Levels Of Estrogen Metabolites Research Articles

Associations of tubal ligation and hysterectomy with serum androgen and estrogen metabolites among postmenopausal women in the Women's Health Initiative Observational Study.

Hysterectomy is associated with subsequent changes in circulating hormone levels, but the evidence of an association for tubal ligation is unclear. We evaluated whether circulating concentrations of androgens and estrogens differ by tubal ligation or hysterectomy status in postmenopausal women from the Women's Health Initiative (WHI)-Observational Study (OS). Serum androgens and estrogens were measured in 920 postmenopausal women who did not use menopausal hormone therapy at the time of blood draw, of whom 139 self-reported a history of tubal ligation and 102 reported hysterectomy (with intact ovaries). Geometric mean hormone concentrations (GMs) and 95% confidence intervals (CIs) associated with a history of tubal ligation or hysterectomy (ever/never), as well as time since procedures, were estimated using adjusted linear regression with inverse probability of sampling weights to account for selection. Circulating levels of 12 androgen/androgen metabolites and 20 estrogen/estrogen metabolites did not differ by tubal ligation status. Among women reporting prior hysterectomy compared to women without hysterectomy, we observed lower levels of several androgens (e.g., testosterone (nmol/L): GMyes 0.46 [95% CI:0.37-0.57] vs. GMno 0.62 [95% CI:0.53-0.72]) and higher levels of estrogen metabolites, for example, 2-hydroxyestrone-3-methyl ether (GMyes 11.1 [95% CI:8.95-13.9] pmol/L vs. GMno 8.70 [95% CI:7.38-10.3]) and 4-methoxyestrone (GMyes 6.50 [95% CI:5.05-8.37] vs. GMno 4.92 [95% CI:4.00-6.05]). While we did not observe associations between prior tubal ligation and postmenopausal circulating hormone levels, our findings support that prior hysterectomy was associated with lower circulating testosterone levels and higher levels of some estrogen metabolites, which may have implications for future hormone-related disease risks.

Reference data on estrogen metabolome in healthy pregnancy

IntroductionEstrogen hormones and their metabolites are implicated in the maintenance of healthy pregnancy and adequate fetal development. Abnormal levels were related to increased risk of pregnancy complications, particularly preeclampsia. Our aims were (1) to develop a methodological platform for the comprehensive assessment of estrogen metabolome in pregnancy; (2) to collect healthy reference data for relevant elements of estrogen metabolome in each trimester; (3) to assess unconjugated fractions of the estrogen metabolome, (4) to assess the dominant metabolic pathways of estrogen compounds. MethodsWe enrolled healthy pregnant mothers between gestational week 5–15 (on the confirmation of pregnancy; 79 samples), gestational weeks 19–27 (70 samples), and gestational week 34–39 (54 samples). A method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to assess estrone, 17-beta-estradiol, estriol levels, and their metabolites as conjugated and unconjugated forms. Descriptive statistics were used to characterize the level of compounds in each trimester. ResultsEstrone, 17-beta-estradiol and estriol levels are dramatically increasing with the advancement of pregnancy. Measured levels were in a very wide range. 17-beta-estradiol is neither glucuronated nor sulphated. To the contrary, estriol and estrone are significantly conjugated; unconjugated fraction is <15% of total hormone levels in any trimester. Regarding metabolism, 4-methoxy-estradiol and 17-epiestriol were not detected. ConclusionWe concluded that (1) the levels of estrogen compounds and metabolites increase with advancing gestational age; (2) the wide ranges of levels challenge the establishment of a healthy reference range for clinical purposes; (3) 17-beta-estradiol is not conjugated significantly; (4) 4-methylation and 17-epimerization pathways of estrogens are negligible with our LC-MS/MS method.

Role of CB1 Cannabinoid Receptors in Vascular Responses and Vascular Remodeling of the Aorta in Female Mice.

Both the endocannabinoid system (ECS) and estrogens have significant roles in cardiovascular control processes. Cannabinoid type 1 receptors (CB1Rs) mediate acute vasodilator and hypotensive effects, although their role in cardiovascular pathological conditions is still controversial. Estrogens exert cardiovascular protection in females. We aimed to study the impact of ECS on vascular functions. Experiments were performed on CB1R knockout (CB1R KO) and wild-type (WT) female mice. Plasma estrogen metabolite levels were determined. Abdominal aortas were isolated for myography and histology. Vascular effects of phenylephrine (Phe), angiotensin II, acetylcholine (Ach) and estradiol (E2) were obtained and repeated with inhibitors of nitric oxide synthase (NOS, Nω-nitro-L-arginine) and of cyclooxygenase (COX, indomethacin). Histological stainings (hematoxylin-eosin, resorcin-fuchsin) and immunostainings for endothelial NOS (eNOS), COX-2, estrogen receptors (ER-α, ER-β) were performed. Conjugated E2 levels were higher in CB1R KO compared to WT mice. Vasorelaxation responses to Ach and E2 were increased in CB1R KO mice, attenuated by NOS-inhibition. COX-inhibition decreased Phe-contractions, while it increased Ach-relaxation in the WT group but not in the CB1R KO. Effects of indomethacin on E2-relaxation in CB1R KO became opposite to that observed in WT. Histology revealed lower intima/media thickness and COX-2 density, higher eNOS and lower ER-β density in CB1R KO than in WT mice. CB1R KO female mice are characterized by increased vasorelaxation associated with increased utilization of endothelial NO and a decreased impact of constrictor prostanoids. Our results indicate that the absence or inhibition of CB1Rs may have beneficial vascular effects.

Urine levels of estrogen and its metabolites in premenopausal middle-aged women with different degrees of obesity and their correlation with glucose and lipid metabolism

ObjectivesTo determine the levels of estrogen and estrogen metabolites in the urine of premenopausal women with obesity and their correlation with glucose and lipid metabolism. Methords135 premenopausal women were selected from the same area. According to the body mass index (BMI), they were divided into four different groups. High performance liquid chromatography-mass spectrometry (HPLC/MS) was adopted to detect the concentrations of estrogen and estrogen metabolites in the urine. The influencing factors of BMI were analyzed, the correlation between the urinary degrees of estrogen and estrogen metabolites and glucose and lipid metabolism levels was assessed. Results(1) The concentrations of 17β-estradiol (E2), estrone (E1), 16α-hydroxyestrone (16α-OHE1) and 2-hydroxyestrone (2-OHE1) gradually increased with increasing BMI (p < 0.05). (2) Stepwise regression analysis displayed that the concentrations of E2, 16α-OHE1 and 2-OHE1 in urine were significantly positively correlated with BMI (p < 0.05). (3) The concentrations of E2, E1, 16α-OHE1, 2-OHE1 and 16α-OHE1/2-OHE1 in urine were greatly positively related to fasting insulin (FIN), Triglyceride (TG), Total Cholesterol (TC) and Low-density lipoprotein (LDL) (p < 0.05). And they were greatly negatively related to High-density lipoprotein (HDL) (p < 0.05). ConclusionsEarly screening can reflect the degree of obesity and glucose and lipid metabolism disorders in premenopausal middle-aged women, thereby providing guidance for improving the prognosis of obese women.

Association of Urinary Levels of Estrogens and Estrogen Metabolites with the Occurrence and Development of Endometrial Hyperplasia Among Premenopausal Women.

Endometrial cancer is known to be an estrogen-dependent cancer, and it is believed that exposure to estrogens increases the risk of developing endometrial cancer in the absence of progesterone. Estrogens and estrogen metabolites may help to predict the risk of endometrial hyperplasia (EH) with atypia. Estrogens and estrogen metabolites were measured by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) in the first morning urine sample collected from 150 patients with EH and 50 healthy premenopausal women in the study. In healthy premenopausal women, the level of 16α-hydroxyestrone (16α-OHE1) in the overweight group was significantly higher than that in the lean group (p < 0.05). The levels of 4-hydroxyestradiol (4-OHE2) and 16α-OHE1 were elevated in the AEH group as compared to that in the control group (p < 0.05). Overweight is related to EH incidence as it causes an imbalance of estrogen metabolites. This study provides identifies potential biomarkers for estrogen-induced AEH.

Establishment and validation of a sensitive LC-MS/MS method for quantification of urinary estrogens in women with gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is not only a threat to the health of pregnant women, but also has profound effects on the health of offspring. Studies have shown that the imbalance of estrogen metabolism is associated with an increased risk of GDM. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established and validated for simultaneous quantification of thirteen estrogens in the urine of GDM women, including estrone (E1), estradiol (E2), estriol (E3), and their hydroxylated and methylated metabolites. The method was achieved on a Waters CORTECS C18 column (2.1 mm × 150 mm, 1.6 µm) within 8.5 min. The linear range of thirteen estrogens in urine was 2–1000 pg·mL−1. Both intra- and inter-day precision for each analyte were less than 15%, with accuracies ranging from 8.3% to 7.3%. The extraction recoveries rate were between 86% and 111%, and stability verification results met the requirements for determination of biological samples. The results suggested that the concentrations of estrogens in all urine samples range from 0.08 to 134.06 (pg·mg−1 creatinine). The mean levels of E1, E2 and most estrogen metabolites in the urine of GDM women were higher than those in healthy pregnant women. Notably, the mean level of 2-hydroxyestrone (2-OHE1) in GDM women was 13.2-fold lower than that in healthy pregnant women. The types of estrogens with the highest mean levels in the urine of GDM and healthy pregnant women were obviously different, which are 2-methoxyestrone (2MeOE1) and E3, respectively. Our results demonstrated that this specific and sensitive method is suitable for quantifying estrogens in human urine and could provide support for further research on estrogen-related pathological mechanisms in GDM and other diseases.

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.

Variations, validations, degradations, and noninvasive determination of pregnancy using fecal steroid metabolites in free-ranging pronghorn

Pregnancy status is a key parameter used to assess reproductive performance of a species as it represents a starting point for measuring vital rates. Vital rates allow managers to determine trends in populations such as neonate survival and recruitment; two important factors in ungulate population growth rates. Techniques to determine pregnancy have generally involved capture and restraint of the animal to obtain blood samples for determining serum hormone levels. Non-invasive pregnancy assessment, via feces, eliminates any hazards between handler and animal, as well as removes handling-induced physiological biases. Using noninvasive fecal sampling, we conducted hormone validations, investigated pregnancy rates, and determined hormone degradation rates across five subpopulations of pronghorn (Antilocapra americana) in Idaho. Samples were collected during April-May of 2018 and 2019 from adult pronghorn of known sex and age class. Metabolites of testosterone, cortisol, 17β-estradiol, and progesterone were measured in fecal samples, and concentrations of estradiol and progesterone were examined for pregnancy determination. Average fecal progesterone metabolite (FPM) levels of pregnant females were more than double compared to levels of nonpregnant females. Fecal estrogen metabolite (FEM) levels did not differ during concurrent sampling. The largest difference in FPM levels between pregnant and nonpregnant females began on 28 April. Pregnancy determination sampling showed average FPM levels for all five subpopulations were significantly different than the nonpregnant female validation group. Nonetheless, pregnancy rates for some subpopulations lacked conclusive estimates due to early fecal sampling. Fecal glucocorticoid metabolites (FGM) levels significantly differed between pregnant females and male pronghorn, but did not differ from nonpregnant females. Degradation rates of FPM and FGM differed across days, with values for FPM from Day 1 being significantly different from all subsequent days, and after Day 9 for FGM, demonstrating the requirement of fresh samples to accurately measure hormone concentrations. We concluded that a noninvasive method to diagnosis pregnancy is possible in pronghorn via progesterone metabolites if fresh samples are collected during late gestation.

Abstract PO029: Oral contraceptive use and postmenopausal sex steroid hormone metabolism

Abstract OBJECTIVE: Oral contraceptive (OC) use is common in the United States and high-profile articles have renewed research interest in OC use and cancer risk. Several mechanisms have been proposed to explain how OC use influences postmenopausal cancer risk; one of the leading hypotheses is that OC use fundamentally alters feedback in the hypothalamic-pituitary-gonad axis, leading to long term changes in sex steroid hormone metabolism. However, women who use OCs are also unique with respect to factors that may affect both hormone metabolism and cancer risk. No studies have comprehensively commented on hormone levels in postmenopausal women who did and did not use OCs. DESIGN: We examined differences in circulating markers of sex steroid hormone metabolism associated with prior use of OCs among a subcohort of postmenopausal women from the Women’s Health Initiative Observational Study (983 women with at least one intact ovary and not currently using menopausal hormone therapy). MATERIALS AND METHODS: Using highly sensitive liquid chromatography–tandem mass spectrometry assays, we measured over 30 markers of estrogen and androgen metabolism in study serum samples. We used linear regression (adjusted and weighted) to estimate geometric mean hormone levels, which were then converted into relative percent differences. We controlled for potential confounders and stratified by: lifetime ovulatory cycles, body mass index, and parity (the focus of this abstract). RESULTS: Women with a history of OC use (n=346) had lower levels of estrogen metabolites than women who never used OCs (n=637), particularly when limiting this comparison among parous women (n=158 nulliparous women, n=825 parous women). In parous women, OC use was associated with reductions in total estrone (-18.5%, 95% confidence interval [CI] -31.1, -3.7%) and in estrogen metabolism overall: 2-hydroxylation metabolites were reduced by -18.5% (CI -28.2, -7.7%), 4-hydroxylation metabolites by -19.9% (CI -29.4, -9.1%), and 16-alpha-hydroxylation metabolites by -24.0% (CI -34.2, -12.2%). Hormone differences associated with OC use among nulliparous women were imprecise. When we made comparisons to nulliparous women who did not use OCs, women who both used OCs and were parous had lower testosterone (-21.2%, CI -33.1, -7.2%) and higher dihydrotestosterone relative to testosterone (its metabolic precursor; 16.5%, CI% -0.6, 36.6). However, aside from lower total estrone (-25.5%, CI -44.6, 0.2%), estrogen metabolism was not different between these groups. CONCLUSIONS: We saw evidence suggesting estrogen metabolism, as reflected in circulating markers, may be lower in postmenopausal women who previously used OCs. These effects were strongest among parous women, likely due to sufficient sample size, rather than any synergistic effect attributable to both using OCs and giving birth. These results support the idea that OC users have lower estrogen levels, but this should be evaluated in other age groups, as differences in hormone metabolism early in the lifecourse may lead women to use OCs. Citation Format: Kara A. Michels, Sally B. Coburn, Garnet Anderson, Louise A. Brinton, Chu Chen, Roni T. Falk, Margery L. Gass, Ashley M. Geczik, JoAnn E. Manson, Ruth M. Pfeiffer, Kerryn Reding, Gloria E. Sarto, Nicolas Wentzensen, Robert A. Wild, Xia Xu, Britton Trabert. Oral contraceptive use and postmenopausal sex steroid hormone metabolism [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO029.

Relationship Between Calving Rate and Concentration of Hormones and Blood Metabolites During Pregnancy in Post-Induction Pasundan Cows GnRH

Twenty Pasundan heifers were used in the study to find out calving rates related to the concentration of hormones and metabolites of the mother's blood during the farm. The experimental mother cow was given a combination of the hormone prostaglandin F2α as much as 5 ml per head and gonadotropin realising hormone (GnRH) of 2.5 ml per head intramuscular to uniformize fertility conditions and improve fertility. The mother cow is immediately carried out artificial insemination 2 times with an interval of 6 hours. All experimental cows were given forage basalt food and adlibitum drinking water. Pregnancy examination is carried out on the 60th and 150th days of post-insemination using the rectal palpation method. The observed variables in the form of calving rate were related to plasma concentrations of the hormones progesterone (P), estrogen (E), and blood metabolites levels β-OH butyric (BHBA), blood urea nitrogen (BUN), and non-esterified fatty acids (NEFA). The results showed that in Garut calving average rate of 90%, the plasma concentration of hormone progesterone 4.73±0.28 ng/ml and estrogen 24.59±1.91 pg/ml higher than Bogor with an average calving rate of 70%, the plasma concentration of progesterone hormone 3.46±0.71ng/ml and estrogen 21.67±1.57 pg/ml. The concentration of BHBA, BUN, and NEFA for Bogor region respectively was 12.33±1.81 mg dl-1; 23.70±2.40 mg dL; 1.85±0.25 mmol L-1) higher than Garut in a row is 10.17±1.25*mg dl-1, 22.70±2.70mg dL; 1.76±0.37mmol L^-1). The results of regression analysis showed that the relationship of calving rate (CR) with the concentration of hormones conceptus estrogen (E) and progesterone (P) in garut region has a real form of linear relationship (P<0.05) by following the equation Calving rate=4772 +0.232 Estrogen + 0.643 Progesterone - 0.074 BHBA + 0.335 NEFA - 0.082 BUN with a coefficient of determination of 43.65%, while for Bogor region according to Calving regression rate=5,590+ 0.010 Estrogen +0.638 Progesterone - 0.231 BHBA + 1.11NEFA – 0.105 BUN with a coefficient of determination of 16.34%. It was concluded that calving rate is strongly influenced by the concentration of the hormones progesterone and estrogen, as well as having a close relationship with the condition of the mother blood metabolite during the pregnancy.

The role of 7-hydroxymatairezinol in modulation of estrogen metabolism and therapy for mastopathy

Quantitative estimates of the effect of 7-hydroxymatairesinol (7-HMR) intake on estrogen balance and clinical aspects of the course of fibrocystic mastopathy have been carried out.&#x0D; Aim. Assessment of effects 7-HMR on metabolism of estrogen (16a-OHE1, 2-OHE2, 2-OHE1, 2-OMEE1, 4-OMEE2, 4-OHE1, a ratio 2-OHE1/16a-OHE1, 2-OHE1/2-OMeE1 and 4-OHE1/4-OMeE1) and progesterone in daily urine at patients with fibrous and cystous mastopathy in the post-menopausal period.&#x0D; Materials and methods. The analysis of metabolites of estrogen (16a-OHE1, 2-OHE2, 2-OHE1, 2-OMEE1, 4-OMEE2, 4-OHE1, ratios 2-OHE1/16a-OHE1, 2-OHE1/2-OMeE1 and 4-OHE1/4-OMeE1) and progesterone in daily urine with use of solid-phase immunofermental set (IFA) of Amerscham International on the analyzer of Amerkard by means of standard sets is carried out. Statistical 10.0 and Microsoft Excel spreadsheets were used in statistical material processing.&#x0D; Results. It was found that taking 7-HMR (60 mg/day, 1 month) contributes to normalizing estrogen metabolism (decreasing estradiol, estrone, estriol, 16a-OHE1) and improves the clinical symptoms of mastopathy.&#x0D; Conclusions. In patients with FCM, 7-HMR contributes to a decrease in hyperestrogenia-related tumor risks by lowering estrogen metabolites levels.

Effect of menopausal hormone therapy on proteins associated with senescence and inflammation.

BackgroundEstrogen may inhibit cell senescence that contributes to age‐related disorders.This study determined the effects of menopausal hormone treatments on circulating levels of markers of cell senescence.MethodsGrowth differentiation factor 15 (GDF15), tumor necrosis factor receptor 1 (TNFR1), FAS, and macrophage inflammatory protein 1α (MIP1α) were measured in serum using multiplexed bead‐based assays and compared among menopausal women participating in the Kronos Early Estrogen Prevention Study randomized to either placebo (n = 38), oral conjugated equine estrogen (oCEE, n = 37), or transdermal 17β‐estradiol (tE2, n = 34). Serum levels of the senescent markers for each treatment were compared to placebo 36 months after randomization using the Wilcoxon rank sum test.ResultsSerum levels of GDF15, TNFR1, and FAS, but not MIP1α, were lower in both the oCEE and tE2 groups compared to placebo. The difference in levels between treatment and placebo for GDF15, TNFR1, and FAS were greater for oCEE [−108 pg/mL (p = .008), −234 pg/mL (p = .0006), and −1374 pg/mL (p < .0001), respectively] than for tE2 [−76 pg/mL (p = .072), −105 pg/mL (p = .076), and −695 pg/mL (p = .036), respectively]. Additionally, TNFR1 showed a positive association with time past menopause (correlation = 0.255, p = .019).ConclusionsCirculating levels of some markers of cell senescence were lower in menopausal women treated with oCEE and tE2 compared to placebo. Differences in the magnitude of effect of the two active treatments may reflect the differences in circulating levels of estrogen metabolites due to formulation and mode of delivery. These data generate new hypotheses with regard to the effects of menopause on the biology of aging.

LC-HRMS of derivatized urinary estrogens and estrogen metabolites in postmenopausal women

In order to undertake an epidemiologic study relating levels of parent estrogens (estrone and estradiol) and estrogen metabolites (EMs) to other breast cancer risk factors, we have optimized methods for EM quantification with ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). A two-step approach was adopted; the first step comprised method development and evaluation of the method performance. The second step consisted of applying this method to quantify estrogens in postmenopausal women and determine if the observed patterns are consistent with the existing literature and prior knowledge of estrogen metabolism. First, 1-methylimidazole-2-sulfonyl chloride (MIS) was used to derivatize endogenous estrogens and estrogen metabolites in urine from study participants. Since C18 reversed phase columns have not been able to separate all the structurally related EMs, we used a C18-pentafluorophenyl (PFP) column. The parent estrogens and EMs were baseline resolved with distinct retention times on this C18-PFP column using a 30 min gradient. This method was used to quantify the parent estrogens and 13 EMs in urine samples collected in an initial pilot study involving males as well as pre- and peri-menopausal females to assess a range of EM levels in urine samples and enable comparison to the previous literature for assay evaluation. Detection limits ranged from 1 − 20 pg/mL depending on the EM. We evaluated matrix effects and interference as well as the intra- and inter-batch reproducibility including hydrolysis, extraction, derivatization and LC-MS analysis using charcoal-stripped human urine as a matrix. Methods were then applied to the measurement of estrogens in urine samples from 169 postmenopausal women enrolled in an epidemiological study to examine relationships between breast cancer risk, the intestinal microbiome, and urinary EMs. The results from our cohort are comparable to previous reports on urinary EMs in postmenopausal women and enabled thorough evaluation of the method.

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

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)? 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. 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. 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. 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. 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. 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. 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. This study was funded by CONICYT/FONDECYT 1140693 and NIH grant R01HD083323. All authors declare no conflict of interest. N/A.

Cognitive markers of dementia risk in middle-aged women with bilateral salpingo-oophorectomy prior to menopause

Oophorectomy prior to menopause is associated with late-life dementia. Memory decline may start within 6 months after oophorectomy in middle-aged women, suggested by lower verbal and working memory performance. Unknown is whether such changes persist beyond 6 months, and whether they are reversed by estradiol. Short-term benefits of estradiol on verbal memory following oophorectomy were observed in one study, but longer term effects remain unknown. In the present study, middle-aged BRCA1/2 mutation carriers with early oophorectomy at least 1 year prior to study onset were tested on verbal and working memory with results stratified by (1) current estradiol use (n = 22) or (2) no history of estradiol use (n = 24), and compared to age-matched premenopausal controls (n = 25). Both memory abilities were adversely affected by oophorectomy, but only working memory was maintained by estradiol. Estrogen metabolite levels correlated with working memory, suggesting a role for estradiol in preserving this ability. Memory decline appears to persist after early oophorectomy, particularly for women who do not take estradiol.

REPEATED FREEZE-THAW CYCLES BUT NOT SHORT-TERM STORAGE OF FECAL EXTRACTS AT AMBIENT TEMPERATURE INFLUENCE THE STABILITY OF STEROID METABOLITE LEVELS IN CRESTED MACAQUES

The objective of this study was to examine the effects of repeated freeze-thaw cycles and short-term storage of fecal extracts at ambient temperature on the stability of fecal glucocorticoid (fGCM) and estrogen metabolite (fEM) levels from crested macaques.In total 100 aliquots of fecal extracts from fecal samples collected from female crested macaques (Macaca nigra) living at the Tangkoko-Batuangus Nature Reserve, North Sulawesi were used. We performed two different experiments: (1) An experiment to investigate if levels of fGCM and fEM measured from fecal extracts that were exposed to two, four, six and eight repeated freeze-thaw cycles (test groups) differ to control samples (i.e. fecal extracts always stored frozen); (2) An experiment to evaluate whether storing fecal extracts at ambient temperature for two, four, six, and eight days (test groups)affects the levels of fGCM and fEM compared to the control group (i.e. fecal extracts frozen immediately).Results showed that hormone levels were significantly increased (P&lt;0.05) after four freeze-thaw cycles for fGCM and after eight freeze-thaw cycles for fEM. By contrast, there was no significant difference (P&gt;0.05) in levels of fGCM and fEM between the test groups and the control group in fecal extracts stored at ambient temperature. In conclusion, our data show that more than two and six repeated freeze-thaw cycles should be avoided when measuring fGCM and fEM in crested macaque fecal extracts, respectively. We also demonstrate that storing fecal extracts at ambient temperature is possible for at least 8 days without taking a risk of affecting the stability of fGCM and fEM levels.

The biological and clinical relevance of estrogen metabolome

Considerable knowledge has been gathered on the physiological role of estrogens. However, fairly little information is available on the role of compounds produced in the breakdown process of estrone and estradiol wich may play a role in various diseases associated with estrogen impact. To date, approximately 15 extragonadal estrogen-related compounds have been identified. These metabolites may exert protective, or, instead, pro-inflammatory and/or pro-oncogenic activity in a tissue-specific manner. Systemic and local estrogen metabolite levels are not necesserily correlated, which may promote the diagnostic significance of the locally produced estrogen metabolites in the future. The aim of the present study is a bibliographic review of the extragonadal metabolome in peripheral tissues, and to highlight the role of the peripheral tissue homeostasis of estrogens as well as the non-hormonal biological activity and clinical significance of the estrogen metabolome. Orv Hetil. 2017; 158(24): 929-937.

Relation of Serum Estrogen Metabolites with Terminal Duct Lobular Unit Involution Among Women Undergoing Diagnostic Image-Guided Breast Biopsy.

Higher levels of circulating estrogens and estrogen metabolites (EMs) have been associated with higher breast cancer risk. In breast tissues, reduced levels of terminal duct lobular unit (TDLU) involution, as reflected by higher numbers of TDLUs and acini per TDLU, have also been linked to elevated breast cancer risk. However, it is unknown whether reduced TDLU involution mediates the risk associated with circulating EMs. In a cross-sectional analysis of 94 premenopausal and 92 postmenopausal women referred for clinical breast biopsy at an academic facility in Vermont, we examined the associations of 15 EMs, quantified using liquid chromatography-tandem mass spectrometry, with the number of TDLUs and acini count/TDLU using zero-inflated Poisson regression with a robust variance estimator and ordinal logistic regression models, respectively. All analyses were stratified by menopausal status and adjusted for potential confounders. Among premenopausal women, comparing the highest vs. the lowest tertiles, levels of unconjugated estradiol (risk ratio (RR) = 1.74, 95% confidence interval (CI) = 1.06-2.87, p trend = 0.03), 2-hydroxyestrone (RR = 1.74, 95% CI = 1.01-3.01, p trend = 0.04), and 4-hydroxyestrone (RR = 1.74, 95% CI = 0.99-3.06, p trend = 0.04) were associated with significantly higher TDLU count. Among postmenopausal women, higher levels of estradiol (RR = 2.09, 95% CI = 1.01-4.30, p trend = 0.04) and 16α-hydroxyestrone (RR = 2.27, 95% CI = 1.29-3.99, p trend = 0.02) were significantly associated with higher TDLU count. Among postmenopausal women, higher levels of EMs, specifically conjugated estrone and 2- and 4-pathway catechols, were also associated with higher acini count/TDLU. Our data suggest that higher levels of serum EMs are generally associated with lower levels of TDLU involution.

Circulating Estrogens and Postmenopausal Ovarian Cancer Risk in the Women's Health Initiative Observational Study.

Hormonal and reproductive factors contribute to the development of ovarian cancer, but few studies have examined associations between circulating estrogens and estrogen metabolites and ovarian cancer risk. We evaluated whether serum estrogens and estrogen metabolite levels are associated with ovarian cancer risk among postmenopausal women in a nested case-control study in the Women's Health Initiative (WHI) Observational Study (OS). We selected all 169 eligible epithelial ovarian cancer cases and 412 matched controls from women enrolled in WHI-OS who were not using menopausal hormones at baseline. Baseline levels of 15 estrogens and estrogen metabolites were measured via liquid chromatography/tandem mass spectrometry. Associations with ovarian cancer risk overall and stratified by histologic subtype (serous/nonserous) were analyzed using logistic regression. The mean time from serum collection to cancer diagnosis was 6.9 years. Overall, we observed modest ovarian cancer risk associations among women with higher levels of estrone [OR (95% confidence interval) quintile (Q)5 vs. Q1: 1.54 (0.82-2.90), Ptrend = 0.05], as well as 2- and 4-methoxyestrone metabolites [2.03 (1.06-3.88), Ptrend = 0.02; 1.86 (0.98-3.56), Ptrend = 0.01, respectively]. Associations of estrogens and estrogen metabolites varied substantially by histologic subtype. Associations with serous tumors were universally null, while estrone [2.65 (1.09-6.45), Ptrend = 0.01, Pheterogeneity = 0.04], unconjugated estradiol [2.72 (1.04-7.14), Ptrend = 0.03, Pheterogeneity = 0.02] and many of the 2-, 4-, and 16-pathway metabolites were positively associated with nonserous tumors. Our study provides novel molecular data showing an association of the parent estrogens and several estrogen metabolites with nonserous ovarian cancers. These findings further support the heterogeneous etiology of ovarian cancer. Cancer Epidemiol Biomarkers Prev; 25(4); 648-56. ©2016 AACR.

Noninvasive monitoring of female reproductive hormone metabolites in the endangered European mink (Mustela lutreola)

This study examined the reproductive physiology of female European mink (Mustela lutreola) to augment the available information on estrus, ovulation, and pregnancy with the long-term goal of supporting ex situ breeding management of this highly endangered species. Fecal reproductive hormone metabolites were measured using EIAs for estrogen and 20-oxo-pregnane metabolites. Seasonal hormone profiles were established. A comparison of hormone fluctuations in pregnant and nonpregnant females showed that both estrogen and 20-oxo-pregnane metabolites were significantly elevated during gestation, which is 42 days in length. Delayed implantation or embryonic diapause does not occur in this species. Litter size was correlated with 20-oxo-pregnane levels but not with estrogen concentrations. During lactation, 20-oxo-pregnane metabolite levels remained higher than in nonpregnant females. The breeding season was characterized by peaks in vaginal cornified cells and fecal estrogen metabolite levels. Up to four peaks in estrogen levels were identified and confirmed that European mink are seasonally polyestrous. The results of 20-oxo-pregnane measurements indicated that hCG can be applied to induce ovulation. With the establishment of this noninvasive method, we present a new tool to support population management of this species.