokines (IFN-g and TNF-a) have been altered in this co-culture system. And this alteration could be due to the IL-10 and TGF-b1 produced by trophoblast. These results may shed some light on the mechanism and result of implantation.

Oxidative stress of the placenta is considered a key intermediary step in the pathogenesis of preeclampsia, but the cause for the stress remains unknown. Ischaemia-reperfusion injury, as a result of intermittent placental perfusion secondary to deficient trophoblast invasion of the endometrial arteries, is a possible mechanism. This thesis therefore tests whether hypoxia-reoxygenation (H/R) <i>in vitro</i> can induce placental oxidative stress, and cause increased apoptosis and production of tumour necrosis factor-α as seen in the preeclamptic placenta. The first aim was to examine the oxidative status of human placental tissues during periods of hypoxia and reoxygenation <i>in vitro</i>. Rapid generation of reactive oxygen species (ROS) was detected using a fluorescent marker when hypoxic villous samples were reoxygenated. The expression of oxidative stress markers including nitrotyrosine residues, 4-hydroxy-2-nonenal adducts, and inducible heat shock protein 72 was greatly increased in villous samples subjected to H/R compared to the controls maintained under constant hypoxia. Furthermore, preloading villous samples with ROS scavengers such as desferrioxamine and α-phenyl-<i>N</i>-<i>tert</i>-butylnitrone significantly reduced the levels of oxidative stress in H/R. Having demonstrated that <i>in vitro</i> H/R is capable of inducing oxidative stress in a reproducible and manipulable manner, investigations were next carried out to study the effects of resultant oxidative stress on apoptosis within the trophoblast. Compared to hypoxic and normoxic controls, there was a significant increase in the release of cytochrome <i>c</i> from mitochondria, activation of caspase , and cleavage of poly (ADP-ribose) polymerase in villous samples subjected to H/R. These events were associated with an increased number of syncytiotrophoblastic nuclei displaying apoptotic changes and increased lactate dehydrogenase release into the medium. The causal relationship between the generation of ROS and these apoptotic changes was revealed by the fact that pre-administration of desferrioxamine attenuated the insult.

The nude mouse model of endometriosis was used to study the longitudinal integration of human endometrium at an ectopic site. Histological, morphometric and immunohistochemical techniques were used to investigate nude mouse lesion development. Seven days after endometrial implantation, central necrosis and glandular degeneration were invariably seen in nude mouse lesions. By day 14, tissue remodelling had occurred and a novel vasculature was apparent. Affymetrix microarray GeneChips were used to ascertain the peritoneal transcriptome of nude mouse lesions. mRNA sequences not previously associated with endometriotic lesion development were detected, including some linked to osteoblast activity. techniques to verify the murine origin of these transcripts were developed. A selective cyclooxygenase-2 (COX-2) inhibitor and anti-angiogenic agents were tested in the nude mouse model of endometriosis. COX-2 can modulate inflammation, angiogenesis and cellular proliferation, which were observed in developing nude mouse lesions. However, the quantity and volume of lesions remained unaltered in mice treated with a COX-2 inhibitor. In contrast, when agents that inhibited vascular endothelial growth factor were administered to nude mice, the number of lesions was reduced. This thesis provides the first comprehensive description of the cellular and molecular events that occur during endometriosis-like lesion development. Disruption of only one aspect of this process (neovascularisation) revealed the therapeutic potential of anti-angiogenic agents in endometriotic disease. Many other transcripts and cells were identified that may be critical to nude mouse lesion establishment. Future research directed towards functional suppression of these factors may inhibit endometriotic lesions and increase treatment options for women with endometriosis.

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