No description available.

Short and long term clinical outcomes following endometrial ablation in women with heavy menstrual bleeding. Randomised trials evaluating second generation endometrial ablation techniques are discussed and critically reviewed in this thesis. Patterns of referral, socio-demographic and clinical details, primary care treatment, of women referred with heavy menstrual loss are also considered as are three randomised trials of surgical treatments for the treatment of heavy menstrual bleeding. Microwave endometrial ablation (MEATM) and thermal balloon ablation (TBall) both achieved high levels of satisfaction (-1%, 95% CI (-11, 9)). Microwave had a significantly shorter operating time, reduced usage of anti-emetics and opiate analgesia, increased discharge by six hours and fewer device failures. Comparing MEA™ in the postmenstrual phase to MEA™ following standard drug based endometrial preparation there was no significant difference at five years in menstrual outcomes, health related quality of life, or need for subsequent treatment Ten years following MEATM or transcervical resection of the endometrium (TCRE), the hysterectomy rate after ten years was significantly different with 17% in the MEATM and 28% in the TCRE arm (95% CI -0.21, -0.13). In conclusion primary care guidelines should be followed whilst establishing patient preference prior to referral to secondary care. Microwave ablation has been shown to be an effective treatment in the short, medium and long-term. It can successfully be performed in an outpatient setting in the post menstrual phase and is acceptable under local anaesthesia.

Introduction: The uterine endometrium is a dynamic tissue that undergoes cycles of proliferation, differentiation breakdown and repair in response to fluctuations in the ovarian-derived sex steroids oestrogen (E) and progesterone (P4). During the P4- dominated secretory phase there is an influx of leukocytes which further increases during the menstrual phase. The second most populous leukocyte within the endometrium is the macrophage. Previous studies have postulated a role for the macrophage in breakdown and repair occurring during the menstrual phase associated with production of MMPs and both pro- and anti-inflammatory cytokines. However the mechanisms responsible for the impact of the endometrial subpopulation on endometrial function remain poorly understood. The specific aims of the studies described in this thesis were to examine: 1) the phenotype of the endometrial macrophage during the different phases of the menstrual cycle. 2) the relationship, if any, between the macrophage and endometrial vasculature and 3) whether platelet factor 4 may be one of the factors involved in the interplay between macrophages and other endometrial cells (endothelium, stroma). Methods/Results: Transmission electron microscopy (TEM) identified macrophages carrying out phagocytosis at all stages of the menstrual cycle. Single and double immunocytochemistry revealed that macrophages were proliferating (Ki67+, PH3+) during the late secretory and menstrual phases; TEM and immunohistochemistry identified macrophages in close proximity to the endometrial vasculature throughout the menstrual cycle. Macrophages were immunopositive for glucocorticoid receptor (GR). They expressed anti-angiogenic factors (ANG-2, THBS-1, TWEAK) during the second half of the menstrual cycle preceding endometrial breakdown and pro-angiogenic factors (ANG-1, CTGF, IL-8) during phases characterised by repair processes. When endometrial endothelial capillary networks were incubated in vitro with peripheral monocyte derived macrophages (PMDM) that were incubated in media promoting differentiation into proinflammatory or anti-inflammatory macrophages ‘breakdown’ of capillaries was observed. Further studies revealed media from cortisol(F)-exposed PMDM significantly upregulated expression of pro-angiogenic factors (CTGF, IL-8 and VEGFC) by human endometrial endothelial cells (HEEC) When human endometrial stromal cells (hESC) were incubated with media conditioned by P4-exposed PMDM they increased expression of the anti-angiogenic factor IL-12a and ANGPTL4, a pro-angiogenic factor reported to be upregulated by hypoxia. Notably expression of platelet factor 4 (PF4) by hESC was significantly upregulated by incubation with media from GM-CSF and IFNγ-treated, E-treated or P4-treated PMDM. Further research into the role of PF4 within the human endometrium found the protein increased as the cycle progressed from the proliferative to the menstrual phase, when levels were maximal. Expression was co-localised with CD68, the macrophage marker, throughout the menstrual cycle, as well as in other endometrial cell types. PF4 was significantly increased within HEECs after treatment with F and hESCs after experiencing “P4 withdrawal” (treatment of P4 and cAMP for 6 days then washed and incubated with treatment-free media for 48 hours). Both conditions occur locally within the endometrium during menstruation. Interestingly, PF4 was found to be chemotactic to macrophages that had been exposed to F. Addition of PF4 to HEEC capillary networks resulted in a significant breakdown of the network; PF4 was found to downregulate bFGF RNA expression within HEECs and hESCS. MMP-1 RNA expression was also downregulated by PF4 after P4 withdrawal within HEECs and hESCs. MMP-3 was downregulated by PF4 within F-primed hESCs. This regulation of bFGF indicates a role in anti-angiogenesis; inhibition of MMP-1 and MMP-3 in these culture conditions proposes a role for PF4 in the downregulation of MMPs at menstruation in order enable regrowth of the endometrium and the start of the next menstrual cycle. Conclusions: These data shed new insights into the importance of macrophages in regulating key events in endometrial tissue function during the normal cycle with strong evidence they play a key role in regulating the vasculature during the breakdown and repair of endometrium at menstruation. Notably, new evidence suggesting PF4 may control expression of genes encoding MMP-1 and MMP-3 during menstruation. Further study on the crosstalk between tissue resident populations of macrophages found within the endometrium and other endometrial cell types may provide novel targets for therapies for reproductive disorders associated with inflammation and aberrant angiogenesis including heavy menstrual bleeding and endometriosis.

The endometrium is a complex multicellular tissue that undergoes dynamic alterations under the control of ovarian-derived sex steroid hormones. During the proliferative phase of the human menstrual cycle, oestrogen induces proliferation of the endometrial epithelium while during the progesterone-dominated secretory phase, the endometrial stromal compartment differentiates in preparation for pregnancy. This differentiation event is termed decidualisation and it is accompanied by immune cell infiltration, vascular remodelling and secretion of cytokines and growth factors, as well as a newfound capacity of active steroid synthesis in the endometrium. Defective decidualisation has been described in several endometrial-associated disorders such as endometriosis, a pathology of ectopic endometrial tissue in the peritoneal cavity, often associated with infertility. Rodent models have been used for the investigation of endometrial physiology and pathology due to the similarity in uterine tissue architecture, appropriate endometrial responses to steroid hormones and the opportunity to inform cellular mechanisms using genetic manipulation. While the impact of 17β-oestradiol and progesterone on endometrial function have been extensively studied, androgens have only recently emerged as potent potential regulators of the endometrium, however, their impact on cell function has not been fully elucidated. The aims of this study were to: Identify the impact of androgens on endometrial function using a mouse model of steroid depletion (ovariectomy) followed by administration of the potent androgen dihydrotestosterone (DHT). Investigate the capacity of endometrial stromal cells to synthesise androgens during decidualisation using human primary endometrial stromal cells (hESCs) decidualised in vitro. Elucidate the decidualisation response of hESCs from women with endometriosis after modulation of androgen receptor (AR) function during decidualisation. Novel results obtained provided evidence of a role for androgens in inducing a trophic effect in the mouse uterus characterised by: pronounced endometrial epithelial proliferation, altered expression pattern of AR, changes in the expression of genes involved in cell-cycle progression and stromal-epithelial cross-talk. In addition, androgen treatment resulted in a striking and unexpected increase in the number of endometrial glands. Decidualisation of hESCs resulted in time-dependent changes in expression of the androgen synthesising enzymes AKR1C3 and 5α-reductase (accompanied by biosynthesis of both testosterone and DHT in a dynamic time-dependent manner). Notably, blocking of AR action arising from local androgen signalling during decidualisation of hESCs culminates in sub-optimal decidualisation as detected by the expression of the classical decidualisation markers IGFBP1 and PRL. Women with endometriosis are reported to exhibit defective decidualisation, which may be accompanied with infertility. Treatment of hESCs from women with endometriosis with an AR agonist (DHT) or antagonist (flutamide) during decidualisation resulted in striking differences in decidualisation response as demonstrated in a case-study approach. Taken together, these findings highlight novel roles of androgens in regulating endometrial function by impacting on cell proliferation, gland formation and decidualisation. These striking new findings have implications for endometrial disorders such as endometriosis. Future studies will focus on the use of selective androgen receptor modulators, a novel class of compounds, with tissue-selective actions and without the undesired side-effects of potent androgens. The use of AR modulators would benefit from a personalised medicine approach, instructed by patient profiling to direct therapeutic targeting of endometrial disorders.

Le rôle important joué par les protéases dans plusieurs processus physiologiques (reproduction, cicatrisation et régénération tissulaire) nexclut pas leurs contributions parfois primordiales dans divers pathologies, telles que la croissance tumorale et les métrorragies dysfonctionnelles. Lors de ce travail, nous nous sommes intéressés à létude des protéases et de langiogenèse au niveau de deux aspects distincts de la reproduction : 1) Une situation physiologique quest la placentation par le biais dun modèle murin ; 2) Une situation pathologique chez les femmes souffrant de métrorragies dysfonctionnelles suite à lutilisation dun système intra-utérin à libération de progestatif comme moyen de contraception

Recurrent early pregnancy loss in the ovine uterine gland knockout (UGKO) ewe model manifests on Day 14 of pregnancy, indicating that endometrial secretions are critical for peri-implantation conceptus development. Therefore, the following studies were conducted with fertile ewes and infertile UGKO ewes to identify candidate endometrial factors essential for normal conceptus survival, utilizing both genomics and proteomics approaches. The first study used transcriptional profiling of endometrium from Day 14 cyclic, pregnant, and bred UGKO ewes, as well as ewes treated with interferon tau (IFN??) and progesterone, to identify genes important for conceptus development. A number of novel and previously known IFN??-stimulated genes, as well as progesterone-stimulated genes were identified that are higher in fertile ewes, such as galectin-15. Interactive effects of progesterone and IFN?? regulate endometrial gene expression in a temporal and cell-type specific manner. The second study characterized the endometrial expression and hormonal regulation of galectin-15, a member of the galectin family of secreted ??-galactoside lectins. Galectin-15 was secreted into the uterine lumen by the lumenal (LE) and superficial glandular epithelium (sGE), where it may promote adhesion during implantation, as well as was phagocytosed by the trophectoderm and formed intracellular crystals. The third study determined the endometrial expression of galectin-15 throughout gestation. Galectin-15 was secreted into the uterine lumen, where it was phagocytosed by the trophectoderm/chorion, transferred through placental vasculature to the fetus, and cleared through the fetal kidney to be stored in allantoic fluid. The fourth study utilized proteomic analysis of uterine flushes and endometrial explant cultures from Day 14 cyclic, pregnant and UGKO ewes to identify differences in uterine secretions. Analyses identified several genes that were expressed by the LE and sGE and may be involved in prostaglandin production and/or pH regulation. Collectively, results of these studies suggest that transcriptional profiling and analysis of uterine secretions are effective tools to determine genes important for early pregnancy. Further, identified genes are expected to reveal novel endometrial factors and metabolic pathways for support of conceptus survival and implantation, as well as provide improvements for embryo culture methods and diagnose endometrial dysfunctions leading to infertility.

Thesis--Auburn University, 2010. / Abstract. Vita. Includes bibliographic references (p.98-114).

Cyclic proliferation and differentiation occur in the human endometrium in each menstrual cycle. Aside from the precise regulation of estrogen and progesterone, the physiology of the human endometrium is also tightly regulated by cytokines. It is therefore not surprising that imbalance of cytokine expression could be observed when women suffer from gynecological disease such as endometriosis, a common gynecological disease associated with altered cytokine profile and chronic inflammatory response. Although the etiology of endometriosis is not yet well elucidated, a commonly accepted Sampson theory suggests reflux of endometrial tissue to other parts of the reproductive tract would be one of the causes. As recent findings demonstrate the presence of somatic stem cells residing in human endometrium and endometriotic cyst, the hypothesis of the thesis was that cytokines confer a regulatory role in the proliferation and self-renewal of the endometrial and endometriotic stem/progenitor cells. In this project the following objectives were studied: 1) To investigate the effect of cytokines interleukin (IL) - 1β, IL-8, IL-10, IL-13, tumor necrosis factor (TNF) – α and interferon (IFN) – γ on the clonogenic ability of endometrial and endometriotic colony forming units (CFUs); 2) To determine the effect of IL-1β and IL-13 on the self-renewal and proliferative potential of the CFUs; 3) To elucidate the expression patterns of IL-1 and IL-13 receptors in endometrial and endometriotic sections and CFUs. Clonogenic analysis of endometrial and endometriotic stem cells showed an increase in clonogenicity in endometrial epithelial cells when treated with IL-1β. Treatment of IL-13 led to a drop in clonogenicity in endometrial epithelial and stromal cells while IFN-γ treatment resulted in a decreased clonogenicity in endometrial epithelial, stromal and endometriotic stromal cells. Other cytokines (IL-8, IL-10, TNF-α) displayed no effect on the clonogenicity of endometrial and endometriotic cells. Functional study by replating IL-1β and IL-13 treated endometrial and endometriotic CFUs revealed that these cytokines did not affect the self-renewal ability of endometrial and endometriotic CFUs. The proliferative potential of CFUs was determined by total cell output assay. The results suggested that IL-1β up-regulated the proliferative potential of the endometriotic stromal CFUs but not the endometrial epithelial and stromal CFUs, while IL-13 did not alter the proliferative potentials of endometrial and endometriotic CFUs. Comparative analysis on the effect of IL-1β and IL-13 between endometrial and endometriotic stromal CFUs demonstrated that IL-1β would preferentially promote the proliferative potential of ectopic stromal CFUs while IL-13 selectively increases that of normal stromal CFUs. Receptor expression analysis by immunostaining demonstrated the presence of IL-1 receptor and IL-13 receptors protein in the glandular epithelium of endometrial tissue. Their expression was more diffuse in the endometriotic tissues. Using reverse transcription–polymerase chain reaction, IL-1RI, IL-13Rα1, IL-13Rα2 mRNA was detected in endometrial and endometriotic stromal cells, while only in endometrial epithelial cells express IL-1RII mRNA as well as IL-1RI, IL-13RαI and IL-13Rα2 mRNA. The present study suggests a role of cytokines on endometrial and endometriotic stem/progenitor cells and further investigation of actions of cytokines would be constructive on the development of endometriosis. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Human endometrium undergoes cyclic tissue breakdown and regeneration throughout a woman of reproductive life. It has been speculated that there is a subpopulation of stem/progenitor cells residing in the endometrium responsible to its remarkable capability. One key feature that distinguishes the adult stem cells is their quiescent state within the stem cell niche. Based on this unique stem cell property, slow-cycled cells from human endometrial tissues were examined and characterized. The first aim of this study was to examine label retaining cells (LRCs) in long-term engrafted human endometrial tissues using a mouse xenotransplantation model in vivo. After various initial approaches, the maximum initial labeling of LRCs was determined and, only stromal LRCs were detected after a 12-week chase. Mesenchymal stem cell (MSCs) and stem cell-like phenotypic appearances were detected in a small proportion of stromal LRCs. The second objective was to establish a novel method for identifying slow-cycled cells from cultivated endometrial epithelial and stromal cells in vitro. The method identified cells that retained fluorescence label after long-term culture. These cells were termed fluorescence retaining cells (FRCs). The variables and the different initial approaches leading to the establishment of the protocol for the identification of FRC were optimized. The third objective further characterized the endometrial stromal FRCs. Endometrial stromal FRCs were enriched with cells having higher colony forming and self-renewal abilities when compared with non-FRCs. At subsequent passage (P2) the adult stem cell characteristics of cells derived from FRCs was more prominent and differentiated into mesenchymal lineages. Clonally derived stromal FRCs expressed higher self-renewal and pluripotent genes (BMI-1, NANGOG, OCT4, and SOX2). They also expressed endometrial mesenchymal stem cell phenotypic surface markers: CD146/PDGFRβ and W5C5, suggesting these cells might be of MSC origin. The fourth objective was to examine the role of epithelial-stromal interactions on regulation of endometrial stem cells. Menstrual cycle day 2 epithelial conditioned medium significantly enhanced the colony forming ability of endometrial stromal cells. Protein expression analysis of the early menstrual phase conditioned medium showed two cytokines: angiogenin and interleukin 8, potentially involved in the regulation of endometrial stromal stem/progenitor cells. The fifth objective was to examine the reconstitution ability of the endometrial clonogenic cells in vivo using a mouse xenotransplantation model. Endometrial clonogenic stromal cells together with epithelial cells reconstituted muscle-like tissue. Although no endometrial-like tissue was detected, the findings provide the first in vivo evidence that stromal stem/progenitor cells are present within clonogenic cells. Overall, the thesis reports the existence of slow-cycled cells from human endometrium. By using various label retention techniques in vitro and in vivo a subpopulation of slow-cycled stromal cells which exhibit characteristics properties of adult stem cells was demonstrated for the first time. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy