Associations of Circulating Estrogens and Estrogen Metabolites with Fecal and Oral Microbiome in Postmenopausal Women in the Ghana Breast Health Study.

The human fecal and oral microbiome may play a role in the etiology of breast cancer through modulation of endogenous estrogen metabolism. This study aimed to investigate associations of circulating estrogens and estrogen metabolites with the fecal and oral microbiome in postmenopausal African women. A total of 117 women with fecal (N = 110) and oral (N = 114) microbiome data measured by 16S rRNA gene sequencing, and estrogens and estrogen metabolites data measured by liquid chromatography tandem mass spectrometry were included. The outcomes were measures of the microbiome and the independent variables were the estrogens and estrogen metabolites. Estrogens and estrogen metabolites were associated with the fecal microbial Shannon index (global P < 0.01). In particular, higher levels of estrone (ß = 0.36, P = 0.03), 2-hydroxyestradiol (ß = 0.30, P = 0.02), 4-methoxyestrone (ß = 0.51, P = 0.01), and estriol (ß = 0.36, P = 0.04) were associated with higher levels of the Shannon index, while 16alpha-hydroxyestrone (ß = -0.57, P < 0.01) was inversely associated with the Shannon index as indicated by linear regression. Conjugated 2-methoxyestrone was associated with oral microbial unweighted UniFrac as indicated by MiRKAT (P < 0.01) and PERMANOVA, where conjugated 2-methoxyestrone explained 2.67% of the oral microbial variability, but no other estrogens or estrogen metabolites were associated with any other beta diversity measures. The presence and abundance of multiple fecal and oral genera, such as fecal genera from families Lachnospiraceae and Ruminococcaceae, were associated with several estrogens and estrogen metabolites as indicated by zero-inflated negative binomial regression. Overall, we found several associations of specific estrogens and estrogen metabolites and the fecal and oral microbiome. IMPORTANCE Several epidemiologic studies have found associations of urinary estrogens and estrogen metabolites with the fecal microbiome. However, urinary estrogen concentrations are not strongly correlated with serum estrogens, a known risk factor for breast cancer. To better understand whether the human fecal and oral microbiome were associated with breast cancer risk via the regulation of estrogen metabolism, we conducted this study to investigate the associations of circulating estrogens and estrogen metabolites with the fecal and oral microbiome in postmenopausal African women. We found several associations of parent estrogens and several estrogen metabolites with the microbial communities, and multiple individual associations of estrogens and estrogen metabolites with the presence and abundance of multiple fecal and oral genera, such as fecal genera from families Lachnospiraceae and Ruminococcaceae, which have estrogen metabolizing properties. Future large, longitudinal studies to investigate the dynamic changes of the fecal and oral microbiome and estrogen relationship are needed.

Fabrication of Ce-doped DUT-52 as a sorbent for dispersive solid phase extraction of estrogens in human urine samples.

A cerium (Ce)-doped metal-organic framework composite (Ce/DUT-52) was prepared by using a solvothermal method and was explored as a sorbent for dispersive solid phase extraction (DSPE) of three estrogens (α-estradiol, estrone, and hexestrol) in human urine samples. After doping with Ce(III), Ce/DUT-52 exhibited more attractive features involving a higher specific surface area (774.7 m2 g-1) and zeta potential (31.4 mV), which made it an efficient adsorbent for the separation and enrichment of estrogens. The factors influencing DSPE efficiency such as the adsorbent amount, extraction time, pH, NaCl concentration, elution solvent and elution volume were investigated in detail. Under the evaluated conditions, Ce/DUT-52 showed good reusability (n = 6, RSDs ≤ 4.8%). Notably, the cofunction of electrostatic interaction, hydrophobic interaction, hydrogen bonding and π-π interaction might play major roles between estrogens and Ce/DUT-52. Finally, coupled with high-performance liquid chromatography (HPLC), a fast and sensitive method was established, which provided low limits of detection (1.5-2.0 ng mL-1), wide linear ranges (3-500 ng mL-1) and satisfactory recoveries (79.8-96.1%). The results demonstrated that Ce/DUT-52 had excellent adsorption ability to the targets and the developed method provided an alternative strategy for the determination of trace estrogens or other compounds with similar chemical structures in urine samples.

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, 17ß-estradiol was enhanced in P-EOSIS treated with OCPs. At PSI, 16-keto-17ß-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 equol levels are positively associated with urinary estradiol excretion in women.

Isoflavones found in soy products are a promising class of nutrients that may have a positive effect on human health. In particular, the phytoestrogen metabolite equol is associated with a reduced risk of developing female hormone-related diseases. However, the effect of equol on estrogen remains unclear. Equol can modify blood and urinary estradiol (E2) levels. The aim of this cross-sectional study was to examine the associations between urinary estrogen levels, equol levels, and equol production status in Japanese women. We analyzed urine samples from 520 women by gas chromatography-mass spectrometry. Urinary E2 and 4-hydroxylated E2 levels were higher in equol producers (EQP) than in non-EQPs (P < 0.0001 and P=0.00112, respectively). After adjusting for age and tobacco use by analysis of covariance, the association remained significant (ß = 0.299, P < 0.0001). Analysis of covariance demonstrated that equol levels in urine were also positively associated with urinary E2 (ß = 0.597, P < 0.0001). The log equol concentration showed a significant, but moderate, negative association with the serum E2 concentration (ß = - 0.0225, P = 0.0462). Our findings suggest that equol may promote urinary E2 excretion and modify blood E2 levels in women.

Optimization and validation of a GC-MS quantitative method for the determination of an extended estrogenic profile in human urine: Variability intervals in a population of healthy women.

An analytical method based on GC-MS was developed for the determination of a wide panel of urinary estrogens, together with their principal metabolites. Because of the low concentration of estrogens in urine, an efficient sample pre-treatment was optimized by a design of experiment (DoE) procedure to achieve satisfactory sensitivity. A second DoE was built for the optimization of the chromatographic run, with the purpose of reaching the most efficient separation of analytes with potentially interfering ions and similar chromatographic properties. The method was fully validated using a rigorous calibration strategy: from several replicate analyses of blank urine samples spiked with the analytes, calibration models were built with particular attention to the study of heteroscedasticity and quadraticity. Other validation parameters, including the limit of detection, intra-assay precision and accuracy, repeatability, selectivity, specificity, and carry-over, were obtained using the same set of data. Further experiments were performed to evaluate matrix effect and extraction recovery. Then the urinary estrogen profiles of 138 post-menopausal healthy women were determined. These profiles provide a representation of physiological concentration ranges, which, in forthcoming studies, will be matched on the base of multivariate statistics with the urinary estrogenic profile of women with breast or ovarian cancer.

Preparation of porous polymers based on high internal phase emulsion for enrichment of estrogens in urine.

In this work, graphene oxide-hybridized high internal emulsion polymers with crosslinking and open-cell structure was prepared and applied for separation and enrichment of estrogens. The prepared graphene oxide-hybridized high internal emulsion polymer monoliths had hydrophobicity, porosity and stability, which were just obtained by one step in-situ emulsion polymerization of 2-ethylhexyl acrylate, glycidyl methacrylate, and divinylbenzene after doping with graphene oxide. Benefit from the advantages of its unique character, the graphene oxide-hybridized high internal emulsion polymers monolith with low background pressure (85 kPa) and high mechanical strength could be applied for efficient separation for trace estrogens in urine. Under the optimized condition, trace estrogens, including estrone, estradiol, and diethylstilbestrol in urine, were detected by high-performance liquid chromatography, all the sample preparation process were carried out in 15 min, the recovery rate was ranged from 85.0 to 106.0% and the relative standard deviation was less than 4.

Estrogen Signaling and Portopulmonary Hypertension: The Pulmonary Vascular Complications of Liver Disease Study (PVCLD2).

BACKGROUND AND AIMS: Portopulmonary hypertension (POPH) was previously associated with a single-nucleotide polymorphism (SNP) rs7175922 in aromatase (cytochrome P450 family 19 subfamily A member 1 [CYP19A1]). We sought to determine whether genetic variants and metabolites in the estrogen signaling pathway are associated with POPH. APPROACH AND RESULTS: We performed a multicenter case-control study. POPH patients had mean pulmonary artery pressure >25 mm Hg, pulmonary vascular resistance >240 dyn-sec/cm-5 , and pulmonary artery wedge pressure ≤15 mm Hg without another cause of pulmonary hypertension. Controls had advanced liver disease, right ventricular (RV) systolic pressure <40 mm Hg, and normal RV function by echocardiography. We genotyped three SNPs in CYP19A1 and CYP1B1 using TaqMan and imputed SNPs in estrogen receptor 1 using genome-wide markers. Estrogen metabolites were measured in blood and urine samples. There were 37 patients with POPH and 290 controls. Mean age was 57 years, and 36% were female. The risk allele A in rs7175922 (CYP19A1) was significantly associated with higher levels of estradiol (P = 0.02) and an increased risk of POPH (odds ratio [OR], 2.36; 95% confidence interval [CI], 1.12-4.91; P = 0.02) whereas other SNPs were not. Lower urinary 2-hydroxyestrogen/16-α-hydroxyestrone (OR per 1-ln decrease = 2.04; 95% CI, 1.16-3.57; P = 0.01), lower plasma levels of dehydroepiandrosterone-sulfate (OR per 1-ln decrease = 2.38; 95% CI, 1.56-3.85; P < 0.001), and higher plasma levels of 16-α-hydroxyestradiol (OR per 1-ln increase = 2.16; 95% CI, 1.61-2.98; P < 0.001) were associated with POPH. CONCLUSIONS: Genetic variation in aromatase and changes in estrogen metabolites were associated with POPH.

Reproductive hormone measurement from minimally invasive sample types: Methodological considerations and anthropological importance.

Energetic investment in human reproduction has long been recognized as costly, influencing developmental, physiological, and behavioral patterns in males and females. These effects are largely coordinated through the actions of reproductive hormones (eg, testosterone, estradiol, and progesterone). Here, the utility and limitations of minimally invasive sampling techniques are explored, providing a novel perspective on how reproductive hormone measurements can enhance reproductive endocrinology research. Salivary steroid measures are most commonly used, although several dried blood spot and urine assays are also available, and researchers continue to explore the efficacy of other sample types. These relatively simple measures have facilitated the collection of multiple samples from a single participant, allowing researchers to more accurately track the diurnal and cyclical variation exhibited by many reproductive hormones. Ultimately, the ability to collect fine-grained participant data allows biological anthropologists to better test questions central to human reproductive ecology, life history theory, and public health. For example, fieldwork using these techniques suggests that testosterone profile variation across populations is influenced by energetic constraints and reproductive status. Moreover, hormone concentrations shape the development of sex characteristics, with implications for evolutionary questions related to sexual selection. Hormone levels also can be used to identify a range of medical concerns (eg, suppressed hormone production levels linked with psychosocial stress). These findings highlight how minimally invasive collection techniques can be applied to test diverse evolutionary hypotheses and identify important health concerns. Still, more work is needed to standardize collection and laboratory analysis procedures, thereby enabling more direct data comparisons between researchers.

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.

An emerging powerful technique for distinguishing isomers: Trapped ion mobility spectrometry time-of-flight mass spectrometry for rapid characterization of estrogen isomers.

RATIONALE: Isomer metabolites are involved in metabolic pathways, and their characterization is essential but remains challenging even using high-performance analytical platforms. The addition of ion mobility prior to mass analysis can help to separate isomers. Here, the ability of a recently developed trapped ion mobility spectrometry system to separate metabolite isomers was examined. METHODS: Three pairs of estrogen isomers were studied as a model of isomeric metabolites under both negative and positive electrospray ionization (ESI) modes using a commercial trapped ion mobility spectrometry-TOF mass spectrometer. The standard metabolites were also spiked into human urine to evaluate the efficiency of trapped ion mobility spectrometry to separate isomers in complex mixtures. RESULTS: The estradiol glucuronide isomers (E2 ß-3G and E2 ß-17G) could be distinguished as deprotonated species, while the estradiol epimers (E2 ß and E2 α) and the methoxyestradiol isomers (2-MeO-E2 ß and 4-MeO-E2 ß) were separated as lithiated adducts in positive ionization mode. When performing analyses in the urine matrix, no alteration in the ion mobility resolving power was observed and the measured collision cross section (CCS) values varied by less than 1.0%. CONCLUSIONS: The trapped ion mobility spectrometry-TOF mass spectrometer enabled the separation of the metabolite isomers with very small differences in CCS values (ΔCCS% = 2%). It is shown to be an effective tool for the rapid characterization of isomers in complex matrices.

Peri-implantation urinary hormone monitoring distinguishes between types of first-trimester spontaneous pregnancy loss.

BACKGROUND: Lutenising hormone (LH) and human chorionic gonadotropin (hCG) hormone are useful biochemical markers to indicate ovulation and embryonic implantation, respectively. We explored "point-of-care" LH and hCG testing using a digital home-testing device in a cohort trying to conceive. OBJECTIVE: To determine conception and spontaneous pregnancy loss rates, and to assess whether trends in LH-hCG interval which are known to be associated with pregnancy viability could be identified with point-of-care testing. METHODS: We recruited healthy women aged 18-44 planning a pregnancy. Participants used a home monitor to track LH and hCG levels for 12 menstrual cycles or until pregnancy was conceived. Pregnancy outcomes (viable, clinical miscarriage, or biochemical pregnancy loss) were recorded. Monitor data were analysed by a statistician blinded to pregnancy outcome. RESULTS: From 387 recruits, there were 290 pregnancies with known outcomes within study timeline. Adequate monitor data for analysis were available for 150 conceptive cycles. Overall spontaneous first-trimester pregnancy loss rate was 30% with clinically recognised miscarriage rate of 17%. The difference to LH-hCG interval median had wider spread for biochemical losses (0.5-8.5 days) compared with clinical miscarriage (0-5 days) and viable pregnancies (0-6 days). Fixed effect hCG profile change distinguished between pregnancy outcomes from as early as day-2 post-hCG rise from baseline. CONCLUSIONS: The risk of first-trimester spontaneous pregnancy loss in our prospective cohort is comparable to studies utilising daily urinary hCG collection and laboratory assays. A wider LH-hCG interval range is associated with biochemical pregnancy loss and may relate to late or early implantation. Although early hCG changes discriminate between pregnancies that will miscarry from viable pregnancies, this point-of-care testing model is not sufficiently developed to be predictive.

Sexual Dimorphism in Titi Monkeys' Digit (2D:4D) Ratio is Associated with Maternal Urinary Sex Hormones During Pregnancy.

The second-to-fourth digit (2D:4D) ratio is a sexually-dimorphic biomarker for prenatal sex hormone exposure. We investigated whether titi monkeys (Plecturocebus cupreus) exhibit sexually-dimorphic 2D:4D ratio, and whether variation in 2D:4D ratio correlates with maternal testosterone and estrogen levels during early pregnancy. Subjects were 61 adult titi monkeys (32 males, 29 females). For 26 subjects, maternal urine samples were collected approximately 15-20 weeks before birth and assayed for testosterone and estrone conjugate (E1 C). Titi monkeys exhibited a human-like pattern of sexual dimorphism in right-hand 2D:4D ratio, with females exhibiting higher 2D:4D ratio than males (ß = -0.29, p = 0.023). For left-hand 2D:4D ratio, high levels of maternal E1 C predicted low offspring 2D:4D ratio (ß = -0.48, p = 0.009). For right-hand 2D:4D ratio, high levels of testosterone (ß = -0.53, p = 0.005) and testosterone-to-E1 C ratio (ß = -0.41, p = 0.028) predicted low offspring 2D:4D ratio. For 2D:4D ratio asymmetry (right-hand - left-hand), high levels of testosterone (ß = -0.43, p = 0.03) and testosterone-to-E1 C ratio (ß = -0.53, p = 0.003) predicted low (right-biased) asymmetry. This is the first report of sexually-dimorphic 2D:4D ratio in New World monkeys, and the results support a growing literature suggesting prenatal sex hormones may modulate offspring 2D:4D ratio.

Development and validation of LC-ESI-MS/MS methods for quantification of 27 free and conjugated estrogen-related metabolites.

An imbalance in the estrogen metabolism has been associated with an increased risk of breast cancer development. Evaluation of the estrogen biotransformation capacity requires monitoring of various estrogen metabolites. Up to now, only some estrogen metabolites could be measured in urine. However, in order to offer tailor made nutritional support or therapies, a complete estrogen metabolite profile is required in order to identify specific deficiencies in this pathway for each patient individually. Here, we focused on this need to quantify as many as possible of the estrogen-related metabolites excreted in urine. The method was developed to quantify 27 estrogen-related metabolites in small urine quantities. This entailed sample clean-up with a multi-step solid phase extraction procedure, derivatisation of the metabolites in the less water-soluble fraction through dansylation, and analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The metabolites accurately quantified by the method devised included parent estrogens, hydroxylated and methylated forms, metabolites of the 16α-hydroxyestrogen pathway, sulphate and glucuronide conjugated forms, precursors and a related steroid hormone. This method was validated and enabled quantification in the high picograms and low nanograms per millilitre range. Finally, analyses of urine samples confirmed detection and quantification of each of the metabolites.

Urinary estrogens as a non-invasive biomarker of viable pregnancy in the giant panda (Ailuropoda melanoleuca).

Female giant pandas show complex reproductive traits, being seasonally monoestrus, displaying a variable length embryonic diapause and exhibiting pseudopregnancy. Currently, there is no confirmatory non-invasive biomarker of blastocyst implantation or pregnancy. This study aimed to monitor urinary estrogens across gestation in pregnancy (n = 4), pseudopregnancy (n = 4) and non-birth cycles (n = 5) in the giant panda. A pregnancy-specific profile of estrogens corrected for urinary specific gravity was identified during the gestation period. Pregnant females showed increasing concentrations of estrogens for 29 days until birth, no increase was observed during pseudopregnancy and the two profiles were distinguishable from each other for the final 2 weeks of the cycle suggesting the estrogens are of placental origin. This allowed a nomogram, starting at a known fixed point during the cycle, to be created and tested with cycles of known outcome, and cycles which were inseminated but did not result in a birth. Non-birth profiles showed deviations from that of pregnancy. We believe these deviations indicate the point of failure of the placenta to support a developing cub. Non-invasive longitudinal monitoring of estrogen concentrations therefore has the potential to be developed as a panda pregnancy test to predict viable cub development.

A Generic Liquid Chromatography-Tandem Mass Spectrometry Exposome Method for the Determination of Xenoestrogens in Biological Matrices.

We are constantly exposed to a variety of environmental contaminants and hormones, including those mimicking endogenous estrogens. These highly heterogeneous molecules are collectively referred to as xenoestrogens and hold the potential to affect and alter the delicate hormonal balance of the human body. To monitor exposure and investigate potential health implications, comprehensive analytical methods covering all major xenoestrogen classes are needed but not available to date. Herein, we describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of multiple classes of endogenous as well as exogenous estrogens in human urine, serum, and breast milk to enable proper exposure and risk assessment. In total, 75 analytes were included, whereof a majority was successfully in-house validated in the three matrices. Extraction recoveries of validated analytes ranged from 71% to 110% and limits of quantification from 0.015 to 5 µg/L, 0.03 to 14 µg/L, and 0.03 to 4.6 µg/L in urine, serum, and breast milk, respectively. The applicability of the novel method was demonstrated in proof-of-principle experiments by analyzing urine from Austrian individuals and breast milk from Austrian and Nigerian individuals. Thereby, we proved the methods' feasibility to identify and quantify different classes of xenoestrogens simultaneously. The results illustrate the general importance of multiclass exposure assessment in the context of the exposome paradigm. Specifically, they highlight the need for estimating total estrogenic burden rather than single analyte or chemical class measurements and its potential impact in endocrine disruption and hormone related diseases including cancers.

Individual and cyclic estrogenic profile in women: Structure and variability of the data.

The concentration of estrogens in the body fluids of women is highly variable, due to the menstrual cycle, circadian oscillations, and other physiological and pathological causes. To date, only the cyclic fluctuations of the principal estrogens (estradiol and estrone) have been studied, with limited outcome of general significance. Aim of the present study was to examine in detail the cyclic variability of a wide estrogens' panel and to interpret it by multivariate statistics. Four estrogens (17α-estradiol, 17ß-estradiol, estrone, estriol) and eleven of their metabolites (4-methoxyestrone, 2-methoxyestrone, 16α-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestrone, 4-methoxyestradiol, 2-methoxyestradiol, 4-hydroxyestradiol, 2-hydroxyestradiol, estriol, 16-epiestriol, and 17-epiestriol) were determined in urine by a gas chromatography - mass spectrometry method, which was developed by design of experiments and fully validated according to ISO 17025 requirements. Then, urine samples collected every morning for a complete menstrual cycle from 9 female volunteers aged 24-35 years (1 parous) were analysed. The resulting three-dimensional data (subjects × days × estrogens) were interpreted using several statistical tools. Parallel Factor Analysis compared the estrogen profiles in order to explore the cyclic and inter-individual variability of each analyte. Principal Component Analysis (PCA) provided clear separation of the sampling days along the cycle, allowing discrimination among the luteal, ovulation, and follicular phases. The scores obtained from PCA were used to build a Linear Discriminant Analysis classification model which enhanced the recognition of the three cycle's phases, yielding an overall classification non-error rate equal to 90%. These statistical models may find prospective application in fertility studies and the investigation of endocrinology disorders and other hormone-dependent diseases.

Mammographic breast density and its association with urinary estrogens and the fecal microbiota in postmenopausal women.

BACKGROUND: Breast density, as estimated by mammography, is a strong risk factor for breast cancer in pre- and postmenopausal women, but the determinants of breast density have not yet been established. The aim of this study was to assess if urinary estrogens or gut microbiota alterations are associated with mammographic density in postmenopausal women. METHODS: Among 54 cancer-free, postmenopausal controls in the Breast and Colon Health study, we classified low- versus high-density women with Breast Imaging Reporting and Data System (BI-RADS, 5th edition) mammographic screening data, then assessed associations with urinary estrogens and estrogen metabolites (determined by liquid chromatography/tandem mass spectrometry), and fecal microbiota alpha and beta diversity (using Illumina sequencing of 16S rRNA amplicons). RESULTS: Multiple logistic regression revealed no significant association between breast density and fecal microbiota metrics (PD_tree P-value = 0.82; un-weighted and weighted UniFrac P = 0.92 and 0.83, respectively, both by MiRKAT). In contrast, total urinary estrogens (and all 15 estrogens/estrogen metabolites) were strongly and inversely associated with breast density (P = 0.01) after adjustment for age and body mass index. CONCLUSION: Mammographic density was not associated with the gut microbiota, but it was inversely associated with urinary estrogen levels. IMPACT: The finding of an inverse association between urinary estrogens and breast density in cancer-free women adds to the growing breast cancer literature on understanding the relationship between endogenous estrogens and mammographic density.

Bract as a novel extraction phase in thin-film SPME combined with 96-well plate system for the high-throughput determination of estrogens in human urine by liquid chromatography coupled to fluorescence detection.

In this study, an environmentally friendly and high-throughput method was developed for the determination of estrone (E1), 17ß-estradiol (E2), 17α-ethinylestradiol (EE2) and estriol (E3) in human urine by liquid chromatography-fluorescence detector (HPLC-FLD). A biosorbent (bract) was proposed as extraction phase for Thin-Film SPME combined with 96-well system. The characterization of the biosorbent was performed by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The optimizations were carried out through univariate and multivariate approaches with optimal conditions comprised of urine samples diluted 40-fold, liquid desorption performed in methanol and addition of 20% (w/v) of NaCl in the sample. Considering an extraction/desorption cycle using the 96-well plate system, the sample preparation time was 1.7 min per sample, which contributes to the high-throughput of the method proposed. The analytical parameters of merit were determined and satisfactory results were achieved, including limits of detection ranging from 0.3 µg L-1 for estradiol to 3 µg L-1 for estrone, while limits of quantification varied from 1 µg L-1 for estradiol to 10 µg L-1 for estrone. The correlation coefficients ranged from 0.9947 for estrone to 0.9999 for estriol. The accuracy and intra-assay and intermediate precisions (RSD) were evaluated through extractions in diluted urine samples (40-fold) spiked with each analyte (1, 200 and 400 µg L-1 for E3; 0.1, 200 and 400 µg L-1 for E2; 0.5, 200 and 400 µg L-1 for EE2 and 10, 200 and 400 µg L-1 for E1). The relative recoveries (n = 3) ranged from 71 to 105%, intra-assay precision (n = 3) varied from 1 to 17% and intermediate precision (n = 9) ranged from 2 to 19%. The method developed can be successfully used for the quantification of estrogens in human urine samples.

Urinary mycoestrogens and age and height at menarche in New Jersey girls.

BACKGROUND: Despite evidence of the endocrine disrupting properties of zearalenone (ZEN) and alpha-zearalanol (zeranol, α-ZAL), they have been minimally studied in human populations. In previous cross-sectional analyses, we demonstrated that 9-10 years old girls with detectable urinary ZEN were of shorter stature and less likely to have reached the onset of breast development than girls with undetectable urinary ZEN. The aim of this study was to examine baseline concentrations of ZEN, (α-ZAL), and their phase-1 metabolites in relation to subsequent growth and timing of menarche using 10 years of longitudinal data. METHODS: Urine samples were collected from participants in the Jersey Girl Study at age 9-10 (n = 163). Unconjugated ZEN, (α-ZAL), and their metabolites were analyzed using high performance liquid chromatography and tandem mass spectrometry. Information on height, weight, and pubertal development was collected at a baseline visit with annual follow-up by mail thereafter. Cox regression was used to evaluate time to menarche in relation to baseline ZEN, (α-ZAL), and total mycoestrogen exposure. Z-scores for height and weight were used in mixed models to assess growth. RESULTS: Mycoestrogens were detectable in urine in 78.5% of the girls (median ZEN: 1.02 ng/ml, range 0-22.3). Girls with detectable urinary concentrations of (α-ZAL) and total mycoestrogens (sum of ZEN, (α-ZAL) and their metabolites) at baseline were significantly shorter at menarche than girls with levels below detection (p = 0.04). ZEN and total mycoestrogen concentrations were inversely associated with height- and weight-z-scores at menarche (adjusted ß = - 0.18, 95% CI: -0.29, - 0.08, and adjusted ß = - 0.10, 95% CI: -0.21, 0.01, respectively). CONCLUSION: This study supports and extends our previous results suggesting that exposure to ZEN, (α-ZAL), and their metabolites is associated with slower growth and pubertal development in adolescent girls.

Reduction in FSH Throughout the Menstrual Cycle After Omega-3 Fatty Acid Supplementation in Young Normal Weight but not Obese Women.

Dietary fish oil restores ovarian function in subfertile rats, which is thought to be associated with decreased transcription of follicle-stimulating hormone (FSH) ß-subunit. We have previously demonstrated a reduction in early follicular serum FSH levels in normal weight but not obese women after treatment with omega-3 polyunsaturated fatty acids (PUFA). Herein, we report the effect of supplementation with omega-3 PUFA on urinary reproductive hormones across the whole menstrual cycle. This interventional study included 17 eumenorrheic women, aged 24-41 years. One month of daily morning urine was collected before and after 1 month of omega-3 PUFA supplementation with 4 g of eicosapentaenoic acid and docosahexaenoic acid daily. Measurements included urinary FSH, luteinizing hormone (LH) and estrogen and progesterone metabolites, plasma fatty acid composition, and markers of endoplasmic reticulum stress. Compliance with dietary supplementation was verified by significantly reduced ratios of omega-6 to omega-3 PUFA for all subjects after treatment (P < .01). After 1 month of omega-3 PUFA supplementation, urinary FSH was significantly decreased in normal weight, but not obese women, in both follicular and luteal phases (-28.4% and -12.6%, respectively, both P = .04). No significant changes were seen in LH or sex steroids for either weight group. The selective and specific decrease in FSH suggests that omega-3 PUFA supplementation merits further investigation in normal weight women with decreased fertility and/or diminished ovarian reserve.