COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Copyright Service. sydney.edu.au/copyright Pain related genes in endometriosis: A meta-analysis by Manika Saxena A thesis submitted to the Sydney Medical School in fulfilment of the requirement for the degree of Master of Philosophy in Science 2015 Queen Elizabeth II Research Institute for Mothers and Infants Department of Obstetrics, Gynaecology and Neonatology Sydney Medical School The University of Sydney, NSW, 2006 Australia © Manika Saxena, 2015 Declaration I, Manika Saxena hereby declare that the contents of this thesis consist of original work carried out by the author unless otherwise stated and duly acknowledged. To the best of my knowledge no part of this thesis has been submitted in whole or part for the award of any other degree of the university or other institution. Signature Manika Saxena Date 5.03.2015 i Abstract Endometriosis is a benign gynaecological disorder characterised by the presence of endometrial-like glands and stroma occurring outside the uterine cavity. It affects 6-10% women of reproductive age and is often associated with chronic pelvic pain which can be extremely debilitating. Chronic pain in endometriosis may be of nociceptive, neuropathic and/or inflammatory origin. Processes involved in pain generation include neuronal development, peripheral sensitisation due to inflammation, signal transduction, conduction and pain modulation. A range of genes are known to play important roles in these processes and their expression levels are altered in other pain conditions. Dysregulated genes may contribute to pain generation in women with endometriosis. Therefore, the aim of this study was to investigate the expression of pain related genes in women with endometriosis compared to women without the disease by conducting a meta-analysis of available microarray gene expression data and associated clinical information. Suitable studies and data were identified from electronic databases, gene expression repositories and within a University Obstetrics and Gynaecology department, by limiting the search to peer-reviewed, English language studies. Sixteen published full-text studies and one unpublished thesis were included. Included studies were case-control or cross-sectional design evaluating gene expression of eutopic endometrium from women with and without endometriosis and/or of endometriotic lesions. Study participants were pre-menopausal with regular menstrual cycles and surgically proven to have or not have endometriosis. RNA hybridisation with whole human genome microarray data were extracted and dysregulated gene expression determined through meta-analyses of microarray data. ii This study has shown that in the eutopic endometrium from women with endometriosis and endometriotic lesions, pain related genes were significantly upregulated. Genes involved in pain generation were also upregulated in the secretory phase of the menstrual cycle in endometriosis. In particular, genes involved in neuronal development, inflammation leading to sensitisation, signal transduction, conduction and modulation were upregulated. Upregulation of genes involved in pain generation likely contributes to pain symptoms in women with endometriosis. Increased neuronal development and sensory innervation in the eutopic endometrium and endometriotic lesions may contribute to increased neuronal sensitisation due to the inflammatory microenvironment. This may lead to enhanced signal transduction and conduction of pain signals as well as promotion of pain by pain modulation. An improved understanding of the underlying molecular pain mechanisms may aid in identifying candidate genes for managing endometriosis-associated pain. iii Acknowledgements I would like to thank my supervisor, Dr. Alison Hey-Cunningham for her knowledge, guidance, constructive criticism and enormous support in all the stages of my project. Her suggestions have greatly enhanced this thesis. I am also thankful to my supervisor Dr. Emily Miller for her constant guidance and support throughout. I am grateful to Dr. Robert Markham for his kind support. I am grateful to Dr. Gillian Begg, my academic editor who spent her holiday time on editing and giving thoughtful comments on my thesis chapters. I am also grateful to Dr. Azmat Riaz for thoughtful discussion, support and encouragement in the last two years. Most importantly, I am forever grateful to my family for their unconditional support and encouragement in all the stages of my life. iv Table of Contents Chapter 1 ................................................................................................ 1 Endometriosis .......................................................................................... 1 1.1 Introduction ..................................................................................................................... 1 1.1.1 Heritability ............................................................................................................... 2 1.2 Symptoms ....................................................................................................................... 3 1.3 Diagnosis ......................................................................................................................... 3 1.4 Theories of pathogenesis ................................................................................................. 4 1.4.1 Coelomic metaplasia ................................................................................................ 4 1.4.2 Retrograde menstruation .......................................................................................... 5 1.5 Eutopic endometrial anomalies ....................................................................................... 6 1.5.1 Decreased apoptosis ................................................................................................. 6 1.5.2 Ineffective immune response ................................................................................... 7 1.5.3 Increased cell adhesion and proliferation ................................................................ 8 1.5.4 Angiogenesis ............................................................................................................ 8 1.5.5 Local oestrogen production ...................................................................................... 9 1.5.6 Neurogenesis .......................................................................................................... 10 1.6 Endometriotic lesions .................................................................................................... 11 1.6.1 Angiogenesis .......................................................................................................... 12 1.6.2 Hormone production and responsiveness .............................................................. 13 1.6.3 Immune alterations ................................................................................................. 14 1.6.4 Neurogenesis .......................................................................................................... 14 Chapter 2 .............................................................................................. 16 Pain in Endometriosis ............................................................................. 16 2.1 Introduction ................................................................................................................... 16 2.2 Nervous system ............................................................................................................. 16 2.2.1 Female pelvic innervation ...................................................................................... 18 2.3 Pain ............................................................................................................................... 20 2.3.1 The pain matrix ...................................................................................................... 21 2.3.1.1 Neuronal development .................................................................................... 23 2.3.1.2 Sensitisation due to inflammation ................................................................... 24 2.3.1.3 Transduction ................................................................................................... 25 v 2.3.1.4 Conduction and transmission .........................................................................