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Insights Into the Urogenial Microbiome of Females Suffering from Infectious

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Insights Into the Urogenial Microbiome of Females Suffering from Infectious From the Department of Infectious Diseases and Microbiology of the University of Lübeck Director: Prof. Dr. med. Jan Rupp The cervical microbiome in female infectious infertility: clinical trial and experimental mouse models Dissertation for Fulfillment of Requirements for the Doctoral Degree of the University of Lübeck from the Department of Natural Sciences Submitted by Simon Graspeuntner from Oberndorf b. Sbg. (Austria) Lübeck 2016 First referee: Prof. Dr. med. Jan Rupp Second referee: Prof. Dr. rer. nat. Ulrich Schaible Date of oral examination: 26.06.2017 Approved for printing: Lübeck, 06.07.2017 Table of content P a g e | I Table of content Abstract ........................................................................................................................... 1 Zusammenfassung .......................................................................................................... 3 1 Introduction .................................................................................................................... 5 1.1 Female infectious infertility ............................................................................................. 5 1.1.1 Female infertility is an important worldwide health issue .............................................. 5 1.1.2 Sexually transmitted diseases cause tubal factor infertility ............................................ 5 1.2 The microbiome of the female urogenital tract .............................................................. 7 1.2.1 Characterization of the vaginal microbiota in health and disease .................................. 7 1.2.2 Next-generation-sequencing drives the knowledge on the urogenital microbiome ...... 8 1.2.3 The urogenital microbiome and sexually transmitted pathogens: recent findings ....... 10 1.2.4 The role of the urogenital microbiome in infectious infertility remains elusive ........... 12 1.3 Mice as model organisms for studying sexually transmitted diseases .......................... 12 1.3.1 Chlamydia trachomatis as a model for sexually transmitted pathogens in mice .......... 13 1.4 Aims of the study ........................................................................................................... 14 2 Material and Methods .................................................................................................. 16 2.1 Material .......................................................................................................................... 16 2.1.1 Devices ........................................................................................................................... 16 2.1.2 Consumable supplies ..................................................................................................... 18 2.1.3 Chemicals ....................................................................................................................... 19 2.1.4 Buffers and solutions ..................................................................................................... 20 2.1.5 Cultivation media ........................................................................................................... 20 2.1.6 Antibodies ...................................................................................................................... 21 2.1.7 Enzymes.......................................................................................................................... 21 2.1.8 Kits .................................................................................................................................. 22 2.1.9 Primer for PCR ................................................................................................................ 22 2.1.10 Organisms, cell lines and human samples ..................................................................... 22 2.1.10.1 Organisms...................................................................................................................... 22 2.1.10.2 Cell lines ........................................................................................................................ 23 2.1.10.3 Human samples ............................................................................................................. 23 2.2 Ethics declaration ........................................................................................................... 23 2.3 Declaration of consent and information of study participants ...................................... 23 2.4 Questionnaire ................................................................................................................. 23 Table of content P a g e | II 2.5 Clinical diagnostic analysis ............................................................................................. 24 2.5.1 Pathogen detection by PCR ............................................................................................ 24 2.5.2 Pathogen detection by cultivation ................................................................................. 24 2.5.3 Serology of C. trachomatis ............................................................................................. 24 2.6 Molecular biological methods ........................................................................................ 25 2.6.1 DNA isolation .................................................................................................................. 25 2.6.2 Polymerase chain reaction ............................................................................................. 25 2.6.3 Agarose gel electrophoresis ........................................................................................... 26 2.6.4 Elution of DNA from agarose gels .................................................................................. 27 2.6.5 Next-generation-sequencing.......................................................................................... 27 2.6.5.1 454-Pyrosequencing (Roche) ........................................................................................ 27 2.6.5.2 Sequencing-by-synthesis (Illumina) .............................................................................. 27 2.7 Animal experiments ....................................................................................................... 27 2.7.1 Chlamydial infection mouse models .............................................................................. 27 2.7.2 C. muridarum model scoring system ............................................................................. 28 2.8 Cell culture ..................................................................................................................... 28 2.8.1 Long term cultivation of HEp-2 cells .............................................................................. 28 2.8.2 Passaging ........................................................................................................................ 28 2.8.3 Freezing and thawing ..................................................................................................... 29 2.8.4 Counting ......................................................................................................................... 29 2.9 Infection biology ............................................................................................................ 29 2.9.1 Preparation of chlamydial stocks ................................................................................... 29 2.9.2 Recovery assay ............................................................................................................... 30 2.9.3 Quantifying Chlamydia ................................................................................................... 30 2.10 Microscopy ..................................................................................................................... 31 2.10.1 Indirect immunofluorescence of Chlamydiae ................................................................ 31 2.11 Bioinformatics and statistics .......................................................................................... 31 2.11.1 Software ......................................................................................................................... 31 2.11.2 Processing of raw sequencing data ................................................................................ 32 2.11.3 Graphical and statistical evaluation ............................................................................... 32 2.11.4 Probability modelling ..................................................................................................... 33 3 Results ........................................................................................................................... 34 3.1 The microbiome of females suffering from infectious infertility ................................... 34 3.1.1 Study inclusion criteria and study groups ...................................................................... 34 Table of content P a g e | III 3.1.2 Clinical characterization of the study groups ................................................................. 35 3.1.3 General characteristics of the cervical microbiome ...................................................... 39 3.1.4 Reduced Lactobacillus abundance with increased Gardnerella abundance in females with infectious infertility and FSW ................................................................... 44 3.1.5 Low alpha diversity is a characteristic displayed
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