Abstract 17β-hydroxysteroid dehydrogenases (17HSDs) catalyze the interconversion of high-activity 17β-hydroxysteroids and low-activity 17-ketosteroids. In the present study, the mRNA of the 17HSD type 1 and 2 enzymes, catalyzing opposite reactions of estrogen metabolism, was analyzed in normal and malignant breast and gastrointestinal tract by in situ hybridization. Further, the activities of these enzymes were measured in normal and adenomatous intestinal cell lines. Markers of the main mesenchymal cell types were also used to study the cell-type specific expression of the 17HSD type 2 enzyme in the gastrointestinal tract. The mRNA of the 17HSD types 1 and 2 was expressed in normal breast tissues of premenopausal, but not postmenopausal women. In breast cancer, varied mRNA expressions of the enzymes were seen in both groups of women. Variable mRNA expressions of the reductive 17HSD type 5 enzyme were also seen in breast cancer tissues. Patients with tumors expressing 17HSD type 1 mRNA had significantly shorter overall survival and disease-free interval than those without 17HSD type 1 expression, suggesting that inhibition of the enzyme may be beneficial in the prevention or treatment of hormone-dependent breast cancers. In normal gastric tissues, 17HSD type 2 mRNA was expressed mainly in the surface and foveolar epithelium. Expression was weak or absent in glandular epithelium. Gender did not have any effect on expression, but there was a decrease with increasing age. Chronic gastritis was associated with decreased expression, while upregulation was observed in intestinal metaplasia. In gastric malignancy, downregulation was observed in most specimens. 17HSD type 2 mRNA was expressed mainly in absorptive epithelia cells and the upper parts of crypts in normal intestinal tissues. In the lamina propria, expression was detected in endothelial cells and mononuclear phagocytes. In colon cancer, the enzyme was downregulated in most, but not all cases. 17HSD type 1 and 2 activity measurements in normal and colon cancer cell lines showed a predominant oxidative activity. Northern analysis also revealed the transcript for the 17HSD type 2 enzyme. Female subjects had significantly more colon cancers with high 17HSD type 2 mRNA than males; however, low 17HSD type 2 mRNA expression was associated with survival in females with cancer of the distal colon and rectum. These data indicate the presence of gender- and location-related differences in the pathogenesis of colon cancer and suggest that low 17HSD type 2 mRNA expression is a marker of a favorable prognosis.

Endometriosis is characterized by aberrant estrogen signaling and chronic inflammation that results in prolonged pelvic pain and infertility. Research from our lab along with others have found that the chronic inflammatory state is maintained by a high ratio of inflammatory/ tolerant (Th17/ Tregs) cells systemically as well as increased Treg localization (tolerance) within endometriotic lesions, allowing endometriotic lesions to escape effector immune clearance. Moreover, this phenotype creates an intolerant environment for successful implantation which poses a risk of infertility and pre-term delivery in this group of women. Our previous research has also found that women with endometriosis have microbial dysbiosis within both gastrointestinal and urogenital (GI/UG) environments that may contribute to abnormal metabolism of parent estrogens. We hypothesized that microbial disruption alters enterohepatic recirculation of endogenous hormones by changing expression of cytochrome P450 (CYPs) enzymes that metabolize parent estrogens. Along with presence of disease, many external factors might also contribute to microbial disruption within the urogenital environment such as sexual partner encounters. Surgical intervention and use of hormonal therapies for treatment of endometriosis, may also contribute to microbial composition within the GI and UG environments in diseased patients. Our goal for this study was to 1) measure several CYP enzyme gene expressions in both eutopic endometrium and ectopic endometriotic lesions of diseased subjects; and 2) investigate the association of sexual partner number on microbial profiles in vaginal, urine and fecal samples of diseased patients, and whether hormonal therapy and surgical intervention affect microbial dynamics within the GI and UG environments. Our data showed that CYP1B1 gene expression was altered in eutopic endometrium of diseased patients. Hormonal therapy (HT) increased CYP1B1 gene expression in both eutopic and ectopic endometrial tissue. Patients who had more than 7 sexual partners had increased microbial dysbiosis indicated by increased composition of anaerobic bacteria in fecal and vaginal samples, indicating that increased number of sexual partners further altered microbial dysbiosis in patients with endometriosis. In conclusion, our long-term goal is to identify a unique microbiome that may serve as a potential marker to detect the early onset of endometriosis. Our study contributes to current knowledge on the expression of CYP enzyme metabolites on endometriosis. Since our patient cohorts are divided based on hormonal and surgical treatment, we hope to contribute additional knowledge on the impact of pharmacological and surgical therapy on the expression of CYP enzymes in endometriosis. Moreover, we hope to gain more understanding about mechanisms that cause alteration of CYPs gene expression in patients with endometriosis. Understanding the pathophysiology of endometriosis is critical for advancement of novel therapeutic targets and treatment of disease. Through investigation of a patient’s immune system, endocrine regulation and microbial profiling, we hope to advance our understanding of the disease and identify potential areas for improving diagnostics and therapeutic interventions.

Dietary components potentially influence breast cancer risk factors, including breast density (BD) and estrogen metabolism (EM). Tamoxifen (TAM) is a commonly prescribed anti-estrogen adjuvant cancer treatment to reduce breast cancer risk, partially through modulation of BD and EM. Epidemiological evidence has suggested a potential protective effect from dietary intakes of fiber and vegetables in breast cancer recurrence in women on TAM as well as an independent influence on BD and EM. The relationship between dietary intake BD and EM in women prescribed TAM is not fully understood. A cross-sectional analysis using baseline data collected from 130 pre- and post-menopausal women taking TAM and enrolled in the Diindolylmethane Efficacy (DIME) Study was conducted. Participants completed the Arizona Food Frequency Questionnaire to assess dietary intake. TAM metabolites were analyzed through HPLC. BD was measured from digital mammograms and urinary EM was analyzed using LC/MS. Unadjusted linear regression between diet and four TAM metabolites indicated significant association between endoxifen and caffeine. 4-hydroxyTAM had significant inverse associations with fat intake, including monounsaturated, polyunsaturated and saturated fats. Linear regression adjusted for BMI revealed a statistically significant positive association with caffeine intake and BD, with no other dietary associations observed. The highest amount of correlations was observed between 2OHE and energy, total fat, MUFA, PUFA, protein and carbohydrate intake, though weak. Correlations were seen between 4OHE, 16αOHE and total isoflavones. Cholesterol was weakly positively correlated with 2mOHE, E1 and approached significance with E2. Dietary intake shows little association with BD or EM in women taking TAM therapy. Alternate preventive mechanisms for diet in women on TAM therapy should be investigated.

Nuclear receptors play crucial roles in the transcriptional regulation of many biological processes such as development and cellular differentiation. ERRalpha is known, along with coactivator PGC-1alpha, to playa central role in the control of energy metabolism in cardiac and skeletal muscle. They activate the expression of many genes involved in mitochondrial oxidative metabolism. Here we identified PROX1, a factor that was previously shown to broadly influence metabolism, as a regulator of this pathway. Indeed, PROX1 interacts in vitro and in vivo with both ERRalpha and PGC-1alpha. To provide more insight on the hepatic functions of ERRalpha and PROX1, we performed ChIP-on-chip using mouse liver, identifying a large number of ERRalpha and PROX1 genomic targets and reinforcing their role in energy metabolism. Over 40% of the target genes were found to be common to both factors and we observed that PROX1 could be recruited to ERRalpha binding sites and act as a negative regulator o fthe ERRalpha/POC-1alpha pathway.

Chronic inflammation is associated with the pathophysiology of obstetrical disorders (e.g. preterm birth [PTB]) and gynecological diseases (e.g. endometriosis); however, the exact mechanism(s) for these conditions are unknown. Numerous immunological conditions and disease states (e.g. inflammatory bowel disease, Crohn’s disease, systemic lupus erythematomus) also disrupt the microbiome homeostasis by inducing a number of changes in the microbial flora when compared to that of healthy individuals. Furthermore, the gastrointestinal (GI) microbiome is one of the principal regulators of circulating estrogens which are known to directly impact the female reproductive disorders endometriosis and PTB. Thus, an alteration of microbial species could indicate a shift in immune balance from homeostatic to pro-inflammatory, and an aberrant estrogen metabolism that precipitates the development of disease stages in endometriosis and/or PTB. The Braundmeier-Fleming lab has developed a systems biology model that investigates the interactions between the immune system, microbial dynamics (in the GI and reproductive system) and estrogen metabolism, in women, as a potential diagnostic tool for endometriosis and PTB. This dissertation, therefore, examined how inflammation triggered by female reproductive disorders (endometriosis or PTB) alter the systemic and localization immune responses, the microbial communities in the urogenital (UG), peritoneal and GI mucosal epithelium, as well as levels of excreted conjugated estrogen. The first specific hypothesis is that inflammation associated with endometriosis alters microbial dynamics and functions that are distinct from those of non-diseased patients. Preliminary data indicated that reproductive tract microbial communities from patients with endometriosis are unique when compared to non-disease patients. Therefore, the central aims of this study are to identify the immune and microbial profiles of patients diagnosed with endometriosis and determine if an alteration of these profiles impact estrogen signaling, thus driving disease pathogenesis. Additionally, I hypothesized that surgery or hormonal therapy will temporarily restore the microbiome and estrogen levels of patients with endometriosis. Differences in systemic (blood) regulatory T cell (Treg) and T-helper 17 (Th17) cell populations (tolerant and inflammatory, respectively) were measured by flow cytometry, and the immune mediators was measured by serum cytokine levels via 10-plex-ELISA kits. Immunohistochemistry was used to identify resident Th17/Treg immune cell distribution within the endometrium and ectopic endometriotic lesions, and RORγt+/FOXP3+ transcripts within these same tissues were analyzed by real-time-qPCR. We implemented high-throughput non genomic sequencing targeting bacterial-V4 16S rRNA and robust bioinformatics analyses to characterize microbial composition/diversity within the GI (fecal swab), vaginal (vaginal swab), and UG (urine) cavities. Alterations in estrogen metabolism, parent estrogens and metabolites, in urine were analyzed via LC-MS/MS. Patients with endometriosis exhibit 1) systemic and localized inflammation within ectopic and endometrial tissues, 2) altered GI/UG microbial dynamics, 3) aberrant levels of endogenous estrogen and estrogen metabolites, 4) dampened inflammation (caused by disease) due to hormonal therapy, 5) altered bacteria populations in the gut and vaginal canal of patients with endometriosis due to hormonal therapy treatment, and 6) increased post-surgical variability in microbial community dynamics. The second specific aims examined the hypothesis that induction of endometriosis in baboons (P. Anubis) results in chronic systemic and tissue specific inflammation through regulation of Th17 and Treg populations. Further, the induction of endometriosis altered GI/UG/peritoneal cavity microbial communities that are distinct from non-diseased animals. Utilizing a non-human primate animal model of induced endometriosis allowed us to characterize factors involved at the early onset of endometriosis and throughout the disease progression. We collected samples from 8 baboons at pre-inoculation (no evidence of disease) and at 3, 6, 9, and 15 months post-induction of the disease. We found that the induction of endometriosis decreased peripheral Tregs cells while Th17 cells increased at all post-induction collections with reduced ratio of total Tregs to Th17 cells indicating systemic inflammation. Microbial community diversities as well as abundances at each sample site (GI, UG [vagina, urine] tracts and peritoneal cavity) were also altered at post-induction. These results therefore suggest that induction of endometriosis in non-human primates caused an inflammatory shift. Disease induction also resulted in altered vaginal, urinary and fecal microbial profiles, which may drive inflammation through the production of inflammatory mediators. The last specific aims studied the hypothesis that patients who deliver preterm have a systemic and placental inflammatory phenotype and abnormal estrogen levels during pregnancy that are distinct from those of patients with term delivery. Biological samples were collected at 8-12 weeks, 20-24 weeks, 32-36 weeks, at delivery and 6 weeks postpartum. Subjects with PTB showed signs of systemic inflammation with an elevation in Th17:Treg ratio, greater Th17 and lower levels of natural Tregs during the 2nd trimester, and lower inducible Tregs during the 3rd trimester and at delivery. Placental tissues from subjects with PTB also had an inflammatory immune phenotype (higher Th17) within the decidua basalis and maternal-fetal interface. Immunological shifts from tolerant to inflammatory were observed in both patient groups, but these shifts occurred early in gestation for subjects with PTB and at a later gestational age for subjects delivering at term. Levels of conjugated parent estrogens and estrogen metabolites were reduced in subjects with PTB, indicative of an abnormal production of estrogen. These analyses gave us a better understanding of the inflammatory cascade with estrogen metabolism associated with pregnancy, and how these effects are correlated with premature labor. The data from this study suggest that the levels of endogenous estrogen and estrogen metabolites of estrogen metabolism were abnormal in PTB and endometriosis disease models of inflammation compared to their respective controls. In the human and non-human primate model of endometriosis studies, we observed that both patients and baboons with endometriosis had systemic and resident inflammatory phenotypes and an alteration in mucosal microbial community dynamics compared to their respective controls. All together, our long-term goal is to identify factors from the microbiome and/or the immune system that would allow us to have early non-invasive diagnostics for endometriosis or to predict which mothers are most at risk to encounter PTB. Furthermore, it would allow us to determine whether the mucosal microbiome may be a good indicator of immune stress, and if alternative therapies can alter microbial community dynamics—thereby eliminating immune stress associated with female reproductive diseases. These findings may have a substantial impact on the obstetrical care and management of patients with endometriosis and women at risk for PTB, as well as provide evidence to support the development of novel therapeutics to treat these diseases.

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Research Doctorate - Doctor of Philosophy (PhD) / Endometrial cancer is one of the most common female cancers in industrialized countries. Traditional risk factors associated with endometrial cancer are well understood and include excessive exposure to estrogen or estrogen unopposed by progesterone. However, variations in the genes that influence these hormones and their association with endometrial cancer have not been well investigated. By studying genetic variation in endometrial cancer, novel markers of risk may be discovered that can be used to identify women at high risk and for the implementation of specialised treatments. Polymorphisms in the genes involved in the following pathways; hormone biosynthesis, hormone receptors, estrogen metabolism, DNA repair and cell cycle control, have been suggested to be involved in the initiation and development of endometrial cancer. The focus of this study was to examine genetic variants in these pathways to assess the existence of an association with the risk of endometrial cancer. In the first part of this study, the COMT V158M polymorphism was examined in a hereditary non-polyposis colorectal cancer (HNPCC) cohort to determine its association with disease expression. The heterozygous genotype was over-represented in women with endometrial/ovarian cancer that did not harbour mismatch repair (MMR) gene mutations. This result suggested that the COMT V158M polymorphism may alter the risk of developing HNPCC related endometrial/ovarian cancer in MMR mutation negative women. Since COMT is involved in the metabolism of estrogen and that estrogen is the main risk factor for endometrial cancer development, closer examination was warranted to determine the association of genetic variation involved in hormone-related pathways and endometrial cancer risk, outside of the context of an inherited predisposition to disease. In the second part of this study, a cohort of 191 women with endometrial cancer and 291 healthy control women were genotyped for polymorphisms in genes involved in hormone biosynthesis, hormone receptors, estrogen metabolism, DNA repair and cell cycle control. The results revealed that variations in estrogen receptor alpha (ESR1) and beta (ESR2), and the androgen receptor (AR), were associated with an increase and decrease in endometrial cancer risk, respectively. Additionally, polymorphisms in CYP1A1, CYP1B1, GSTM1 and GSTP1 were related to a decrease in endometrial cancer risk. A trend was observed for the cyclin D1 870 G>A polymorphism and an increase in endometrial cancer risk, however, this result did not reach significance. Taken together, these results revealed that perturbations in the hormone receptors and estrogen metabolism genes, may aid in the identification of women at high risk of developing endometrial cancer. Interestingly, stratification of the women with endometrial cancer revealed that combinations of polymorphisms in TP53 and MDM2 were associated with higher grades of cancer. This finding may possibly have significant implications as women with reduced apoptotic ability, due to combinations of polymorphisms in these genes, have an increased risk of presenting with higher grades of endometrial cancer, that are associated with lower survival rates. In summary, the results of this thesis showed that variation in the estrogen and androgen receptors, and estrogen metabolism genes, may alter the risk of developing endometrial cancer. Moreover, polymorphisms in the cell cycle control genes, TP53 and MDM2, appear to be associated with higher grades of endometrial cancer. This study of polymorphisms may help explain genetic differences in individual susceptibility to endometrial cancer and are markers of risk that aid in the development of effective and personalised strategies to prevent disease development. This study has improved the understanding of genetic variation associated with endometrial cancer risk. It has the potential to enhance our ability to treat women with endometrial cancer through improved identification and treatment strategies, by virtue of the genetic variation identified, that appears to predispose to disease.

Introduction Estrogen-induced cholestasis is a disease characterized by a failure of bile flow and bile production. It can develop in women after oral contraceptives use, hormone replacement therapy or during pregnancy. The estrogen metabolism is a complex process leading to formation of metabolites with different biological activities. It takes place primarily in the liver (Phase I and Phase II including hydroxylation, methylation, sulfation and glucuronidation). The enzymes from UDP-glucuronosyltransferases family , abbreviated UGT, are responsible for the glucuronidation of estrogens. Aims The objective of my work is to define estrogen metabolism and gene expression of UGT1A1, CYP1A2 and SULT1A1 and characterize cholestatic liver damage in the UGT1A1 deficient rat strain (Gunn rats) compared to rats with normal enzyme activity and try to define possible mechanisms responsible for the liver damage. Methods Adult female Gunn and corresponding heterozygous rats were treated with ethinylestradiol (EE, 5 mg/kg body weight SC) for 5 days, while control rats received propanediol (vehicle). Day six, the animals were sacrificed and plasma and liver tissue were collected for analysis. Markers of cholestasis and liver damage ALP, AST, ALT and bilirubin were determined using an automatic analyzer, total...

Our lab has previously reported the identification of a novel endogenous 19-nor steroid, estradienolone (ED), in pregnant women that strongly bound to sex hormone binding globulin. Estrogen-receptor related receptors (ERRs), which have no known natural ligands, are a family of orphan receptors consisting of 3 isoforms: ERRalpha, ERRbeta and ERRgamma. The ERRs have been shown to actively modulate estrogenic responses, to play an essential role in pregnancy, and are implicated in breast cancer prognosis. My results show that ED acts as an antagonist of the ERRalpha confirming preliminary results obtained by our group. Studies of cellular responses demonstrate that ED has strong anti-mitogenic properties. ED inhibited the growth of both estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells in a dose-dependent manner but did not have any effects on the proliferation of the non-cancerous immortalized epithelial breast MCF-10A cells. The finding that ED inhibits proliferation of both ER negative and ER positive breast cancer cells, and regulate ERR transcriptional activity may have important ramifications in breast cancer therapy.

No description available.