Uterine inflammation is one of the causes of a poor uterine environment. This can result in early embryonic loss in the mare due to an inhibition of or an increased secretion of prostaglandin F2α (PGF2α ). Oxytocin binds to endometrial cell receptors to activate prostaglandin synthesis. Increased secretion or accumulation of PGF2α within the uterus due to uterine inflammation can cause luteolysis and result in early embryonic loss. Supplementation with polyunsaturated fatty acids (PUFAs) has been shown to influence prostaglandin production in many species, although the effects on the mare remain unknown. Equine endometrial biopsies were collected and used to establish endometrial epithelial cell and explant cultures to determine the release of PGF2α and PGFM in response to oxytocin stimulation. Endometrial explant cultures were used to determine the inhibitory effects of Atosiban, an oxytocin receptor antagonist, and Indomethacin, a cyclooxygenase –2 inhibitor, on PGF2α secretion. Endometrial explant cultures were challenged with oxytocin (250 nM) and PGF2α concentrations were measured over time. The effects of PUFAs on equine endometrial prostaglandin production were determined using endometrial biopsies harvested on day two of behavioral estrus. Equine endometrial cells were established and shown to replicate in culture and on a basement membrane matrix. Equine endometrial explants stimulated with oxytocin had increased secretion of PGF2α and PGE2 and the secretion of PGF2α was inhibited through an oxytocin receptor antagonist and Cox inhibition. Endometrial explants stimulated with lipopolysaccharide had increased secretion of PGF2α and PGE2, however oxytocin stimulated to a greater extent than LPS. Supplementation with PUFAs, specifically DHA, decreased the secretion of PGF2α and PGE2, however AA and EPA failed to influence this response. Expression of mRNA was not influenced by fatty acid supplementation, however was altered by stimulus. Therefore DHA influences the inflammatory response in vitro through mechanisms other than enzyme expression. Decreased PGF2α production associated with PUFA supplementation in vivo, creates a likely approach for decreasing early embryonic loss associated with post breeding inflammation commonly seen in the equine industry.

No description available.

Cheung Ka Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 83-93). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Publications --- p.ii / Awards --- p.iii / List of abbreviations --- p.iv / List of figures --- p.vi / List of tables --- p.vii / Abstract in English --- p.viii / Abstract in Chinese --- p.ix / Table of Contents / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- literature Review / Chapter 2.1 --- Anatomy and Physiology of Endometrium --- p.2 / Chapter 2.2 --- Endometrial cancer --- p.5 / Chapter 2.2.1 --- Epidemiology --- p.6 / Chapter 2.2.2 --- Etiologies and Risk Factors --- p.7 / Chapter 2.3 --- Pathology --- p.11 / Chapter 2.3.1 --- Grading of endometrial cancer --- p.14 / Chapter 2.3.2 --- Staging of endometrial cancer --- p.14 / Chapter 2.4 --- Prevention and Treatment --- p.16 / Chapter 2.5 --- Molecular alterations in endometrial cancer --- p.17 / Chapter 2.5.1 --- Genetic alterations in endometrial cancer --- p.18 / Chapter 2.5.1.1 --- Oncogene activation --- p.19 / Chapter 2.5.1.2 --- Tumor suppressor gene inactivation --- p.20 / Chapter 2.5.1.2.1 --- Mutation and loss of heterozygosity of tumor suppressor genes in endometrial cancer --- p.22 / Chapter 2.5.2 --- Epigenetic alterations --- p.27 / Chapter 2.5.2.1 --- CpG islands methylation --- p.29 / Chapter 2.5.2.2 --- de novo methylation --- p.29 / Chapter 2.5.2.3 --- Detection of gene promoter hypermethylation --- p.31 / Chapter 2.5.2.4 --- Epigenetic alteration in endometrial cancer --- p.31 / Chapter 2.5.2.5 --- Promoter hypermethylation of tumor suppressor genes in other cancers --- p.39 / Chapter 2.5.3 --- Microsatellite instability --- p.42 / Chapter Chapter 3 --- The objectives of study --- p.45 / Chapter Chapter 4 --- Materials and Methods --- p.46 / Chapter 4.1 --- Samples --- p.46 / Chapter 4.1.1 --- Formalin fixed paraffin embedded tissues --- p.46 / Chapter 4.1.2 --- Cell lines --- p.46 / Chapter 4.2 --- Histological grading and staging of samples --- p.47 / Chapter 4.3 --- Microdissection on tissue sections --- p.47 / Chapter 4.4 --- Extraction of nucleic acid / Chapter 4.4.1 --- Extraction of DNA from paraffin-embedded tissues --- p.48 / Chapter 4.4.2 --- Extraction of DNA from cell lines --- p.49 / Chapter 4.5 --- DNA methylation analysis --- p.49 / Chapter 4.5.1 --- Overview of Methylation-Specific PCR (MSP) --- p.49 / Chapter 4.5.2 --- Bisulfite modification of DNA --- p.50 / Chapter 4.5.3 --- Methylation specific PCR (MSP) --- p.51 / Chapter 4.6 --- Microsatellite Analysis --- p.53 / Chapter 4.7 --- Statistical analysis --- p.56 / Chapter Chapter 5 --- Results / Chapter 5.1 --- Clinical-pathological features of endometrioid adenocarcinoma --- p.57 / Chapter 5.2 --- Promoter hypermethylation in endometrial cancer --- p.57 / Chapter 5.3 --- Microsatellite status (MSI) analysis --- p.65 / Chapter Chapter 6 --- Discussion / Chapter 6.1 --- Promoter hypermethylation in endometrial cancer --- p.71 / Chapter 6.1.1 --- Concurrent hypermethylation of multiple genesin endometrioid adenocarcinoma and its precursor lesions --- p.72 / Chapter 6.1.1.1 --- Promoter hypermethylation of E-cad --- p.73 / Chapter 6.1.1.2 --- Promoter hypermethylation of APC --- p.73 / Chapter 6.1.1.3 --- Promoter hypermethylation of MGMT --- p.74 / Chapter 6.1.1.4 --- Promoter hypermethylation of RASSF1A --- p.75 / Chapter 6.1.1.5 --- Promoter hypermethylation of hMLH-1 --- p.76 / Chapter 6.1.1.6 --- Promoter hypermethylation in ECA coexisting with hyperplasia and not coexisting with hyperplasia --- p.77 / Chapter 6.1.2 --- Promoter hypermethylation in SCA --- p.77 / Chapter 6.2 --- Microsatellite status analysis --- p.78 / Chapter 6.2.1 --- MSI in endometrial cancer --- p.78 / Chapter 6.2.2 --- MSI and concurrent promoter hypermethylation --- p.79 / Chapter 6.2.3 --- MSI and promoter hypermethylation of hMLH-1 --- p.80 / Chapter Chapter 7 --- Conclusion --- p.81 / Further studies --- p.82 / References --- p.83

Introduction: The incidence of endometrial cancer is rising steeply, with the obesity epidemic believed to be the cause. Women with a BMI > 42kg/m2 have a 9-fold increase in their relative risk of endometrial cancer. Few studies have investigated the endometrial effects of obesity or weight loss. I hypothesised that morbidly obese women had a high prevalence of undiagnosed endometrial cancer and pre-cancer, and that major weight loss would result in measurable systemic and endometrial effects. Methods: 118 morbidly obese women undergoing weight loss surgery or non-surgical weight management were recruited into a prospective cohort study. Blood and endometrial samples were taken at baseline, 2 and 12 months. Results: 80 women have undergone baseline assessment (mean age 44 years, median BMI 52kg/m2). Menstrual and reproductive dysfunction was common (15% pre-menopausal amenorrhoea, 31% oligomenorrhoea) and less than one third reported regular menstrual cycles. Four cases of endometrial cancer and six of atypical endometrial hyperplasia were detected at baseline (prevalence 12.5%, 95% CI 6.2-21.8), and women with abnormal endometrium had significantly higher HbA1c and pAKT levels. Undiagnosed diabetes was found in 6%, and overall more than 38% were diabetic and up to 40% more had raised HOMA-IR levels. Significant serial improvements were seen in insulin resistance, adipokines, inflammation and androgens after bariatric surgery. In endometrium significant reductions were seen in Ki-67, pAKT, ER and PR expression. In samples matched for cycle timing and not affected by exogenous hormone treatment Ki-67 reduced by 11% and 17% at 2 and 12 months post-surgery. AEH resolved with weight loss alone in 3/6 patients and with weight loss and LNG-IUS in 2/6 women. Ki-67 expression correlated weakly with pAKT, serum oestradiol, HOMA-IR, FAI and adipokines. Conclusions: Such a high prevalence of endometrial cancer and pre-cancer in morbidly obese women supports targeted screening in this high-risk group and highlights the importance of diagnosing and managing insulin resistance. Reduction in proliferation appears to be mediated by the PI3K/AKT pathway and through changes in insulin resistance, reproductive hormones and inflammation. Ki-67 may have a use as a marker of the 'high-risk' endometrium or in the future surveillance of endometrial abnormality being managed by fertility-sparing means.

Orientador: Ilza Maria Urbano Monteiro / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-11-07T13:24:13Z (GMT). No. of bitstreams: 1 Gomes_DanielaAngerameYela_D.pdf: 1084078 bytes, checksum: 7a9455d9433e04415da9abe38a3a7a0f (MD5) Previous issue date: 2008 / Resumo: Introdução: As doenças intra-uterinas são freqüentes entre as mulheres. Entre estas doenças podemos citar os pólipos endometriais, miomas, sinéquias intrauterinas, malformações uterinas, hiperplasias endometriais e câncer de endométrio. Para seu diagnóstico dispõe-se de métodos como o ultra-som e a histeroscopia diagnóstica, considerada padrão-ouro. O ultra-som, que surgiu na ginecologia na década de 70, avalia a espessura do endométrio, sua alteração de ecogenecidade e seus limites. Através destas características pode sugerir a doença, mas muitas vezes deixa dúvidas sobre o diagnóstico definitivo presente no útero. Apesar disso, é um método de fácil realização e com alta sensibilidade para alterações uterinas. A histeroscopia, por sua vez, é um exame mais preciso, pois permite uma melhor identificação das tumorações intracavitárias, embora para o diagnóstico definitivo seja freqüentemente necessário que se lance mão de biópsias. A dificuldade de aprendizado desta técnica tem atrapalhado a difusão da técnica. Objetivo: Avaliar a eficácia do ultra-som transvaginal e da histeroscopia ambulatorial no diagnóstico das alterações intra-uterinas. Sujeitos e Métodos: Foram realizados dois estudos, um deles com mulheres após a menopausa e outro na menacme. Os estudos foram retrospectivos, tipo teste diagnóstico. Dentre as 469 mulheres submetidas à histeroscopia ambulatorial diagnóstica no ano de 2006 no Centro de Atenção à Saúde da Mulher - Caism/Unicamp, foram excluídas 79 por não possuírem ultra-sonografia. Cento e quarenta e sete não estavam menopausadas e 243 já tinham apresentado menopausa. Foram calculados a sensibilidade, especificidade, valor preditivo positivo, valor preditivo negativo e a acurácia. O padrão-ouro para a ultra-sonografia foi à histeroscopia diagnóstica e para a histeroscopia diagnóstica foi o anatomopatológico. As fichas do estudo foram preenchidas através da análise de seus prontuários. Análise dos dados: Os dados coletados foram registrados em uma Planilha Excel e transferidos para o software SAS versão 9.1.3, considerando um nível de significância (a) de 0,05 e um poder (1-ß) de 0,80. Resultados: O grupo das mulheres na menopausa teve média de idade de 61± 9,4 anos. Observamos 6,6% de casos de hiperplasia endometrial e câncer de endométrio e o diagnóstico mais freqüente foi de pólipo endometrial (54%). O ultra-som apresentou sensibilidade de 95,6%, especificidade de 7,4% e acurácia de 53,7%, enquanto que a histeroscopia apresentou sensibilidade de 95,7%, especificidade de 83% e acurácia de 88,7%. No outro grupo, a média de idade foi de 40±8,2 anos. Não encontramos nenhum caso de câncer endometrial, mas houve três casos de hiperplasia de endométrio. A histeroscopia foi normal em 44% dos casos e observamos 34% de pólipos endometriais. A sensibilidade do ultra-som no diagnóstico de pólipo foi de 52,9% e a especificidade de 68,4%, com acurácia de 61,2%, enquanto que na histeroscopia a sensibilidade foi de 78,8%, a especificidade de 67,6% e a acurácia de 73,1%. No diagnóstico de mioma temos 70,6% e 64,3% de sensibilidade, 44,3% e 98,1% de especificidade e 63,3% e 91,2% de acurácia, respectivamente, para o ultra-som e para a histeroscopia. Conclusão: A histeroscopia apresentou maior acurácia que o ultra-som no diagnóstico das patologias intra-uterinas em ambos os grupos / Abstract: Introduction: Intrauterine diseases are common morbid disorders. Endometrial polyps, myomas, synechiae, uterine malformations, endometrial hyperplasia and endometrial cancer are cited among intrauterine pathology. The investigations using ultrasonography and outpatient hysteroscopy had been a gold standard. Ultrasonography has been utilized for pelvic examination in the early 1970's. It shows endometrial thickness and heterogeneous variations within the echogenecity of the endometrium uterine pathology. Ultrasonography is easy to apply for evaluation of intrauterine pathology and it has high sensitivy to diagnostic for intrauterine disorders. Hysteroscopy was used the gold standard control. It permited the better identification of intrauterine pathology but the histologic examination has been used for definitive diagnostic. Difficulty apprenticeship this technique had been perturbed technique diffusion. Objectives: To evaluate the efficiency of transvaginal ultrasonography and outpatient hysteroscopy in the diagnosis of intrauterine pathology. Subjects and methods: Two studies were done, one with postmenopausal women and another with premenopausal women. The studies conducted were a retrospective diagnostic-type test. They involved a total of 469 women underwent diagnostic hysteroscopy in 2006 in Women's Integral Healthcare Center - CAISM/Unicamp. Seventy-nine women were excluded due to lack of ultrasound results in their medical charts. One-hundred and forty-seven premenopausal women and two-hundred and forty-three postmrnopausal women. For statistical analysis, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy. The gold standard of the ultrasonography was the hysteroscopy and the gold standard of the hysteroscopy was the endometrium biopsy. The study chips were performed after analysis medical charts. Data analysis: The collected data were registered by means of the Microsoft Excel and transferred to SAS version 9.1.3 statistics program, considering a significance level (a) of 0.05 and 0.80 power (1-ß). Results: The mean age of postmenopausal women was 61±9.4 yaers. We observed 6.6% of endometrial hyperplasia and cancer and 54% of endometrial polyps. Ultrasonography had a sensitivity of 95.6%, a specificity of 7.4% and an accuracy of 53.7%, while hysteroscopy had a sensitivity of 95.7%, a specificity of 83% and an accuracy of 88.7%. The mean age of premenopausal women was 40±8.2 years. Endometrial cancer was not observed and two cases of endometrial hyperplasia were found. Hysteroscopy was normal in 44% and we observed 34% of endometrial polyps. Sensibility was 52.9%, specificity was 68.4% and the accuracy was 61.2% for polyps on ultrasonography while in hysteroscopy was 78.8%, 67.6% and 73.1% respectively. For myoma, sensitivily was 70.6% and 64.3%, specificity was 44.3% and 98.1% and accuracy was 63.3% and 91.2% in ultrasonography and hysteroscopy respectively. Conclusion: Hysteroscopy had better diagnostic accuracy than ultrasonography for the detection of intrauterine pathology. / Doutorado / Tocoginecologia / Doutor em Tocoginecologia

子宮内膜癌是最常见的妇科恶性肿瘤，其中有80%属于子宮内膜腺样癌，这一癌症发病的分子机制尚未清楚。研究表明，癌基因表达异常或者功能异常在肿瘤的发生发展过程中具有重要的作用。另一方面，抑癌基因在肿瘤细胞中特异性甲基化失活通常导致细胞恶性转变和肿瘤的发生。本实验将阐明癌基因阴阳1和抑癌基因PCDH10在人类子宮内膜腺样癌发病中的作用。 / 本实验第一部分研究多功能转录因子阴阳1（YY1）在子宮内膜腺样癌发病过程中的作用。首先本实验证实YY1在子宮内膜腺样癌临床标本和癌细胞系中均明显表达上调，并且上调的程度与肿瘤的FIGO分期相关。接着体外细胞培养和裸鼠荷瘤模型的实验均提示抑制YY1 表达可抑制癌细胞增殖和体外迁移，而过表达YY1则促进癌细胞增殖。这些结果表明YY1在子宮内膜腺样癌发病中具有促进作用。进一步全细胞基因组转录谱分析提示YY1 介入子宮内膜腺样发病的各个方面，并通过抑制抑癌基因APC的表达发挥发挥重要作用。深入的分子机制研究发现一个新的表观抑制作用模型：YY1可募集EZH2等多梳蛋白到APC启动子区并导致后者组蛋白3赖氨酸27上三甲基化，从而抑制APC基因转录。此外，本实验还发现YY1在子宮内膜腺样癌的表达增高是由于微小RNA，miR-193a-5p，在此癌中表达下降所导致的。所以，本实验第一部分的结果揭示了miR-193a-5p-YY1-APC这条全新的信号通路在子宮内膜腺样癌发病中发挥重要作用，并可作为潜在的治疗靶点。 / 本实验第二部分鉴定出PCDH10作为子宮内膜腺样癌一个新的抑癌基因。通过5-氮杂-2'-氧胞嘧啶处理和亚硫酸氢钠测序的方法，我们证实抑癌基因PCDH10在子宮内膜腺样癌中失活是由于其启动子区DNA甲基化所致，并且这种DNA甲基化介导的PCDH10表达沉默在子宮内膜腺样癌临床标本和癌细胞系中很常见，但不存在于正常子宮内膜组织。另外，在子宮内膜腺样癌细胞系体外实验中恢复PCDH10的表达可抑制细胞增殖、单细胞克隆形成，促进细胞凋亡。 同时在体实验荷瘤模型中恢复PCDH10的表达也可抑制肿瘤细胞增殖，这些结果与其肿瘤抑制功能相符。 / 总之，本实验结果阐明了YY1和PCDH10在子宮内膜腺样癌发病过程中新的作用，拓展了子宮内膜腺样癌发病分子机制的研究并为其药物治疗提供了潜在的靶点。 / Endometrial cancer is the most common gynecologic malignancy and about 80% of these cancers are endometrial Endometrioid carcinoma (EEC). The molecular mechanisms underlying EEC tumorigenesis are under-explored. Aberrant expression and function of oncogenes promote tumor development by modulating many aspects of tumor cell growth. On the other hand, tumor specific promoter methylation on tumor suppressor genes (TSG), which are generally unmethylated in normal cells, usually initiate and promote malignant transformation and cancer initiation. Our study aims to characterize the functions of an oncogenic transcription factor Yin Yang 1 (YY1) and a novel tumor suppressor gene PCDH10 in Human Endometrioid Endometrial Adenocarcinoma. / In the first part of our study, we investigated the function of a multifunctional TF, YY1 in EEC tumorigenesis. We demonstrated YY1 is up-regulated in EEC cell lines and primary tumors and its expression is associated with FIGO stages. Depletion of YY1 inhibits EEC cell proliferation and migration both in vitro and in vivo whereas over-expression of YY1 promotes EEC cell growth. These results suggest that YY1 functions as an onocogenic factor in EEC. Transcriptome analysis revealed a significant effect of YY1 on critical aspects of EEC tumorigenesis and its down-regulation of APC transcripts. Further mechanistic investigation uncovered a new epigenetic silencing mode of Adenomatosis Polyposis Coli (APC) by YY1 through recruitment of EZH2 and trimethylation of histone 3 lysine 27 in its promoter region. Additionally, YY1 over-expression was found to be a consequence of miR-193a-5p down-regulation through direct miR-193a-5p-YY1 interplay. Our results therefore established a novel miR-193a-5p-YY1-APC regulatory axis contributing to EEC development, which may serve as future intervention target. / In the second part of our study, we identified PCDH10 as a novel tumor suppressor gene in EEC. By using bisulfate genomic sequencing combined with pharmacologic demethylation drug treatment, we elucidated that PCDH10 inactivation in EEC is a consequence of DNA hypermethylation on its promoter region. Further study suggested that hypermethylation-mediated PCDH10 silencing was a common event in EEC cell lines and clinical samples, but not in normal endometrial tissues. Restoration of PCDH10 expression in EEC cells suppressed cell proliferation, inhibited single cell colony formation and induced cell apoptosis; moreover, overexpression of PCDH10 inhibited EEC xenograft tumor growth in vivo.These results suggest PCDH10 acts as a tumor suppressor. / Together, our results reveal the novel functions of YY1 and PCDH10 in EEC. These findings add novel insights into the molecular mechanisms of EEC development and progression, which may serve as potential therapeutic targets for this disease. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yang, Yihua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 170-186). / Abstracts also in Chinese. / TITLE --- p.I / ABSTRACT --- p.III / ACKNOWLEDGEMENTS --- p.VII / PUBLICATION --- p.IX / ABBREVIATIONS --- p.X / LIST OF FIGURES --- p.XIII / LIST OF TABLES --- p.XVI / TABLES OF CONTENT --- p.XVII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Endometrioid Endometrial Adenocarcinoma (EEC) --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.2 / Chapter 1.1.2 --- Etiology and risk factors --- p.3 / Chapter 1.1.3 --- Treatment and prognosis --- p.8 / Chapter 1.1.4 --- Molecular Mechanisms --- p.9 / Chapter 1.1.5 --- APC and Wnt/β-catenin signaling pathway --- p.12 / Chapter 1.1.6 --- Summary --- p.18 / Chapter 1.2 --- Epigenetic modifications and EEC --- p.20 / Chapter 1.2.1 --- Epigenetic modifications --- p.20 / Chapter 1.2.2 --- Epigenetic and cancer --- p.21 / Chapter 1.2.5 --- Summary --- p.30 / Chapter Chapter 2 --- Material and Method --- p.31 / Chapter 2.1 --- Tissue samples --- p.31 / Chapter 2.2 --- Cell culture --- p.32 / Chapter 2.3 --- Cell proliferation assays --- p.33 / Chapter 2.4 --- Cell migration assay --- p.34 / Chapter 2.5 --- 3-deazaneplanocin A (Dznep) or 5-aza-2'-deoxycytidine (5-aza) treatment --- p.34 / Chapter 2.6 --- Computational prediction --- p.35 / Chapter 2.7 --- Cell cycle assay --- p.35 / Chapter 2.8 --- Apoptosis assay --- p.36 / Chapter 2.9 --- Total RNAs, Total proteins and Genomic DNA extraction --- p.36 / Chapter 2.10 --- Bisulfite Genomic Sequencing --- p.38 / Chapter 2.11 --- Oligonucleotides --- p.39 / Chapter 2.12 --- RT-PCR, Semi-quantitative PCR and Real-time PCR --- p.41 / Chapter 2.13 --- microRNA validation --- p.43 / Chapter 2.14 --- Plasmid construction --- p.43 / Chapter 2.15 --- Transfection --- p.45 / Chapter 2.16 --- Luciferase reporter assay --- p.45 / Chapter 2.17 --- Western blotting --- p.46 / Chapter 2.18 --- Immunofluorescence ( IF ) --- p.48 / Chapter 2.19 --- Immunohistochemistry (IHC) --- p.50 / Chapter 2.20 --- ChIP assay --- p.53 / Chapter 2.21 --- Sequencing and base calling --- p.55 / Chapter 2.22 --- Read mapping to genome with splice-aware aligner sequenced --- p.55 / Chapter 2.23 --- Xenograft mouse model --- p.55 / Chapter 2.24 --- Statistical analysis --- p.57 / Chapter Chapter 3 --- Yin Yang 1 Plays an Oncogenic Role in Human Endometrioid Endometrial Adenocarcinoma --- p.58 / Chapter 3.1 --- YIN YANG 1(YY1) --- p.58 / Chapter 3.1.1 --- YY1 structure --- p.58 / Chapter 3.1.2 --- YY1 function --- p.59 / Chapter 3.1.3 --- YY1 and epigenetic --- p.61 / Chapter 3.1.4 --- YY1 and cancer --- p.62 / Chapter 3.1.5 --- Regulation of YY1 expression and activity --- p.66 / Chapter 3.2 --- Results --- p.68 / Chapter 3.2.1 --- YY1 is up-regulated in EEC lines and localizes in nuclei of EEC cells --- p.68 / Chapter 3.2.2 --- YY1 expression level is associated with EEC clinicopathological features --- p.72 / Chapter 3.2.3 --- Knock-down of YY1 by RNAi inhibits EEC cell proliferation and migration --- p.77 / Chapter 3.2.4 --- Ectopic expression of YY1 promotes EEC cell proliferation --- p.84 / Chapter 3.2.5 --- YY1 does not affect EEC cell cycle and cell apoptosis --- p.91 / Chapter 3.2.6 --- Genome-wide characterization of YY1-mediated transcriptome changes --- p.94 / Chapter 3.2.7 --- Gene Ontology analysis of YY1 targets on EEC tumorigenesis --- p.98 / Chapter 3.2.8 --- YY1 inhibits APC gene expression and functions --- p.101 / Chapter 3.2.9 --- YY1 inhibits APC expression through recruiting EZH2 and causing H3K27me3. --- p.105 / Chapter 3.2.10 --- Knock-down of YY1 does not change DNA methylation status of CpG island of APC gene --- p.117 / Chapter 3.2.11 --- SiYY1 oligo injection inhibits tumor grows in vivo --- p.119 / Chapter 3.2.12 --- miR-193a-5p is down-regulated in EEC cell lines and clinical samples --- p.126 / Chapter 3.2.13 --- miR-193a-5p targets YY1 3’UTR and inhibits YY1 expression --- p.128 / Chapter 3.2.14 --- miR-193a-5p inhibits tumor grow in vivo --- p.133 / Chapter 3.3 --- Discussion --- p.136 / Chapter 3.3.1 --- YY1 oncogenic functions in EEC --- p.136 / Chapter 3.3.2 --- YY1 epigenetically silences APC --- p.138 / Chapter 3.3.3 --- miR-193a-5p down-regulates YY1 in EEC --- p.139 / Chapter 3.4 --- Conclusion --- p.141 / Chapter Chapter 4 --- The tumor suppressive functions of PCDH10 in Human Endometrioid Endometrial Adenocarcinoma --- p.143 / Chapter 4.1 --- Introduction --- p.143 / Chapter 4.1.1 --- PCDH10 structure and function --- p.143 / Chapter 4.1.2 --- PCDH10 and tumor --- p.146 / Chapter 4.1 --- Results --- p.149 / Chapter 4.2.1 --- PCDH10 is down-regulated in EEC cell lines and clinical samples --- p.149 / Chapter 4.2.2 --- PCDH10 is hypermethylated in EEC cell lines and clinical samples --- p.150 / Chapter 4.2.3 --- Pharmacologic demethylation restores PCDH10 expression in EEC cell lines --- p.152 / Chapter 4.2.4 --- Ectopic over-expression of PCDH10 inhibits EEC cell proliferation --- p.154 / Chapter 4.2.5 --- PCDH10 over-expression induces EEC cell apoptosis --- p.161 / Chapter 4.2.6 --- PCDH10 over-expression inhibits tumor grows in vivo --- p.166 / Chapter 4.3 --- Discussion and future plan --- p.169 / REFERENCE --- p.170

The endometrium undergoes recurrent cycles of dynamic remodelling, involving breakdown and scarless repair, proliferation and differentiation, including decidualisation of the stroma, during the menstrual cycle. Extensive studies have characterised how the steroid hormones oestrogen and progesterone acting via their nuclear receptors coordinate these remarkable changes. Although a few previous studies have postulated a role for androgens the impact of androgens on endometrial function remains understudied. The studies described in this thesis aimed to 1) identify cellular processes, pathways and networks regulated by androgens in human androgen receptor-positive endometrial stromal cells (hESCs), 2) investigate the potential for regulation and determine the regulation of putative dihydrotestosterone (DHT)-regulated gene expression by androgen in hESCs, 3) investigate the expression and regulation of putative androgen-regulated genes in the human endometrium across the menstrual cycle and in early pregnancy and 4) explore the role of androgens in modulating metformin-induced gene expression associated with decidualisation of hESCs. Analysis of data from a whole genome array conducted previously in the laboratory using primary hESCs treated with DHT for 2 or 8 hours identified time dependant putative androgen-regulated mRNAs (34 and 268 genes, respectively). Thereafter, all work was completed by the author. Gene ontology and functional based bioinformatic analyses of the putative androgen-regulated gene sets revealed potential androgen regulation of a variety of cell processes, pathways and networks including those associated with gene transcription, signal transduction pathways (such as phosphatidylinositol, oestrogen receptor alpha (ERα) and Wnt signalling), cancer pathways, metabolism, cell cycle, development, apoptosis/survival. In addition, various transcription factors (e.g. AR, c-Myc, SP1, ERα, p53, E2F1, RUNX2, CREB1 and STAT3) were associated with androgen regulation in hESCs. Consensus androgen receptor binding sites were identified in the promoter sequences of 18 genes by transcription factor binding site sequence analysis. Direct DHT regulation of ten of 15 of these genes was validated in endometrial stromal cells using qRTPCR. Of these genes, RGS2, SIK1, and SNCAIP mRNAs were confirmed as DHT-regulated in hESCs by use of an AR inhibitor (flutamide) and in addition, were not found to be regulated by oestradiol. Discovery bioinformatics predicted these genes may interact in a gene network involving AR and the cAMP transduction pathway. Expression of the 15 putative androgen-regulated genes was confirmed by qRTPCR in intact human endometrial tissue (13 novel) and 9 of these genes were regulated in association with decidualisation i.e. either in the secretory phase, the time at which decidualisation begins and/or in first trimester decidua. Protein expression of RGS2, SIK1 and Synphilin-1 (encoded by SNCAIP) was confirmed by immunohistochemistry in endometrial tissues and protein expression also appeared greater in decidua. Regulation of putative androgen-regulated gene expression by decidualisation was confirmed in 4 out of 8 genes by employing a model of reduced in vivo decidualisation i.e. decidua from ectopic pregnancies. Regulation of 5 out of 7 genes was confirmed in decidualised hESCs (RGS2, SIK1, SLC6A6, SNCAIP and AXIN2) but expression of these genes was not altered by DHT inclusion during decidualisation. Finally, only a high metformin concentration enhanced hESC decidualisation and putative androgen-regulated gene expression (4 genes) in decidualised hESCs. In comparison, in the presence of DHT, a lower clinically relevant metformin concentration (100μM) did enhance decidualisation marker expression but did not alter expression of putative androgen-regulated genes. In summary, these studies have revealed new insights into androgen action in the human endometrium. Studies in hESCs 1) predicted the pathways and interacting transcription factor regulatory networks that may be androgen-dependent in this cell type, these were associated with cell differentiation, apoptosis and proliferation, 2) identified novel putative androgen-regulated genes expressed in hESCs and in endometrial tissues, 3) showed putative androgen-regulated genes are regulated by DHT (possibly via AR) in endometrial stromal cells, some of which are also regulated in association with decidualisation and 4) showed that androgens may enhance decidualisation during exposure to the commonly used drug metformin. Collectively, these new findings support a physiological role for androgens in endometrial function and provide a series of new avenues for further studies of the regulation of differentiation and proliferation.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Pathology / Master / Master of Medical Sciences

The endocrine disrupting chemicals (EDCs) are exogenous compounds that mimic natural hormones and disrupt endocrine functions in humans and animals. Accumulating evidence suggests that EDCs may have detrimental impact on human reproduction including infertility, distortion of sex ratios and menstrual problems. TCDD, one of the most toxic man-made chemicals, was found to affect embryo maturity, implantation and reproduction. Bisphenol A (BPA), another EDC commonly used nowadays in plastic products, exhibits weak estrogenic activity in human bodies. However, TCDD and BPA have distinct chemical structures, chemical properties and receptor binding, and their mechanistic actions on human body remain largely unknown. The present study is to delineate the effects of EDCs on embryo implantation and development under in vitro exposure. It is hypothesized that EDCs (TCDD and BPA) may modulate fertility of animals by affecting early pre-implantation embryo development and attachment onto uterus. The effect of EDCs on the expression of receptors (AhR, ERE, ERR,,and ERa) and downstream reporter genes (CYP1A1 and C3) were studied by real-time PCR and Western blotting. An in vitro spheroid (JEG-3)-endometrial cells (Ishikawa) co-culture assay was established to study the effect of EDCs on spheroid attachment. Since microRNA, Wnt-signaling and adhesion molecules play important roles in implantation process, the expression of selected miRNA, Wnt-signaling and adhesion molecules was studied after EDCs treatment. Specific activities of the EDCs receptors were confirmed by inhibitor treatment or siRNA knockdown studies. Furthermore, EDC-treated embryos were transferred to pseudo-pregnant surrogate mice to determine the effect of EDCs on implantation outcome on day 8. It was found that Ishikawa and JEG-3 cells expressed AhR, ERIIand ERRE. TCDD treatment induced CYP1A1 expression in both cell lines (P<0.05). TCDD at 10nM significantly suppressed (P<0.005) spheroid attachment (74% compared to control 97%). When both cell lines were treated with AhR antagonists DMF (10 aaa/ANF (1 /////with TCDD (10nM), the suppressive effect of TCDD on spheroid attachment in co-culture assay was nullified, suggesting that TCDD acts through AhR receptor. In BPA exposure, BPA (0.001 to 10 tt) induced the expressions of ER))and C3ain Ishikawa but reduced the expressions of EReeand C3ain JEG-3 cells. BPA (10 iiiisuppressed spheroids attachment (74% vs Control 94%, P<0.005); while co-treatment with ER antagonists (ICI 182,780), ERaaantagonistaaMPP), ERMMantagonist (PTHPP) or ERE///siRNA, the suppressive effect of BPA (10sM) on spheroid attachment was nullified, suggesting that BPA acts through ER receptors. Moreover, TCDD and BPA suppressed the expressions of B-catenin and E-cadherin for cell-cell adhesion. Activation of Wnt-signaling pathway by Wnt3a conditioned medium or LiCl, rescued the low spheroid attachment rate induced by EDCs. Both EDCs reduced embryo implantation rate in pseudo-pregnant mice, suggesting the adverse effect of EDCs on embryo implantation and/or endometrial receptivity. Taken together, TCDD and BPA affected the JEG-3 spheroid attachment onto the Ishikawa endometrial cells and mouse embryo implantation. These effects might be mediated through the action of receptors and Wnt/β-catenin signaling pathway. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy