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Uterine Natural Killer (Unk) Cells

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Uterine Natural Killer (Unk) Cells University of Warwick institutional repository: http://go.warwick.ac.uk/wrap A Thesis Submitted for the Degree of PhD at the University of Warwick http://go.warwick.ac.uk/wrap/62785 This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. Role of Decidual Corticosteroid production in Reproductive Failure Radha Viriyur Venkatakrishnan Submitted to the University of Warwick Faculty of Reproductive Medicine For the Degree of Doctor of Medicine May 2014 Table of Contents Declaration and Acknowledgement ......................................................... 5 Acknowledgements ..................................................................................... 6 Abstract ........................................................................................................ 7 List of Abbreviations ................................................................................... 9 List of Figures and Tables......................................................................... 13 1.0 INTRODUCTION ................................................................................... 16 1.1 The Human Endometrium ...................................................................................... 16 1.1.1 Structure of the endometrium ....................................................................... 17 1.1.2 Cyclical changes of the human endometrium ............................................... 19 1.2 Menstruation ......................................................................................................... 22 1.2.1 Purpose of menstruation ............................................................................... 23 1.3 Decidualization ....................................................................................................... 24 1.3.1 Functions of the decidua ................................................................................ 26 1.3.2 Progesterone and cAMP signalling Pathways ................................................ 27 1.4 Endometrial receptivity and window of implantation ........................................... 28 1.5 Implantation ........................................................................................................... 32 1.5.1 Pre-implantation embryo development ........................................................ 32 1.5.2 Implantation process ..................................................................................... 34 1.6 Immunomodulation ............................................................................................... 35 1.6.1 Immune cells in the endometrium................................................................. 35 1.6.2 Peripheral Natural Killer (NK) cells ................................................................. 36 1.6.3 Human uterine Natural Killer (uNK) cells ....................................................... 36 1.6.4 HLA compatibility ........................................................................................... 45 1.6.5 CD3+ T cells .................................................................................................... 45 1.6.6 Macrophages.................................................................................................. 46 1.6.7 Cytokines ........................................................................................................ 46 1.6.8 Th1/Th2 balance in pregnancy ....................................................................... 48 1.7 Steroid hormone regulation of endometrium ....................................................... 48 1.7.1 Steroid receptor expression in the endometrium ......................................... 49 1.7.2 Glucocorticoids .............................................................................................. 50 1.8 Clinical Perspective ................................................................................................ 53 1 1.8.1 Reproductive failure ....................................................................................... 54 1.8.2 Infertility ......................................................................................................... 55 1.8.3 Recurrent Implantation Failure (RIF) ............................................................. 55 1.8.4 Recurrent miscarriage .................................................................................... 56 1.8.5 Endometrium in RM ....................................................................................... 57 1.8.6 Glucocorticoids in RM .................................................................................... 58 1.9 Hypothesis and Aims .............................................................................................. 61 2.0 Materials and Methods ....................................................................... 63 2.1 Materials ................................................................................................................ 63 2.1.1 Antibodies ...................................................................................................... 63 2.1.2 Cells ................................................................................................................ 64 2.1.3 Small interfering RNA (SiRNA) ........................................................................ 64 2.1.4 Culture media and materials .......................................................................... 64 2.1.5 Reagents ......................................................................................................... 65 2.1.6 Buffers and solutions ..................................................................................... 65 2.1.7 Immunohistochemistry .................................................................................. 69 2.1.8 Enzyme Linked Immunoassay (ELISA) ............................................................ 69 2.2 Methods ................................................................................................................. 71 2.2.1 Ethics Approval and Patient Recruitment ...................................................... 71 2.2.2 Timed Endometrial samples........................................................................... 71 2.2.3 Endometrial samples for primary cell culture ................................................ 74 2.3 Cell Culture ............................................................................................................. 75 2.3.1 General maintenance of cell cultures ............................................................ 75 2.3.2 Preparation of Dextran coated charcoal (DCC) stripped fetal calf serum (FBS) 75 2.3.3 Primary human endometrial stromal cell culture .......................................... 76 2.3.4 Hormone treatment of cells ........................................................................... 77 2.3.5 Transient transfection .................................................................................... 78 2.4 Tissue specimens ................................................................................................... 78 2.4.1 Protein Analysis .............................................................................................. 79 2.4.2 RNA isolation and extraction ......................................................................... 83 2.4.3 Gene Expression Analysis using Real Time Quantitative PCR (qRT-PCR) ....... 84 2.4.4 Isolation of genomic DNA and cDNA synthesis .............................................. 86 2.4.5 Amplification of Target cDNA ........................................................................ 87 2 2.5 Microarray analysis ................................................................................................ 88 2.6 Enzyme-linked immunosorbent assay (ELISA) ....................................................... 89 2.7 Confocal immunofluorescence microscopy ........................................................... 90 2.8 Immunohistochemistry (IHC) ................................................................................. 90 2.8.1 Principle ......................................................................................................... 91 2.8.2 Procedure ....................................................................................................... 91 2.8.3 Construction of Tissue Micro Array (TMA) .................................................... 93 2.8.4 Image J Analysis ............................................................................................. 94 2.9 Radio-thin layer chromatography .......................................................................... 97 2.10 Statistical analysis .................................................................................................. 98 3.0Human Endometrial Stromal Cells ................................................................................................................... 100 3.1 Introduction ......................................................................................................... 100 3.2 Results .................................................................................................................. 103 3.2.1 Expression patterns of 11βHSD1 and 2, GR, and MR in undifferentiated and decidualized HESCs & impact of progesterone
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