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Effect of administration of selective progesterone receptor modulators (SPRMs) on uterine and endometrial morphology Lucy Harriet Ravenscroft Whitaker Thesis for the degree of Doctor of Medicine The University of Edinburgh 2017 1 2 Table of Contents Declaration ….…………….…………………………………………………………….………………………… 3 Abstract ………………………...…………………………………………………………………………………… 4 Lay summary ……………………………………………………………………………………………………… 6 Acknowledgements ……………………..…………………………………………………………………….. 8 List of abbreviations ….………………..…………………………………………………………………... 10 Chapter contents ….……………...…………………….………………………………...……………......... 16 List of Figures ….……………..…….………………………..………………………………………………... 20 List of tables .……………..…………………………………….….…………………………………………… 24 Bibliography .…………...…..………………………………………………………………………………... 259 Appendices .……………..…………………………………………………………………………………….. 286 Declaration In accordance with the requirements of the University of Edinburgh, I hereby declare that the studies undertaken in this thesis were the unaided work of the author, except where due acknowledgement is made. All work described herein has not previously been submitted or accepted for another degree or professional qualification. Lucy Harriet Ravenscroft Whitaker July 2017 3 Abstract Introduction: The human menstrual cycle is regulated by sex-steroid hormones, including oestrogen (E), progesterone (P4) and androgens which act by ligand binding to their cognate receptors. Perturbation of the complex series of events governing the menstrual cycle may lead to heavy menstrual bleeding (HMB). This is a common debilitating condition and often associated with uterine fibroids. There remains an unmet need for effective, long-term medical treatment so women avoid surgery and preserve their fertility. Selective progesterone receptor modulators (SPRMs, e.g. ulipristal acetate, UPA) are synthetic ligands that bind the progesterone receptor (PR). Many SPRMs have been developed but only mifepristone (for the management of unwanted pregnancy) and UPA are in current clinical use. UPA is licensed for the intermittent treatment of symptomatic fibroids. SPRMs have potential utility for treatment of HMB as administration rapidly induces amenorrhoea but the mechanisms by which this is achieved are unknown. SPRM administration results in unique endometrial morphological changes (progesterone receptor modulator-associated endometrial changes; PAEC). Despite endometrial unopposed estradiol exposure these morphological changes do not appear to be associated with malignancy or pre-malignancy risk. Indeed endometrial cell proliferation appears reduced despite relative progesterone-antagonism. Based upon findings with other SPRMs it was hypothesised that: (i) administration of UPA would have an endometrial specific effect upon the reproductive tract, with regard to alteration in morphology, localisation of sex steroid receptors (SSR) and cell proliferation.; (ii) administration of UPA would impact upon progesterone-regulated (P- regulated) genes in the endometrium. Methods: The data presented within this thesis are derived from biopsies obtained at hysterectomy from the endometrium, fallopian tubes and cervices of women with symptomatic fibroids administered UPA for 8-15 weeks. Samples were obtained for histological assessment, immunohistochemistry and RNA extraction for subsequent quantitative RT-qPCR of sex-steroid receptors (SSR) and proliferation markers. In addition key P-regulated genes within the endometrium were investigated by RT-qPCR and selected protein expression. To further interrogate the anti-proliferative effect, RNA 4 was extracted from “paired” endometrial biopsies from the same woman in the proliferative phase of the menstrual cycle and following subsequent treatment with UPA for at least eight weeks and microarray gene analyses undertaken. Results: Morphological alteration of the endometrium with UPA administration was consistent with previously published data, but with a higher prevalence than previously described. There was a striking alteration in expression and localization of SSRs, particularly PR and androgen receptor (AR), and alteration of many P-regulated genes, consistent with UPA acting with low progesteroneagonism within the endometrium. There was no alteration of SSR expression within the cervix and proliferation was unchanged. Fallopian tube morphology and SSR expression was consistent with proliferative phase but cell proliferation was reduced following UPA administration, consistent with secretory phase levels. Microarray analyses identified multiple transcripts altered relative to proliferative phase, with GREM2 the most significantly down-regulated gene and MUC1 one of the most significantly upregulated genes. Consistent with low levels of mitotic figures and cell proliferation, the most down regulated KEGG pathway was the cell cycle. Multiple elements within this were subsequently validated (RT-qPCR) and included key regulators of all elements of the mitotic cell cycle, many of which were novel to those previously described following administration of another SPRM, mifepristone. In summary the novel data presented in this thesis considerably extend the data available to date concerning the actions of the SPRM, UPA, on the female reproductive tract, and increases knowledge regarding a compound with promising utility for the management of the debilitating complaint of HMB. 5 Lay summary Heavy periods are a common problem for women of child-bearing age and impact significantly on quality of life. A common cause of heavy periods is fibroids (non- cancerous tumours of the muscle layer of the womb). Current medical treatments often fail and many women eventually undergo surgical removal of the uterus (hysterectomy). Ulipristal acetate (UPA) is a new tablet treatment for fibroids which reduces bleeding but how it works is unknown. The menstrual cycle of women is regulated by two key hormones oestrogen and progesterone. These are known as sex-steroids and act by binding to receptors. These receptors frequently occur in the endometrium (lining of the womb) but can also be found elsewhere in the body, including in the fallopian and tube and cervix. UPA acts by binding with the receptor for progesterone. Our early work suggested that UPA reduces the replication of cells in the endometrium but we don’t know exactly how this happens, nor which cell type in the endometrium is most significantly affected. The endometrial cells look different when seen under a microscope compared to untreated women but so far there has been no evidence that women taking UPA have an increased risk of cancer of the endometrium. We also do not know what the effect of treatment with UPA has upon the fallopian tube and cervix. The work presented in this thesis has examined the effect of UPA on the appearance of cells in the endometrium, fallopian tube and cervix, and whether there was an alteration in the appearance of cells and a change in the number and location of the receptors for key sex-steroid hormones. This was done using samples taken from the womb of women with heavy periods and fibroids after hysterectomy. Some of these had had treatment with UPA before their operation. Other samples of endometrium were obtained in the gynaecology clinic from women before and after treatment with UPA. The effect of treatment with UPA on levels of genes regulated by progesterone in the endometrium was also investigated. Finally in order to try and further understand how UPA treatment reduces cell replication in the lining of the womb endometrial samples from women before and after UPA treatment were compared. I found that treatment with UPA changes the appearance of the endometrium. This is in 6 keeping with other people’s findings. New data presented here describes how the patterns of sex-steroid receptors were altered and genes controlled by progesterone were also altered. I found that cervix samples from women treated with UPA were unaltered, and that the fallopian tubes from these women looked similar to those of women in the first half of their menstrual cycle, but had a lower level of cell replication. In keeping with our earlier findings I found samples from women taking UPA had a lower level of cell replication in the lining of the womb than before they started treatment. New data generated during my studies revealed that genes involved in cell replication were also altered. Many of these have not been demonstrated before with other medications similar to UPA. In summary, the findings presented in this thesis extend the knowledge base about the effects of UPA on the lining of the womb, fallopian tubes and cervix. This is important as UPA appears to be an