This work is protected by copyright and other intellectual property rights and duplication or sale of all or part is not permitted, except that material may be duplicated by you for research, private study, criticism/review or educational purposes. Electronic or print copies are for your own personal, non- commercial use and shall not be passed to any other individual. No quotation may be published without proper acknowledgement. For any other use, or to quote extensively from the work, permission must be obtained from the copyright holder/s. Changes in advanced glycation content, structural and mechanical properties of vaginal tissue during pregnancy and in prolapse Homayemem Kinikanwo Weli Institute for Science and Technology in Medicine Thesis submitted to Keele University for the degree of Doctor of Philosophy June, 2018. ABSTRACT Introduction: Pelvic organ prolapse is present in up to 50% of women. It is commoner in older women, often above 50 years of age and associated with hormonal e.g. oestrogen decline, and stiffer vaginal tissue. Pregnancy, on the contrary is a physiologic condition with higher oestrogen level and exhibits reversible structural and mechanical changes in pelvic tissues. Advanced glycation products, the ageing markers, stiffen connective tissues. There has been no previous systematic study on the relationship and action mechanisms of oestrogen, glycation level and mechanical property of vaginal tissues. Aim: The aim of this project was to study the ultrastructural and mechanical properties of vaginal tissues, and understand the relationship and possible mechanisms of accumulation of glycation (pentosidine), vaginal wall mechanics and oestrogen receptor (ER- expression in the vaginal tissues in both pregnancy and prolapse. It was hypothesised that prolapse is a disease of accelerated ageing and that mechanical and ultrastructural changes in prolapsed tissues and oestrogen decline are related to the elevation of glycation content in the tissues. Methods: Following ethical approval, vaginal tissues from 49 women with prolapse and 16 controls were obtained and proformas containing information on known and suspected prolapse associations were completed for each participant. Female Sprague Dawley rats‘ vaginal tissues were used for the pregnancy study. Nano-scale, micro-scale and tissue level mechanical characterizations of the tissues were performed using ball indentation technique, scanning electron microscopy, peakforce nanomechanical property mapping atomic force microscopy, and optical coherence elastography, which was applied for the first time to the study of vaginal tissues. The glycation contents of vaginal tissues in pregnancy and prolapse were quantified by high performance liquid chromatography and values obtained were analysed in comparison to medical comorbidities. Tissues were qualified by histological and immunological staining for structure (haematoxylin &eosin, trichrome and picrosirus red stainings), glyoxalase I, ER- elastin and neural stain. A sulphated glycosaminoglycan (sGAG)-collagen model was used to study the role of sGAG in collagen fibrillogenesis. ii Results: Rat vaginal tissues in pregnancy contained significantly lower amounts of pentosidine, higher oestrogen receptor- and glyoxalase I (antioxidant enzyme) expression with larger creep, lower elastic modulus, larger fibril diameter and higher sGAG content than their non-pregnant counterparts. Observed morphological changes of the collagen fibrils in pregnancy were attributed to sGAG, which was noted to influence collagen fibril aggregation and bundling. Skin pentosidine content was reflective of vaginal tissue pentosidine in the same subjects. Pentosidine was significantly higher in prolapsed tissues and increased with age; with more age-dependent increase observed in the prolapse population and also significantly different between the 6th and 7th decades. Glyoxalase I and ER- were poorly expressed in the prolapsed tissues in comparison to controls. Prolapsed tissues had notably disorganized ultrastructure and higher collagen fibril modulus. At all levels of tissue organization, prolapsed tissues were stiffer than controls, with increased stiffening at the more superficial layers of the tissue. Hypertension and smoking were associated with higher glycation and prolapse. In both pregnancy and prolapse, higher expression of glyoxalase I and ER- were associated with lower glycation content of the vaginal tissues and lower modulus. These observations have led to the suggestions that oestrogen plays an important role in increase or reduction of glycation through an oestrogen-gluthathione-glyoxalase (antioxdant) pathway, which directly affects vaginal tissues‘ mechanics. Future implications: These findings have implications to the current understanding of how prolapse may occur and can inspire future translational research on improved treatment of women with prolapse. Oestrogen may significantly influence the temporary and permanent mechanics of pelvic tissues such as the vaginal wall through its modulation of glycation accumulation within the tissues. Oestrogen thus shows promise of a potential future medical treatment for early stages of prolapse. The knowledge of new prolapse comorbidities can aid the early detection and possible prevention of prolapse through a high index of suspicion. iii CONTENTS ABSTRACT .............................................................................................................................. ii FIGURES.............................................................................................................................. xiii TABLES .............................................................................................................................. xxii ABBREVIATIONS ................................................................................................................ xxiii PUBLICATIONS .................................................................................................................. xxvii ACKNOWLEDGEMENT ........................................................................................................ xxix CHAPTER 1: BACKGROUND .................................................................................................... 0 1.1 STRUCTURAL AND FUNCTIONAL ASPECTS OF THE FEMALE PELVIS ....... 1 1.1.1 OVERVIEW.......................................................................................................... 1 1.1.2 BONY PELVIS ..................................................................................................... 1 1.1.3 PELVIC DIAPHRAGM, LIGAMENTS AND FASCIA ...................................... 2 1.1.4 LEVELS OF PELVIC ORGAN SUPPORT ......................................................... 3 1.2 VAGINAL CONNECTIVE TISSUE STRUCTURE AND BIOMECHANICS ......... 6 1.2.1 HISTOLOGY OF THE VAGINA ........................................................................ 6 1.2.2 VAGINAL MECHANICS .................................................................................... 7 1.2.3 THE VAGINA AS A CENTRAL PELVIC SUPPORT ....................................... 7 1.2.4 VAGINAL TISSUE EXTRACELLULAR MATRIX (ECM) ............................. 9 1.3 PREGNANCY AND OESTROGEN EFFECTS ON THE VAGINAL WALL CONNECTIVE TISSUE ....................................................................................................... 11 1.3.1 OVERVIEW........................................................................................................ 11 1.3.2 THE RELATIONSHIP BETWEEN COLLAGEN, MATRIX REMODELLING ENZYMES AND OESTROGEN ...................................................................................... 13 iv 1.4 CONNECTIVE TISSUE AGEING ............................................................................ 14 1.4.1 OVERVIEW........................................................................................................ 14 1.4.2 AGEING THEORIES AND GLYCATION MARKERS OF AGEING ............ 15 1.4.3 FORMATION OF ADVANCED GLYCATION END PRODUCTS (AGEs) ... 17 1.4.4 TYPES OF AGEs ................................................................................................ 18 1.4.5 ACCUMULATION AND REMOVAL OF AGEs IN VIVO .............................. 19 1.4.6 AGEs REACTION MECHANISM .................................................................... 20 1.4.7 EFFECT OF AGEs ON THE ECM .................................................................... 21 1.5 PELVIC ORGAN PROLAPSE .................................................................................. 22 1.5.1 OVERVIEW........................................................................................................ 22 1.5.2 EPIDEMIOLOGY ............................................................................................... 24 1.5.3 GRADING OF DISEASE SEVERITY AND SYMPTOMS .............................. 24 1.5.4 CURRENT TREATMENT ................................................................................. 26 1.5.5 KNOWN AND POTENTIAL RISK FACTORS ................................................ 27 1.5.6 CONNECTIVE TISSUE CHANGES IN PROLAPSED TISSUE ..................... 31 1.6 VAGINAL WALL MECHANICS IN PREGNANCY AND PROLAPSE ............... 35 1.6.1 PREGNANCY .................................................................................................... 35 1.6.2 PELVIC ORGAN PROLPASE ........................................................................... 37 1.6.3 AGE AND MENOPAUSE.................................................................................