Association of microbial dynamics with urinary estrogens and estrogen metabolites in patients with endometriosis

Endometriosis is an estrogen dependent gynecological disease associated with altered microbial phenotypes. The association among endogenous estrogen, estrogen metabolites, and microbial dynamics on disease pathogenesis has not been fully investigated. Here, we identified estrogen metabolites as well as microbial phenotypes in non-diseased patients (n = 9) and those with pathologically confirmed endometriosis (P-EOSIS, n = 20), on day of surgery (DOS) and ~1–3 weeks post-surgical intervention (PSI). Then, we examined the effects of surgical intervention with or without hormonal therapy (OCPs) on estrogen and microbial profiles of both study groups. For estrogen metabolism analysis, liquid chromatography/tandem mass spectrometry was used to quantify urinary estrogens. The microbiome data assessment was performed with Next generation sequencing to V4 region of 16S rRNA. Surgical intervention and hormonal therapy altered gastrointestinal (GI), urogenital (UG) microbiomes, urinary estrogen and estrogen metabolite levels in P-EOSIS. At DOS, 17β-estradiol was enhanced in P-EOSIS treated with OCPs. At PSI, 16-keto-17β-estradiol was increased in P-EOSIS not receiving OCPs while 2-hydroxyestradiol and 2-hydroxyestrone were decreased in P-EOSIS receiving OCPs. GI bacterial α-diversity was greater for controls and P-EOSIS that did not receive OCPs. P-EOSIS not utilizing OCPs exhibited a decrease in UG bacterial α-diversity and differences in dominant taxa, while P-EOSIS utilizing OCPs had an increase in UG bacterial α-diversity. P-EOSIS had a strong positive correlation between the GI/UG bacteria species and the concentrations of urinary estrogen and its metabolites. These results indicate an association between microbial dysbiosis and altered urinary estrogens in P-EOSIS, which may impact disease progression.

Urinary estrogen metabolites and breast cancer risk in Chinese population

Background: In China, the association between estrogen metabolism and breast cancer risk and the differences in metabolic pattern between breast cancer patients and controls are poorly understood. Methods: A total of 84 patients with invasive breast cancer and 47 controls with benign breast diseases were included in this study. Estrogen metabolites from their morning urine were determined by HPLC-MS/MS and evaluated in both groups, and the predictive value of each estrogen metabolite in the malignant group according to their menstrual status was analyzed. Results: Urinary concentration of estrogen metabolites 2-OHE1, 2-OHE2, 4-OHE2, 4-MeOE1, and 16ɑ-OHE1 were lower in postmenopausal patients with breast cancer, compared with benign controls, In logistic regression model, breast cancer risk increased with the decline in the levels of 4-OHE2 and 4-MeOE1. In premenopausal patients,, a difference in the level of 2-OHE2 was observed between both groups, and 2-OHE2 was found to have predictive value for breast cancer. Additionally, urinary 2-OHE2 level in premenopausal HR+ patients was considerably higher compared with HR- patients. Conclusions: We found that lower urinary levels of 4-OHE2 and 4-MeOE1had predictive value for breast cancer, and higher 2-OHE1 were associated with HR+ breast cancer in premenopausal women.

Significance of pro-angiogenic estrogen metabolites in normal follicular development and follicular growth arrest in polycystic ovary syndrome

STUDY QUESTION Do alterations in pro- and anti-angiogenic estrogen metabolites in follicular fluid (FF) contribute to the follicular growth arrest and anovulation associated with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER FF of PCOS women with anovulation have reduced levels of pro-angiogenic estrogen metabolites (EMs) and vascular endothelial growth factor (VEGF) compared to that of fertile women with regular menstrual cycles, but exogenous gonadotropins increase the pro-angiogenic EMs and VEGF levels in PCOS women. WHAT IS KNOWN ALREADY PCOS is characterized by the arrest of follicular development that leads to chronic anovulation. Follicular arrest is generally associated with elevated plasma levels of luteinizing hormone (LH), androgens and anti-Mullerian hormone (AMH). There is also reduced angiogenesis in the follicles of PCOS women compared to those of normal cycling women. It is known that angiogenesis is a critical factor during follicular development. We and other investigators have explored the role of EMs in ovarian angiogenesis, particularly in human corpus luteum function, showing that 4-hydroxyestrone (4-OHE1) and 16-ketoestradiol (16-kE2) have pro-angiogenic effects while 2-methoxyestradiol (2-ME2) and 2-methoxyestrone (2-ME1) have anti-angiogenic effects. Additionally, 2-hydroxyestradiol (2-OHE2), which is produced in the ovary, has proliferative and pro-angiogenic properties. We hypothesized that EMs could be involved in angiogenesis necessary for ovarian follicular development in fertile women, and that dysregulation of these factors may contribute to follicular arrest in PCOS. The relationship between EMs, VEGF and AMH in the pathophysiology of follicular arrest in PCOS has not been previously studied at a follicular level in anovulatory women without ovulation induction. STUDY DESIGN, SIZE, DURATION This is a comparative experimental study of serum and FF collected from different sized follicles (antral ˂10 mm and dominant ˃16 mm) of women with and without ovarian stimulation. The study included women with regular menstrual cycles who were proven to be fertile (n = 20) and PCOS women with follicular arrest who were candidates for ovarian drilling (n = 17), as well as other patients requiring ovarian stimulation, i.e. control women undergoing IVF for male factor infertility (n = 12) and PCOS women undergoing IVF (n = 17). In vitro studies were carried out on granulosa-lutein cells (GCs) obtained from subsets of women undergoing IVF for male factor infertility (n = 6) and PCOS women undergoing IVF (n = 6). GCs were maintained in culture for up to 6 days. PARTICIPANTS/MATERIALS, SETTING, METHODS Intrafollicular estradiol, estrone and EMs concentrations were determined by high performance liquid chromatography–mass spectrometry. Testosterone in serum was measured by RIA, and LH, FSH and sex hormone-binding globulin in serum were measured with IRMA kits. AMH was determined in serum and FF by enzyme linked immunosorbant assay (ELISA). VEGF levels were measured in FF and conditioned medium by ELISA. Conditioned medium were obtained from cultured GCs. The angiogenic potential was assessed by in vitro angiogenic assays. MAIN RESULTS AND THE ROLE OF CHANCE Pro-angiogenic EMs (4-OHE1, 16-kE2 and 2-OHE2) and VEGF were lower in FF of antral follicles of PCOS women with follicular arrest compared those of fertile women with ovulatory cycles (P < 0.05). In contrast, higher concentrations of AMH were found in FF of antral follicles from PCOS women with follicular arrest compared to those of fertile women with ovulatory cycles (P < 0.05). Exogenous gonadotropins used in IVF increased pro-angiogenic EMs and VEGF production in PCOS women, reaching similar profiles compared to control women receiving gonadotropins in their IVF treatment for male factor infertility. The pro-angiogenic EM 2-OHE2 increased the angiogenic potential and VEGF levels of GCs from PCOS women compared to the basal condition (P < 0.05). These findings suggest that there is a role for pro-angiogenic EMs in the control of follicular VEGF production. LIMITATIONS, REASONS FOR CAUTION The limitations include the possibility that in vitro analysis of GCs might not reflect the in vivo mechanisms involved in the pro-angiogenic action of 2-OHE2 since GCs obtained at the time of oocyte retrieval belong to a very early stage of the luteal phase and might not be representative of GCs during follicular growth. Therefore, our findings do not conclusively rule out the possibility that other in vivo mechanisms also account for defective angiogenesis observed in PCOS. WIDER IMPLICATIONS OF THE FINDINGS The present study highlights the significance of EMs, angiogenic factors and AMH and their interaction in the pathophysiology of follicular development in PCOS. This study provides new insights into the role of pro-angiogenic factors in follicular arrest in PCOS. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by CONICYT/FONDECYT 1140693 and NIH grant R01HD083323. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A

Urinary Estrogen Metabolites and Long-Term Mortality Following Breast Cancer

Background Estrogen metabolite concentrations of 2-hydroxyestrone (2-OHE1) and 16-hydroxyestrone (16-OHE1) may be associated with breast carcinogenesis. However, no study has investigated their possible impact on mortality after breast cancer. Methods This population-based study was initiated in 1996–1997 with spot urine samples obtained shortly after diagnosis (mean = 96 days) from 683 women newly diagnosed with first primary breast cancer and 434 age-matched women without breast cancer. We measured urinary concentrations of 2-OHE1 and 16-OHE1 using an enzyme-linked immunoassay. Vital status was determined via the National Death Index (n = 244 deaths after a median of 17.7 years of follow-up). We used multivariable-adjusted Cox proportional hazards to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the estrogen metabolites-mortality association. We evaluated effect modification using likelihood ratio tests. All statistical tests were two-sided. Results Urinary concentrations of the 2-OHE1 to 16-OHE1 ratio (>median of 1.8 vs ≤median) were inversely associated with all-cause mortality (HR = 0.74, 95% CI = 0.56 to 0.98) among women with breast cancer. Reduced hazard was also observed for breast cancer mortality (HR = 0.73, 95% CI = 0.45 to 1.17) and cardiovascular diseases mortality (HR = 0.76, 95% CI = 0.47 to 1.23), although the 95% confidence intervals included the null. Similar findings were also observed for women without breast cancer. The association with all-cause mortality was more pronounced among breast cancer participants who began chemotherapy before urine collection (n = 118, HR = 0.42, 95% CI = 0.22 to 0.81) than among those who had not (n = 559, HR = 0.98, 95% CI = 0.72 to 1.34; Pinteraction = .008). Conclusions The urinary 2-OHE1 to 16-OHE1 ratio may be inversely associated with long-term all-cause mortality, which may depend on cancer treatment status at the time of urine collection.

Steroidomics for the Prevention, Assessment, and Management of Cancers: A Systematic Review and Functional Analysis

Steroidomics, an analytical technique for steroid biomarker mining, has received much attention in recent years. This systematic review and functional analysis, following the PRISMA statement, aims to provide a comprehensive review and an appraisal of the developments and fundamental issues in steroid high-throughput analysis, with a focus on cancer research. We also discuss potential pitfalls and proposed recommendations for steroidomics-based clinical research. Forty-five studies met our inclusion criteria, with a focus on 12 types of cancer. Most studies focused on cancer risk prediction, followed by diagnosis, prognosis, and therapy monitoring. Prostate cancer was the most frequently studied cancer. Estradiol, dehydroepiandrosterone, and cortisol were mostly reported and altered in at least four types of cancer. Estrogen and estrogen metabolites were highly reported to associate with women-related cancers. Pathway enrichment analysis revealed that steroidogenesis; androgen and estrogen metabolism; and androstenedione metabolism were significantly altered in cancers. Our findings indicated that estradiol, dehydroepiandrosterone, cortisol, and estrogen metabolites, among others, could be considered oncosteroids. Despite noble achievements, significant shortcomings among the investigated studies were small sample sizes, cross-sectional designs, potential confounding factors, and problematic statistical approaches. More efforts are required to establish standardized procedures regarding study design, analytical procedures, and statistical inference.