Further Work

Given the paucity of statistically significant findings in this study there is considerable potential for further work. Refinements could be made to the kidney chimera model to improve study of the human cell population. Vybrant staining could be optimised to improve visualisation of the cells as high intensity staining presented difficulty in image interpretation. Further investigation into the fate of the endometrial cells in the chimera could be performed by immunostaining for either markers of cell death e.g. Caspases or mouse specific leukocyte markers to attempt to identify if an immune response was occurring. If a neonatal mouse immune response were identified then further experimentation could include a chimera culture using neonatal kidney obtained from SCID

mice as this would allow endometrial cell survival.

Refinements could be made to the chimera model of endometriosis by considering the culture medium used. It is known that the angiogenic activity of endometrial cells is increased if cultured in peritoneal fluid of women with endometriosis (Oosterlynck et al., 1993). It is possible then that this aspect of the microenvironment might be important in the cells proliferative potential. The model could be refined to include conditioned medium

136 from primary culture of endometrial cells from patients with endometriosis; however immune aspects would have to be considered and possibly embryos or SCID mice used. While the findings from the chimera experiment seem to indicate a limited use for this model in the investigation of endometriosis one avenue for further work may be considered. Previous models have investigated the attachment of endometrial cells in the process of lesion formation and have demonstrated the in both epithelial and stromal populations attachment occurs within 1 hour with invasion a similarly quick process completed within 24 hours (Witz et al., 2001). This is consistent with the findings of this study which found endometrial cells throughout the full thickness of the chimera by day 3 of culture. Further characterisation of this model could be undertaken investigating these processes at time points within the first 24 hours of culture which may yield some findings before a large increase in the endometrial population occurs.

Analysis of neonatal kidney explants seemed to demonstrate the formation of glomerular structures on culture. To elucidate with greater accuracy the processes occurring, an increased number of biological and technical repeats would be useful in RT-qPCR analysis and quantitation of immunofluorescence images could be performed. As it is hypothesised that cell populations capable of forming mature glomerular basement membrane are surviving in culture, one further experiment would be to attempt to characterise these structures using both immunofluorescence and RT-qPCR for markers of mature GBM e.g. Laminin 5II and Type IV collagen (Takemoto et al., 2006).

When investigating the survival of endothelial cells in the culture one explanation is that the disaggregation-reaggregation protocol could be responsible for their survival, as previous investigation into this cell population in explant culture did not use this method (Loughna et al., 1998). A useful further investigation would be to compare endothelial cell survival in explants generated from embryonic kidneys with those from neonatal kidneys in order to help establish whether the neonatal environment in some way responsible for the survival of this population.

In this preliminary in silico analysis normal endometrium and endometrium from patients with endometriosis has been investigated. The next step in this work would be to combine these analyses, overlaying pathways of interest, in order to find upstream mediators which may be in some way responsible for the aberrant proliferation seen in endometriosis and

137 which could be targeted for wet laboratory investigation and eventually clinical intervention.

In analysis of ‘Normal Endometrium’ the canonical pathway ‘Superoxide Radicals Degradation’ was upregulated within which ‘SOD2’ was identified as a molecule of interest. A ‘wet’ laboratory experiment may be designed to investigate the role of SOD2 in preventing ROS accumulation in secretory phase endometrium. Tissue samples from ‘SPCN’ patients gathered at the Liverpool Womens Hospital could be immunostained for SOD2 and ROS to assess correlation of these two molecules. Telomerase could also be stained for and quantitative analysis may help to demonstrate a potential correlation.

In analysis of endometrium from patients with endometriosis transcriptomic datasets from comparable microarray platforms were not obtained for the Late Secretory Phase endometrium. This represents a deficiency in the literature and one possible further investigation would be to perform a microarray experiment on a Late Secretory phase endometrial sample from a patient with endometriosis fulfilling the inclusion/exclusion criteria outlined in the ‘Methods’ section. Obtaining this data would allow further investigation into the cellular and molecular mechanisms associated with this proliferative disease.

Endometriosis can be considered a benign disease showing metastatic behaviour as the eutopic endometrium spreads to distant sites (Impey, 2012). To investigate this further transcriptomic datasets from microarray experiments may be analysed to identify common molecular pathways between patients with endometriosis and patients with endometrial cancer. A literature search has already been carried out and a suitable dataset identified. Early limiting factors have been identified in this analysis as cancer samples are almost always collected from post-menopausal patients and as such cannot be separated according to menstrual cycle phase. This introduces a large confounding factor into any analysis. One possible way around this would be to compare both cancer and endometriosis datasets to a post menopausal dataset used as a control.

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