Hereditary endometrial cancer (EC) is a Lynch syndrome (LS) related cancer variant and 2-10% of all EC are hereditary. The aim of this study was to develop a method for analysis of microsatellite instability (MSI) as such analysis would assist in identifying potential LS patients with EC at an early state of their disease, before a possible second cancer is developed in another organ. Twenty-six patients with adenocarcinoma in the endometrium, diagnosed at Uppsala University Hospital in Sweden between 1993 and 2012, were included in the study. Seven of these patients were also diagnosed with LS and the rest were sporadic EC. DNA was extracted from the patients’ formalin-fixed and paraffin-embedded tissues. The extracted DNA was subjected to a multiplex PCR with fluorescently labelled primers and then analysed by using capillary electrophoresis. Of the sporadic EC, 26% was MSI-High, which correlates well with published data. Of the LS patients, 83% was MSI-High. The outcome of this project resulted in that MSI analysis is now a validated and established method used in the process of identifying potential LS among patients with EC.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 162-182) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 162-182) Also available online.

Made available in DSpace on 2014-06-11T19:35:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-08-03Bitstream added on 2014-06-13T19:06:58Z : No. of bitstreams: 1 dias_ds_dr_botfm.pdf: 489085 bytes, checksum: ef210bdb9a4ce05e40c231f4eea53b91 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Pólipos endometriais são neoformações resultantes da hipertrofia focal da camada basal do endométrio associada a um hiperestímulo hormonal. Sua etiopatogenia ainda não está bem estabelecida, não havendo consenso sobre sua história natural, seu real significado como entidade patológica e sua relação com a neoplasia endometrial. Os pólipos endometriais são a principal indicação de histeroscopia cirúrgica, sem que haja, no entanto, protocolo estabelecido para seu melhor manejo. Apresentar um panorama sobre esta condição pode auxiliar na busca em traçar indicadores para seu tratamento adequado. Avaliar os dados histeroscópicos e o perfil imuno-histoquímico dos pólipos endometriais de mulheres acompanhadas no Setor de Endoscopia Ginecológica da Faculdade de Medicina de Botucatu - UNESP. Trata-se de estudo clínico de avaliação prospectiva e transversal com amostra de conveniência de 82 mulheres, submetidas à polipectomia histeroscópica cirúrgica, e 20 mulheres, submetidas à cirurgia oncológica devido à neoplasia endometrial, entre o período de janeiro de 2010 a dezembro de 2011. Foram analisados parâmetros clínicos, ultrassonográficos, histeroscópicos, histopatológicos e imuno-histoquímicos dos pólipos endometriais e do câncer de endométrio. Para estudo estatístico das variáveis quantitativas considerou-se o teste t de Student para amostras independentes, e o teste não paramétrico de Mann-Whitney. Para as variáveis qualitativas utilizou-se o teste de Goodman para contrastes entre e dentro de populações multinomiais, tendo sido adotado o nível de 5% de significância. Na comparação entre biópsias endometriais ambulatoriais, orientada e dirigida, a sensibilidade da biópsia dirigida no diagnóstico dos pólipos endometriais variou de 35,3% a 36,8%, quando realizada no ápice e na base da lesão... / Endometrial polyps are focal neoformation resulting from hypertrophy of the basal layer of the endometrium associated with a hormonal hyperstimulation. Its pathogenesis is not well established, there is no consensus about its natural history, its real significance as a disease and its relationship to endometrial neoplasia. The endometrial polyps are the main indication of hysteroscopic surgery, without a defined protocol for its better management. Presenting an overview of this condition can aid in tracing indicators for its proper treatment. To evaluate hysteroscopic features and the immunohistochemical profile of endometrial polyps in women followed by the Gynecological Endoscopy Sector from Botucatu Medical School - UNESP. This is a clinical study with a prospective and cross-sectional analysis with convenience sample of 82 women who underwent surgical hysteroscopic polypectomy and 20 women undergoing oncological surgery due to endometrial cancer, between January 2010 to December 2011. We analyzed the clinical, sonographic, hysteroscopic, histological and immunohistochemical parameters of endometrial polyps and the cancer of endometrium. For statistical analysis of quantitative variables we considered the Student's t test for independent samples and nonparametric Mann-Whitney test. For qualitative variables we used the Goodman test for contrasts between and within multinomial populations, being adopted the 5% level of significance. In comparison of outpatient endometrial biopsies, guided and directed, the sensitivity of directed biopsy in the diagnosis of endometrial polyps ranged from 35.3% to 36.8% when performed at the apex and base of the lesion, while for the guided biopsy was 29.2%. Specificity was 33.3%, 50% and 60% respectively for each type of biopsy. The positive predictive values were 75%, 77.8% and... (Complete abstract click electronic access below)

Orientador: Jorge Nahás Neto / Coorientador: Eliana Aguiar Petri Nahás / Banca: Nilton José Leite / Banca: Reginaldo Guedes Coelho Lopes / Banca: Waldir Pereira Modotte / Banca: Ricardo Bassif Lasmar / Resumo: Pólipos endometriais são neoformações resultantes da hipertrofia focal da camada basal do endométrio associada a um hiperestímulo hormonal. Sua etiopatogenia ainda não está bem estabelecida, não havendo consenso sobre sua história natural, seu real significado como entidade patológica e sua relação com a neoplasia endometrial. Os pólipos endometriais são a principal indicação de histeroscopia cirúrgica, sem que haja, no entanto, protocolo estabelecido para seu melhor manejo. Apresentar um panorama sobre esta condição pode auxiliar na busca em traçar indicadores para seu tratamento adequado. Avaliar os dados histeroscópicos e o perfil imuno-histoquímico dos pólipos endometriais de mulheres acompanhadas no Setor de Endoscopia Ginecológica da Faculdade de Medicina de Botucatu - UNESP. Trata-se de estudo clínico de avaliação prospectiva e transversal com amostra de conveniência de 82 mulheres, submetidas à polipectomia histeroscópica cirúrgica, e 20 mulheres, submetidas à cirurgia oncológica devido à neoplasia endometrial, entre o período de janeiro de 2010 a dezembro de 2011. Foram analisados parâmetros clínicos, ultrassonográficos, histeroscópicos, histopatológicos e imuno-histoquímicos dos pólipos endometriais e do câncer de endométrio. Para estudo estatístico das variáveis quantitativas considerou-se o teste t de Student para amostras independentes, e o teste não paramétrico de Mann-Whitney. Para as variáveis qualitativas utilizou-se o teste de Goodman para contrastes entre e dentro de populações multinomiais, tendo sido adotado o nível de 5% de significância. Na comparação entre biópsias endometriais ambulatoriais, orientada e dirigida, a sensibilidade da biópsia dirigida no diagnóstico dos pólipos endometriais variou de 35,3% a 36,8%, quando realizada no ápice e na base da lesão... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Endometrial polyps are focal neoformation resulting from hypertrophy of the basal layer of the endometrium associated with a hormonal hyperstimulation. Its pathogenesis is not well established, there is no consensus about its natural history, its real significance as a disease and its relationship to endometrial neoplasia. The endometrial polyps are the main indication of hysteroscopic surgery, without a defined protocol for its better management. Presenting an overview of this condition can aid in tracing indicators for its proper treatment. To evaluate hysteroscopic features and the immunohistochemical profile of endometrial polyps in women followed by the Gynecological Endoscopy Sector from Botucatu Medical School - UNESP. This is a clinical study with a prospective and cross-sectional analysis with convenience sample of 82 women who underwent surgical hysteroscopic polypectomy and 20 women undergoing oncological surgery due to endometrial cancer, between January 2010 to December 2011. We analyzed the clinical, sonographic, hysteroscopic, histological and immunohistochemical parameters of endometrial polyps and the cancer of endometrium. For statistical analysis of quantitative variables we considered the Student's t test for independent samples and nonparametric Mann-Whitney test. For qualitative variables we used the Goodman test for contrasts between and within multinomial populations, being adopted the 5% level of significance. In comparison of outpatient endometrial biopsies, guided and directed, the sensitivity of directed biopsy in the diagnosis of endometrial polyps ranged from 35.3% to 36.8% when performed at the apex and base of the lesion, while for the guided biopsy was 29.2%. Specificity was 33.3%, 50% and 60% respectively for each type of biopsy. The positive predictive values were 75%, 77.8% and... (Complete abstract click electronic access below) / Doutor

A mouse in vitro co-culture model between endometrial epithelial cells (EEC) and peripheral blood lymphocytes and monocytes (PBLM) immune cells was established, and Ct lipopolysaccharide (LPS) was added to the cells to mimic Ct bacterial infection and to stimulate an immune response. This model enabled us to study the cross-talk between EEC and PBLM and the physiological changes that occur in the endometrium upon Ct LPS stimulation. Results showed that EEC-PBLM co-culture and Ct LPS stimulation caused changes in transepithelial resistance (TER) as well as the expression and function of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel of epithelial cells. CFTR gene transcription was up-regulated at early hours after Ct LPS stimulation, while its channel activity was down-regulated at later hours. These results suggested the possible involvement of CFTR acting as a receptor for the internalization of Ct, which may ultimately lead to the disappearance of CFTR on the apical membrane. The EEC-PBLM co-culture showed that cross-talk was important for host defense in the endometrium. Direct cross-talk by cell-cell contact between EEC and PBLM was vital for immune cell survival as well as strengthening epithelials' barrier function. (Abstract shortened by UMI.) / Chlamydia trachomatis (Ct) infection is one of the most prevalent causes for sexually transmitted disease (STD) in the female reproductive tract. Ct is unique in that it is a bacterium, but infects and replicates like a virus inside a host cell. Ct infection can lead to a variety of reproductive diseases, such as pelvic inflammatory diseases (PID), tubule scarring, salpingitis, endometriosis, ectopic pregnancy and infertility. Effective immune defense in the uterus is necessary to eliminate these bacteria and to ensure optimal uterine environment for sperm motility, fertilization, and embryo implantation to occur. The immune system of the endometrium responds to Ct infection by the recruitment of many types of leukocytes, such as T-lymphocytes, B-lymphocytes, monocytes, macrophages and neutrophils, to the site of infection. Cross-talk between endometrial epithelial cells and immune cells may alter the activities of epithelial cells causing changes in channels function and anion secretion. / Ho Alice. / "August 2005." / Adviser: Chan Hsiao Chang. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3532. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 155-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Endometrial adenocarcinoma, originating from the glandular epithelial cells of the uterine endometrial lining, is one of the most prevalent cancers amongst women in the Western world. The prostaglandin F2α (PGF2α) receptor (FP) is upregulated in endometrial adenocarcinoma. A previous microarray analysis of endometrial adenocarcinoma cells (Ishikawa) identified numerous targets of PGF2α-FP signalling including angiogenic factors, VEGF-A, FGF-2, CXCL1 and CXCL8 and antiangiogenic factors ADAMTS1. The regulation of VEGF-A, FGF-2, CXCL1 and CXCL8 was confirmed by previous studies using an in vitro model system, of Ishikawa cells stably expressing the FP receptor to levels observed in cancer (FPS cells). In this thesis, ADAMTS1 expression was found to be upregulated in endometrial adenocarcinoma samples compared to normal endometrium. Using FPS cells, ADAMTS1 expression was regulated in an extracellular signal regulated kinase 1/2 (ERK1/2) independent manner involving activation of nuclear factor of activated T cells (NFAT). Angiogenic and antiangiogenic proteins secreted by epithelial cells, in response to PGF2α-FP receptor signalling, could therefore regulate vascular function in a paracrine manner. Hence this thesis examines the role of angiogenic factors FGF2, CXCL1 and CXCL8, secreted into PGF2α-treated FPS cell conditioned medium (P CM), in the regulation of endothelial cell function in vitro. Firstly, using an in vitro model system, treatment of human umbilical vein endothelial cells (HUVECs) with P CM increased endothelial network formation and proliferation, compared to control CM. Immunoneutralisation of FGF2, CXCL1 and CXCL8 from the P CM reduced endothelial cell network formation and proliferation (P<0.05). In addition, inhibition of their receptors (FGFR1 and CXCR2) with chemical antagonists decreased endothelial cell network formation and proliferation (P<0.05) in response to treatment with P CM. This indicates that FGF2, CXCL1 and CXCL8 are paracrine effectors of FP-mediated endothelial cell network formation and proliferation. Next, the mechanisms by which FGF2 regulates P CM-induced endothelial cell network formation and proliferation were investigated. Using specific inhibitors of cell signalling, FGF2-FGFR1 was found to regulate endothelial cell proliferation via the mTOR pathway. In contrast, FGF2-FGFR1 signalling mediated endothelial cell network formation via the regulation of COX-2 expression and PGF2α synthesis in endothelial cells. Endometrial adenocarcinoma, originating from the glandular epithelial cells of the uterine endometrial lining, is one of the most prevalent cancers amongst women in the Western world. The prostaglandin F2α (PGF2α) receptor (FP) is upregulated in endometrial adenocarcinoma. A previous microarray analysis of endometrial adenocarcinoma cells (Ishikawa) identified numerous targets of PGF2α-FP signalling including angiogenic factors, VEGF-A, FGF-2, CXCL1 and CXCL8 and antiangiogenic factors ADAMTS1. The regulation of VEGF-A, FGF-2, CXCL1 and CXCL8 was confirmed by previous studies using an in vitro model system, of Ishikawa cells stably expressing the FP receptor to levels observed in cancer (FPS cells). In this thesis, ADAMTS1 expression was found to be upregulated in endometrial adenocarcinoma samples compared to normal endometrium. Using FPS cells, ADAMTS1 expression was regulated in an extracellular signal regulated kinase 1/2 (ERK1/2) independent manner involving activation of nuclear factor of activated T cells (NFAT). Angiogenic and antiangiogenic proteins secreted by epithelial cells, in response to PGF2α-FP receptor signalling, could therefore regulate vascular function in a paracrine manner. Hence this thesis examines the role of angiogenic factors FGF2, CXCL1 and CXCL8, secreted into PGF2α-treated FPS cell conditioned medium (P CM), in the regulation of endothelial cell function in vitro. Firstly, using an in vitro model system, treatment of human umbilical vein endothelial cells (HUVECs) with P CM increased endothelial network formation and proliferation, compared to control CM. Immunoneutralisation of FGF2, CXCL1 and CXCL8 from the P CM reduced endothelial cell network formation and proliferation (P<0.05). In addition, inhibition of their receptors (FGFR1 and CXCR2) with chemical antagonists decreased endothelial cell network formation and proliferation (P<0.05) in response to treatment with P CM. This indicates that FGF2, CXCL1 and CXCL8 are paracrine effectors of FP-mediated endothelial cell network formation and proliferation. Next, the mechanisms by which FGF2 regulates P CM-induced endothelial cell network formation and proliferation were investigated. Using specific inhibitors of cell signalling, FGF2-FGFR1 was found to regulate endothelial cell proliferation via the mTOR pathway. In contrast, FGF2-FGFR1 signalling mediated endothelial cell network formation via the regulation of COX-2 expression and PGF2α synthesis in endothelial cells. Angiogenesis is maintained by a balance of pro-and antiangiogenic factors. Hence, concomitantly with the upregulation of proangiogenic factors, antiangiogenic proteins ADAMTS1 and regulator of calcineurin 1 (RCAN1) were upregulated by P CM treatment of HUVECs. They were subsequently shown to limit endothelial cell network formation and proliferation in response to P CM. Finally, the role of PGF2α in angiogenesis was investigated using two in vivo models. PGF2α treatment did not increase angiogenesis in a sponge matrigel mouse model. In a xenograft mouse model, PGF2α-FP signalling increased expression of angiogenic factors in human epithelial cells and mouse stroma but this did not enhance microvessel density. Taken together, this thesis had highlighted that PGF2α-FP receptor signalling stimulates expression of pro-and antiangiogenic factors that in turn regulate endothelial cell function. However, in vivo studies demonstrate that PGF2α-FP receptor interaction does not impact on the level of angiogenesis but may control other aspects of vascular function.

Introduction: The human endometrium has a remarkable capacity for efficient cyclical repair following the inflammatory process of menstruation. Defective postmenstrual repair may contribute to the common complaint of heavy menstrual bleeding (HMB). The mechanisms and factors involved in endometrial repair are still to be fully elucidated. Endometrial function is governed by the ovarian hormones and pre-menstrually progesterone levels decline as the corpus luteum regresses. Consequently, the synthesis of prostaglandins (PG) is increased, namely PGE2 and the potent vasoconstrictor PGF2α. Subsequent vasoconstriction of endometrial spiral arterioles is believed to result in a transient hypoxic episode in the upper endometrial layer. Therefore, the aims of this thesis were to determine (i) the endometrial expression of putative repair factors across the menstrual cycle (ii) the regulation of these factors by hypoxia, PGE2 and PGF2α (ii) the role of hypoxia inducible factor (HIF)-1α in endometrial repair and (iii) differences in endometrium from women with objectively measured HMB (>80ml) and normal controls (<80ml). Methods/Results: Putative repair factors, with known angiogenic, mitogenic and proliferative functions, were identified in human endometrial samples by quantitative reverse transcription PCR and immunohistochemistry. Interleulin-8 (IL-8), vascular endothelial growth factor (VEGF), adrenomedullin (AM), connective tissue growth factor (CTGF) and endothelin-1 (ET-1) were all maximally expressed during the menstrual and/or proliferative phases of the cycle, consistent with the onset of endometrial repair. Endometrial cells and tissue explants treated with 100nM PGE2/F2α and/or hypoxia (0.5% O2) revealed up-regulation of IL-8, VEGF, AM and CTGF. An in vitro progesterone antagonism model revealed that progesterone withdrawal, hypoxia and prostaglandins are all necessary for significant increases in repair factor expression in endometrial tissue. HIF-1α was detected in human endometrium but exclusively in the late-secretory and menstrual phases. Using shorthairpin RNA against HIF-1α, it was determined that hypoxia up-regulated these factors via HIF-1α, whereas PGF2α acted in a HIF-1α independent manner to increase repair factor expression. Finally, whole genome array analysis was performed on menstrual endometrium from women with objectively measured heavy and normal menstrual bleeding to provide an unbiased comparison of gene expression. 259 transcripts displayed significant changes between the two groups. Five candidate genes were validated using Q-RT-PCR. Bioinformatic analysis of the differentially expressed gene set identified bioprocesses that included positive regulation of biological and cellular processes, leukocyte differentiation, regulation of apoptosis and response to stress/hypoxia. The presence of HIF-1α protein was examined in menstrual endometrial tissue nuclear protein extracts by Western blot, revealing significantly decreased levels in women with HMB versus normal controls. Furthermore, the mRNA expression of known target genes of HIF-1α (VEGF, CXCR4) was also significantly decreased in these women. The functional impact of endometrial HIF-1α was assessed using an in vitro angiogenic assay. Silencing of HIF-1α in endometrial cells significantly reduced the angiogenic potential of culture supernatants when compared to untransfected cells or cells transfected with a scrambled sequence. Conclusions: Repair factors are significantly increased in the human endometrium following the onset of menstruation. Progesterone withdrawal, hypoxia via HIF-1α and prostaglandins appear necessary for the regulation of these factors at this time. Menstrual endometrium displays significant differences in gene expression and HIF- 1α protein levels between women with HMB and normal controls. The findings of this thesis contribute to the existing literature on both the physiological process of endometrial repair and the pathogenesis of HMB. Extension of this work may allow the identification of novel therapeutic targets for the treatment of this common, debilitating condition.

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

The novel emergency contraceptive Ulipristal acetate (UPA) belongs to the progesterone receptor modulator family. A single oral dosage of 30mg UPA within 120 hours of unprotected intercourse could delay ovulation and differentiation of endometrium. Yet, whether UPA affect embryo implantation remains largely unknown. This study aims to investigate whether UPA affect endometrial gene expressions and embryo attachment onto endometrial epithelial cells. The PR-expressing human endometrial carcinoma cell line Ishikawa was used and treated with 10nM estrogen, 1μM progesterone or 4μM UPA for 24 hours. Changes in transcriptome profiles were analyzed by Affymetrix Human Gene 1.0 ST array GeneChip. Gene clustering showed the gene expression pattern after UPA treatment was similar to control (0.1% ethanol); while estrogen treated group was different from all the other groups. Totally, 8 genes were significantly increased and 1 was decreased (≥2-fold, p<0.05) after UPA treatment. All except one of the 8 up-regulated genes were also up-regulated by estrogen; while only one of them increased after progesterone treatment. Most genes that were altered by UPA were involved in angiogenesis and vascular remodeling. The effect of UPA on human embryo-endometrium attachment was carried out using an in vitro multi-cellular spheroids-endometrial epithelial cell co-culture model. Human choriocarcinoma cell line JAR and Ishikawa were used. UPA (0.04-4μM) treatment for up to 48 hours did not affect the proliferation of JAR or Ishikawa cells. Similarly, the attachment of JAR spheroids onto Ishikawa cells after 1 hour co-culture was not affected by UPA treatment. The molecules of Wnt/β-catenin signaling pathway, a pathway that is actively involved in embryo implantation, such as the β-catenin and GSK-3β, and endometrial receptive marker E-cadherin were not changed after UPA treatment. In Ishikawa cells, the expression of PR-A was induced after UPA (0.04-4μM) treatment; while PR-B increased when 0.04 or 4μM UPA used. However, the PR-A/PR-B ratio remained unchanged after all concentration of UPA treatment. The effect of UPA on spheroids attachment was further investigated with cultured human primary endometrial epithelial cells. Endometrial glandular epithelial cells were digested and isolated from endometrial biopsy taken from IVF patients on day 7 after luteinizing hormone surge (LH+7). A co-culture assay was optimized with JAR spheroids and endometrial epithelial cells that were growing on Matrigel. The attachment rate of JAR spheroids is approximately 60% after 3 hours incubation. However, after 24 hours of exposure to 4μM UPA, the attachment remained comparable to that of the control group. In conclusion, UPA could alter the expression of genes in Ishikawa cells mainly related to angiogenesis. It is likely that UPA may affect stromal decidualization and blastocyst invasion after attachment. However, UPA did not affect the expression of Wnt-signaling molecules and attachment of JAR spheroids onto either Ishikawa or human primary endometrial epithelial cell. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy