Comparison of Day-Specific Serum LH, Estradiol, and Progesterone with MiraTM Monitor Urinary LH, Estrone-3-glucuronide, and Pregnanediol-3-glucuronide Levels in Ovulatory Cycles.

Background and Objectives: Fertility tracking apps and devices are now currently available, but urinary hormone levels lack accuracy and sensitivity in timing the start of the 6-day fertile window and the precise 24 h interval of transition from ovulation to the luteal phase. We hypothesized the serum hormones estradiol (E2) and progesterone (P) might be better biomarkers for these major ovulatory cycle events, using appropriate mathematical tools. Materials and Methods: Four women provided daily blood samples for serum E2, P, and LH (luteinizing hormone) levels throughout their entire ovulatory cycles, which were indexed to the first day of dominant follicle (DF) collapse (defined as Day 0) determined by transvaginal sonography; therefore, ovulation occurred in the 24 h interval of Day -1 (last day of maximum diameter DF) to Day 0. For comparison, a MiraTM fertility monitor was used to measure daily morning urinary LH (ULH), estrone-3-glucuronide (E3G), and pregnanediol-3-glucuronide (PDG) levels in three of these cycles. Results: There were more fluctuations in the MiraTM hormone levels compared to the serum levels. Previously described methods, the Fertility Indicator Equation (FIE) and Area Under the Curve (AUC) algorithm, were tested for identifying the start of the fertile window and the ovulation/luteal transition point using the day-specific hormone levels. The FIE with E2 levels predicted the start of the 6-day fertile window on Day -7 (two cycles) and Day -5 (two cycles), whereas no identifying signal was found with E3G. However, both pairs of (E2, P) and (E3G, PDG) levels with the AUC algorithm signaled the Day -1 to Day 0 ovulation/luteal transition interval in all cycles. Conclusions: serum E2 and (E2, P) were better biomarkers for signaling the start of the 6-day fertile window, but both MiraTM and serum hormone levels were successful in timing the [Day -1, Day 0] ovulatory/luteal transition interval. These results can presently be applied to urinary hormone monitors for fertility tracking and have implications for the direction of future fertility tracking technology.

Urinary oestrogen steroidome as an indicator of the risk of localised prostate cancer progression.

BACKGROUND: Prostate cancer (PCa) is the most common cancer in North American men. Beyond the established contribution of androgens to disease progression, growing evidence suggest that oestrogen-related pathways might also be of clinical importance. The aim of this study was to explore the association of urinary oestrogen levels with clinical outcomes. METHODS: Urine samples from the prospective multi-institutional PROCURE cohort were collected before RP for discovery (n = 259) and validation (n = 253). Urinary total oestrogens (unconjugated + conjugated), including oestrone and oestradiol, their bioactive and inactive catechol and methyl derivatives (n = 15), were measured using mass spectrometry (MS). RESULTS: The median follow-up time for the discovery and replication cohorts was 7.6 and 6.5 years, respectively. Highly significant correlations between urinary oestrogens were observed; however, correlations with circulating oestrogens were modest. Our findings indicate that higher levels of urinary oestriol and 16-ketoestradiol were associated with lower risk of BCR. In contrast, higher levels of 2-methoxyestrone were associated with an increased risk of development of metastasis/deaths. CONCLUSIONS: Our data suggest that urinary levels of oestriol and 16-ketoestradiol metabolites are associated with a more favourable outcome, whereas those of 2-methoxyestrone are associated with an elevated risk of metastasis after RP. Further studies are required to better understand the impact of oestrogens on disease biology and as easily accessible urine-based risk-stratification markers.

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.

Seasonal transfer and quantification of urinary estradiol in the big brown bat (Eptesicus fuscus).

Growing evidence shows that sex steroids not only act within the individual whose glands produce them; they can also act on proximate conspecifics. Previous studies show that exogenous 17ß-estradiol (E2) can be absorbed both nasally and percutaneously, arriving in blood, neural, reproductive, and peripheral tissues. When male bats were injected with radiolabeled E2 (3H-E2) and housed with females during the mating season, radioactivity was reliably measured in the females' tissues. The present study was designed to compare E2 transfer from male to female bats at three time points in the annual reproductive cycle: spring (ovulation and fertilization), summer (maternal season), and autumn (mating season). Pairs of mature female bats were housed with a mature 3H-E2-treated male (50 µCi). Following 48 h of communal housing, radioactivity was measured in the tissues of female bats. Higher levels of radioactivity were present in the uterus and other tissues during the spring and autumn seasons compared to the summer season. We also measured natural levels of bioactive, unconjugated E2 in the urine of male bats using enzyme immunoassays, and found that it was present in all three seasons but at lower levels during the summer. Male-excreted E2 could transfer to females within the close confines of a roost, potentially influencing their reproductive physiology and behavior. These results suggest increased E2 transfer coincides with female reproduction, with urine as a likely vector. We suggest that sex steroid transfer among interacting individuals may explain several mammalian phenomena historically viewed as "pheromonal".

A multichannel system integrating molecularly imprinted conductive polymers for ultrasensitive voltammetric determination of four steroid hormones in urine.

This work reports on a modularized electrochemical method for the determination of the hormones cortisol, progesterone, testosterone and 17ß-estradiol in urine. These hormones were employed as templates when generating molecular imprints from aniline and metanilic acid by electropolymerization on the surface of screen-printed electrodes. The electrically conductive imprint was characterized by SEM, AFM and cyclic voltammetry. A four-channel system was then established to enable simultaneous determination of the hormones by cyclic voltammetry. The detection limits for cortisol, progesterone, testosterone and 17ß-estradiol are as low as 2, 2.5, 10 and 9 ag·mL-1 (for S/N = 3). Graphical abstract A four-channel system was established to enable simultaneous determination of 4 steroid hormones by cyclic voltammetry and by using moleculalry imprinted polymers.

Sexual dimorphism in postnatal gonadotrophin levels in infancy reflects diverse maturation of the ovarian and testicular hormone synthesis.

BACKGROUND: The postnatal gonadotrophin surge is sexually dimorphic: FSH levels predominate in girls and LH levels in boys. However, in preterm (PT) girls, both gonadotrophin levels are higher than in PT boys. OBJECTIVE: To evaluate how gonadal maturation contributes to the sex differences in FSH and LH. DESIGN: Monthly follow-up of 58 full-term (FT, 29 boys) and 67 PT (33 boys) infants from 1 week (D7) to 6 months of age (M1-M6). Analyses were also carried out according to postmenstrual (PM) age in PT infants. METHODS: Urinary LH, FSH, oestradiol (E2), testosterone (T) and serum inhibin B (InhB) levels. RESULTS: High gonadotrophin levels in PT girls abruptly decreased (P < .001) by M2, corresponding to a PM age of 38-42 weeks, and LH levels fell below the levels found in boys. This decrease was parallel to a steep increase in E2 levels (P < .001), and, from M4 to M6, LH and E2 correlated positively in PT girls (P < .01). T levels in PT boys increased earlier than E2 levels in PT girls. In addition, InhB levels were high in PT boys already at D7, in contrast to low InhB in PT girls. InhB and FSH correlated negatively in the whole group (P < .001). CONCLUSIONS: Ovarian hormone synthesis is immature and incapable of responding to gonadotrophin stimulus before 38-42 PM weeks in PT girls, which may explain their highly elevated FSH and LH levels. The higher InhB levels in boys compared to girls may explain sexual dimorphism in FSH levels.

Multiplex detection of quality indicator molecule targets in urine using programmable hairpin probes based on a simple double-T type microchip electrophoresis platform and isothermal polymerase-catalyzed target recycling.

Recently, it has been crucial to be able to detect and quantify small molecular targets simultaneously in biological samples. Herein, a simple and conventional double-T type microchip electrophoresis (MCE) based platform for the multiplex detection of quality indicator molecule targets in urine, using ampicillin (AMPI), adenosine triphosphate (ATP) and estradiol (E2) as models, was developed. Several programmable hairpin probes (PHPs) were designed for detecting different targets and triggering isothermal polymerase-catalyzed target recycling (IPCTR) for signal amplification. Based on the target-responsive aptamer structure of PHP (Domain I), target recognition can induce PHP conformational transition and produce extension duplex DNA (dsDNA), assisted by primers & Bst polymerase. Afterwards, the target can be displaced to react with another PHP and initiate the next cycle. After several rounds of reaction, the dsDNA can be produced in large amounts by IPCTR. Three targets can be simultaneously converted to dsDNA fragments with different lengths, which can be separated and detected using MCE. Thus, a simple double-T type MCE based platform was successfully built for the homogeneous detection of multiplex targets in one channel. Under optimal conditions, the assay exhibited high throughput (48 samples per hour at most, not including reaction time) and sensitivity to three targets in urine with a detection limit of 1 nM (ATP), 0.05 nM (AMPI) and 0.1 nM (E2) respectively. The multiplex assay was successfully employed for the above three targets in several urine samples and combined the advantages of the high specificity of programmable hairpin probes, the excellent signal amplification of IPCTR, and the high through-put of MCE which can be employed for screening in biochemical analysis.

Magnetic ionic liquids as versatile extraction phases for the rapid determination of estrogens in human urine by dispersive liquid-liquid microextraction coupled with high-performance liquid chromatography-diode array detection.

In this study, a rapid and straightforward approach based on magnetic ionic liquids (MIL) as extraction phases and dispersive liquid-liquid microextraction (DLLME) was developed to analyze the hormones estriol, 17-ß-estradiol, 17-α-ethynylestradiol, and estrone in human urine samples. This is the first report of an application of manganese-based MILs compatible with HPLC to extract compounds of biological interest from urine samples. The hydrophobic MILs trihexyltetradecylphosphonium tetrachloromanganate (II) ([P6,6,6,14+]2[MnCl42-]) and aliquat tetrachloromanganate (II) ([Aliquat+]2[MnCl42-]) were employed and the optimized extraction conditions were comprised of 5 mg of MIL ([P6,6,6,14+]2[MnCl42-]), 5 µL of methanol (MeOH) as disperser solvent, and an extraction time of 90 s at sample pH 6. The analytical parameters of merit were determined under optimized conditions and very satisfactory results were achieved, with LODs of 2 ng mL-1 for all analytes, determination coefficients (R2) ranging from 0.9949 for 17-ß-estradiol to 0.9998 for estrone. In addition, good results of method precision were achieved with the intraday (n = 3) varying from 4.7% for 17-ß-estradiol to 19.5% for estriol (both at 5 ng mL-1) and interday precision (evaluated at 100 ng mL-1) ranging from 11.4% for estrone to 17.7% for 17-α-ethynylestradiol and analyte relative recovery evaluated in three real samples ranged from 67.5 to 115.6%. The proposed DLLME/MIL-based approach allowed for a reliable, environmentally friendly and high-throughput methodology with no need for a centrifugation step. Graphical abstract An overview of the rapid and straightforward extraction procedure using DLLME/MIL-based approach.

Interrelationship among annual cycles of sex steroids, corticosterone and body condition in Nyctibatrachus humayuni.

Synergism between extrinsic and intrinsic factors is crucial for the seasonality of reproduction. Environmental factors such as photoperiod and temperature activate the hypothalamus-pituitary-gonadal axis leading to the secretion of steroid hormones that are crucial for reproduction. Sex steroids are not only essential for the maturation of gonads, but also for development of secondary sexual characters in males and reproductive behaviour of both the sexes. In the present study, we quantified the urinary testosterone (UTM) and corticosterone (UCM) metabolites in males and urinary estradiol metabolites (UEM) and UCM in females of Nyctibatrachus humayuni for two consecutive years to determine annual and seasonal variation in the levels of sex steroids, corticosterone and body condition index (BCI). The results show that sex steroids were highest during the breeding season and lowest during the non-breeding season in both the sexes. An increase in UTM and UEM was observed in males and females respectively during the breeding season. Testicular histology showed the presence of all stages of spermatogenesis throughout the year indicating that spermatogenesis is potentially continuous. Ovarian histology showed the presence of vitellogenic follicles only during the breeding season indicating that oogenesis is strictly seasonal. In males, UCM levels were highest during the breeding season, while in females their levels were highest just prior to the breeding season. In males, BCI was highest during the pre-breeding season, declined during the breeding season to increase again during the post-breeding season. In females, BCI was comparable throughout the year. In males, UTM levels were positively correlated with UCM levels but negatively correlated with BCI. Interestingly, UEM, UCM and BCI were not correlated in females. These results indicate that N. humayuni exhibits an associated pattern of reproduction. Quantification of urinary progesterone metabolites (UPM) during the breeding season showed UPM levels were higher in post-spawning females, suggesting the significance of progesterone in ovulation. Further, non-invasive enzyme immunoassay has been successfully standardized in N. humayuni for the quantification of urinary metabolites of steroid hormones.

Butyl paraben and propyl paraben modulate bisphenol A and estradiol concentrations in female and male mice.

People are routinely exposed to the antimicrobial preservatives butyl paraben (BP) and propyl paraben (PP), as well as the monomer of polycarbonate plastics, bisphenol A (BPA). These chemicals are reliably detected in human urine and potentially interact. We investigated whether BP or PP exposure can modulate the concentrations of 14C-BPA and 17ß-estradiol (E2). Female and male CF1 mice were each given a subcutaneous injection of oil containing 0 (vehicle), 1, 3, or 9mg BP or PP, then given a dietary supplement containing 50µg/kg 14C-BPA. Radioactivity was measured in tissues through liquid scintillation counting. Significantly elevated 14C-BPA concentrations were observed following BP treatment in blood serum of both sexes, as well as the lungs, uterus, and ovaries of females and the testes and epididymides of males. Treatment with PP significantly elevated 14C-BPA concentrations in the uterus only. In another experiment, female and male CF1 mice were each injected with vehicle, 3mg BP, or 3mg PP, and E2 was measured in urine 2-12h later. Whereas PP did not affect E2, BP significantly elevated E2 6-10h after injection in females and 8h after injection in males. These data indicate that BP and PP can alter the pharmacokinetics of BPA in vivo, and that BP can modulate E2 concentrations. These results are consistent with evidence that parabens inhibit enzymes that are critical for BPA and E2 metabolism, and demonstrate the importance of considering concurrent exposure to multiple chemicals when determining regulatory exposure limits.

Progesterone transfer among cohabitating female big brown bats (Eptesicus fuscus).

Experiments using female mice and bats have demonstrated that tritium-labeled 17ß-estradiol (3H-E2) can be absorbed via cutaneous and intranasal routes and distributed to reproductive and neural tissues. Radioactivity has also been measured in tissues of untreated females after 48h cohabitation with 3H-E2 injected males. The present study was designed to quantify steroid transfer among female bats. Radioactive quantification via liquid scintillation counting revealed absorption of tritium-labeled progesterone (3H-P4) in adult females 1h after cutaneous and intranasal application (10µCi). Subsequently, pairs of mature females were each housed for 48h with a single mature female that had been administered 3H-P4 (50µCi) via intraperitoneal injection. Radioactivity was observed in all collected tissues of all non-injected females at levels significantly greater than the control group. Following the same paradigm, radioactivity was not observed in the tissues of untreated female bats that were housed with stimulus females treated with 3H-E2 (50µCi). Enzyme immunoassays revealed measurable levels of unconjugated progesterone and estradiol in the urine of female bats, suggesting urine as a vector for steroid transfer. Given that bats of this species live in predominantly female roosts in very close contact, progesterone transfer among individuals is likely to occur in natural roosts.

Dispersive liquid-liquid microextraction as an effective preanalytical step for the determination of estradiol in human urine.

In this work, a fast and effective dispersive liquid-liquid microextraction was developed for the isolation and preconcentration of free 17 ß-estradiol, the main human estrogen, from real human urine samples. To optimize the extraction technique, few important parameters such as type and volume of extraction and dispersive solvents, centrifugation conditions, effect of salt addition, and extraction time were studied. Optimal conditions were obtained when injecting 600 µL mixture of tetrachloromethane as extraction solvent and ethanol as dispersive solvent (1:5, v/v) into 2 mL of urine containing 8% NaCl and following centrifugation at 10 000 rpm, thus reaching enrichment factor 28 and extraction recovery 98% for estradiol. Procedure was evaluated by means of high-performance liquid chromatography with UV detection (λ = 280 nm) using a C-18 column and methanol/water (60:40, v/v) as the mobile phase. The method was linear within the concentration range 1.0-250.0 mg/L (r = 0.9997) and provided a limit of detection of 0.25 mg/L. The proposed method was applied to the determination of free estradiol in real human pregnancy urine.

Case Study: Impact of Inter- and Intra-Day Energy Parameters on Bone Health, Menstrual Function, and Hormones in an Elite Junior Female Triathlete.

This observational case study examined the association of inter- and intraday energy intake and exercise energy expenditure with bone health, menstrual status and hematological factors in a female triathlete. The study spanned 7 months whereby energy intake and exercise energy expenditure were monitored three times (13 d); 16 blood samples were taken, urinary hormones were assessed for 3 months, and bone mineral density was measured twice. Energy availability tended to be sustained below 30 kcal/kg FFM/d and intraday energy intake patterns were often "back-loaded" with approximately 46% of energy consumed after 6 p.m. Most triiodothyronine values were low (1.1-1.2nmol/L) and supportive of reduced energy availability. The athlete had suppressed estradiol (105.1 ± 71.7pmol/L) and progesterone (1.79 ±1.19nmol/L) concentrations as well as urinary sex-steroid metabolites during the entire monitoring period. Lumbar spine (L1-L4) bone mineral density was low (age-matched Z-score -1.4 to -1.5). Despite these health related maladies the athlete was able to perform typical weekly training loads (swim: 30-40 km, bike: 120-300 km, run 45-70 km) and was competitive as indicated by her continued improvement in ITU World Ranking during and beyond the assessment period. There is a delicate balance between health and performance that can become blurred especially for endurance athletes. Education (athletes, coaches, parents) and continued monitoring of specific indicators will enable evidence-based recommendations to be provided and help reduced the risk of health related issues while maximizing performance gains. Future research needs to longitudinally examine how performance on standardized tests in each discipline (e.g., 800-m swim, 20-km time trial, 5-km run) is impacted when aspects of the female athlete triad are present.

Disruption of the cytochrome P-450 1B1 gene exacerbates renal dysfunction and damage associated with angiotensin II-induced hypertension in female mice.

Recently, we demonstrated in female mice that protection against ANG II-induced hypertension and associated cardiovascular changes depend on cytochrome P-450 (CYP)1B1. The present study was conducted to determine if Cyp1b1 gene disruption ameliorates renal dysfunction and organ damage associated with ANG II-induced hypertension in female mice. ANG II (700 ng·kg(-1)·min(-1)) infused by miniosmotic pumps for 2 wk in female Cyp1b1(+/+) mice did not alter water consumption, urine output, Na(+) excretion, osmolality, or protein excretion. However, in Cyp1b1(-/-) mice, ANG II infusion significantly increased (P < 0.05) water intake (5.50 ± 0.42 ml/24 h with vehicle vs. 8.80 ± 0.60 ml/24 h with ANG II), urine output (1.44 ± 0.37 ml/24 h with vehicle vs. 4.30 ± 0.37 ml/24 h with ANG II), and urinary Na(+) excretion (0.031 ± 0.016 mmol/24 h with vehicle vs. 0.099 ± 0.010 mmol/24 h with ANG II), decreased osmolality (2,630 ± 79 mosM/kg with vehicle vs. 1,280 ± 205 mosM/kg with ANG II), and caused proteinuria (2.60 ± 0.30 mg/24 h with vehicle vs. 6.96 ± 0.55 mg/24 h with ANG II). Infusion of ANG II caused renal fibrosis, as indicated by an accumulation of renal interstitial α-smooth muscle actin, collagen, and transforming growth factor-ß in Cyp1b1(-/-) but not Cyp1b1(+/+) mice. ANG II also increased renal production of ROS and urinary excretion of thiobarburic acid-reactive substances and reduced the activity of antioxidants and urinary excretion of nitrite/nitrate and the 17ß-estradiol metabolite 2-methoxyestradiol in Cyp1b1(-/-) but not Cyp1b1(+/+) mice. These data suggest that Cyp1b1 plays a critical role in female mice in protecting against renal dysfunction and end-organ damage associated with ANG II-induced hypertension, in preventing oxidative stress, and in increasing activity of antioxidant systems, most likely via generation of 2-methoxyestradiol from 17ß-estradiol.

Colorimetric Detection of Small Molecules in Complex Matrixes via Target-Mediated Growth of Aptamer-Functionalized Gold Nanoparticles.

A versatile and sensitive colorimetric assay that allows the rapid detection of small-molecule targets using the naked eye is demonstrated. The working principle of the assay integrates aptamer-target recognition and the aptamer-controlled growth of gold nanoparticles (Au NPs). Aptamer-target interactions modulate the amount of aptamer strands adsorbed on the surface of aptamer-functionalized Au NPs via desorption of the aptamer strands when target molecules bind with the aptamer. Depending on the resulting aptamer coverage, Au NPs grow into morphologically varied nanostructures, which give rise to different colored solutions. Au NPs with low aptamer coverage grow into spherical NPs, which produce red-colored solutions, whereas Au NPs with high aptamer coverage grow into branched NPs, which produce blue-colored solutions. We achieved visible colorimetric response and nanomolar detection limits for the detection of ochratoxin A (1 nM) in red wine samples, as well as cocaine (1 nM) and 17ß-estradiol (0.2 nM) in spiked synthetic urine and saliva, respectively. The detection limits were well within clinically and physiologically relevant ranges, and below the maximum food safety limits. The assay is highly sensitive, specific, and able to detect an array of analytes rapidly without requiring sophisticated equipment, making it relevant for many applications, such as high-throughput drug and clinical screening, food sampling, and diagnostics. Furthermore, the assay is easily adapted as a chip-based platform for rapid and portable target detection.

Variation in maternal urinary cortisol profiles across the peri-conceptional period: a longitudinal description and evaluation of potential functions.

STUDY QUESTION: How do women's first morning urinary cortisol levels, a marker of stress axis activity, vary during the peri-conceptional period (the 12 weeks around conception)? SUMMARY ANSWER: First morning urinary cortisol follows an overall increasing trajectory across the peri-conceptional period, interrupted by 2 week-long decreases during the week preceding conception and the fifth week following conception. WHAT IS KNOWN ALREADY: Later gestational stages (i.e. second and third trimesters) are characterized by increasing levels of circulating cortisol. This increase is hypothesized to constitute a response to the energy demands imposed by fetal growth, and the development of energy reserves in preparation for nursing and performing regular activities while carrying pregnancy's extra weight and volume. STUDY DESIGN, SIZE, DURATION: This study is based on a data set collected as part of a longitudinal, naturalistic investigation into the interactions between the stress (hypothalamic-pituitary-adrenal axis (HPAA)) and reproductive (hypothalamic-pituitary-gonadal axis (HPGA)) axes. Biomarkers of HPAA and HPGA function were quantified in first morning urinary specimens collected every other day from 22 healthy women who conceived a pregnancy during the study. We analyzed the longitudinal within- and between-individual variation in first morning urinary cortisol levels across the 12-week peri-conceptional period. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were recruited from two rural, aboriginal, neighboring communities in Guatemala. Cortisol, estradiol and progesterone metabolites (estrone-3-glucuronide and pregnanediol glucuronide, respectively) and hCG levels were quantified in first morning urinary specimens using immunoassays to determine time of conception and confirm pregnancy maintenance. Linear mixed-effects models with regression splines were used to evaluate the magnitude and significance of changes in cortisol trajectories. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, maternal first morning urinary cortisol increased from 6 weeks prior to conception (geometric mean ± SD = 58.14 ± 36.00 ng/ml) to 6 weeks post-conception (89.29 ± 46.76 ng/ml). The magnitude of the increase between the pre- and post-conception periods varied significantly between women (likelihood ratio test statistic = 8.0017, P = 0.005). The peri-conceptional period is characterized by an increasing cortisol trajectory (+1.36% per day; P = 0.007) interrupted by a week-long decline immediately prior to conception (-4.02% per day; P = 0.0013). After conception cortisol increased again (+1.73% per day; P = 0.0008) for 4 weeks, fell in the fifth week (-6.60% per day; P = 0.0002) and increased again in post-conceptional week 6 (+8.86% per day; P = 0.002). Maternal urinary cortisol levels varied with sex of the gestating embryo. During gestational week 2, mothers carrying female embryos (N = 10) had higher mean cortisol levels than those carrying male embryos (N = 9) (t(17) = 2.28, P = 0.04). LIMITATIONS, REASONS FOR CAUTION: Our results are based on a relatively small sample (n = 22) of women. However, our repeated-measures design with an average of 27 ± 8 (mean ± SD) data points per woman strengthens the precision of estimates resulting in high statistical power. Additionally, our study population's high degree of ethnic and cultural homogeneity reduces the effects of confounders compared with those found in industrialized populations. This higher level of homogeneity also increases our statistical power. However, since there may be small differences in absolute cortisol values among ethnic groups, the social and biological background of our sample may affect the generalizability of our results. General patterns of HPAA activity, however, are expected to be universal across women. Finally, as there is, to the best of our knowledge, no evidence to the contrary, we assumed that urinary cortisol levels reflect HPAA activity and that changes in gonadal steroids across the menstrual cycle do not affect the levels of free cortisol measured in urine. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first longitudinal profile of basal maternal HPAA activity across the peri-conceptional period. A basic understanding of the normative (basal as opposed to stress-induced) changes in HPAA activity across this period is needed to accurately assess women's stress at this juncture. Importantly, changes in HPAA activity are likely to play a critical role in ovulation, fertilization, implantation, placentation and embryonic programing. Thus, this novel information should aid in the development of interventions aimed at preventing or moderating undesired effects of maternal physiological stress during the peri-conceptional period on reproductive outcomes as well as embryonic development. STUDY FUNDING/COMPETING INTERESTS: This research was funded by a CIHR IGH Open Operating grant (CIHR 106705) to P.A.N. and L.Z.; a Simon Fraser University (SFU) President's Start-up grant, a Community Trust Endowment Fund grant through SFU's Human Evolutionary Studies Program and a Michael Smith Foundation for Health Research Career Investigator Scholar Award to P.A.N.; an NSERC Discovery grant to L.Z.; a CIHR Post-Doctoral Fellowship to C.K.B. and an NSERC Undergraduate Student Research Award to H.M. and J.C.B. The funding agencies had no role in the design, analysis, interpretation or reporting of the findings. There are no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Dietary Fat and Fiber Intakes Are Not Associated with Patterns of Urinary Estrogen Metabolites in Premenopausal Women.

BACKGROUND: Interindividual differences in the bioavailability of potentially carcinogenic estrogen and estrogen metabolites (EMs) may play a role in the risk of breast cancer. OBJECTIVE: We examined whether dietary intakes of fiber and fat influence premenopausal EM profiles through effects on estrogen synthesis, metabolism, or excretion. METHODS: We conducted a cross-sectional analysis of 598 premenopausal women who participated in a reproducibility study (n = 109) or served as controls in a nested case-control study of breast cancer (n = 489) within the Nurses' Health Study II. Dietary intakes of fiber and fat were assessed via semiquantitative food frequency questionnaires in 1995 and 1999. Midluteal urine samples were collected between 1996 and 1999 and EMs were quantified with the use of HPLC-tandem mass spectrometry. Linear mixed models were used to estimate creatinine-adjusted geometric means for individual EMs and their pathway groups across categories of dietary intake while controlling for total energy intake and potential confounders. RESULTS: Higher total dietary fiber intake (>25 g/d vs. ≤15 g/d) was associated with significantly higher concentrations of 4-methoxyestradiol (50% difference, P-difference = 0.01, P-trend = 0.004) and lower concentrations of 17-epiestriol (-27% difference, P-difference = 0.03, P-trend = 0.03), but was not associated with any other EMs. The associations did not vary by fiber intake from different sources. Total fat intake (>35% energy vs. ≤25% energy) was suggestively positively associated with 17-epiestriol (22.6% difference, P-difference = 0.14, P-trend = 0.06); the association was significant for polyunsaturated fatty acid (37% difference, P-difference = 0.01, P-trend = 0.01) and trans fat (36.1% difference, P-difference = 0.01, P-trend = 0.01) intakes. CONCLUSION: Fiber and fat intakes were not strongly associated with patterns of estrogen metabolism in premenopausal women. Our data suggest estrogen metabolism is not a major mechanism through which dietary fiber and fat may affect breast or other hormone-related cancer risks.

An ultrasensitive detection of 17ß-estradiol using a gold nanoparticle-based fluorescence immunoassay.

A novel ultrasensitive amplification immunoassay for the determination of 17ß-estradiol (E2) is reported based on the nanoparticle signal amplification platform. It involves two types of particles: magnetic microparticles (MMPs) functionalized with an anti-E2 antibody produced in rabbit as a capture probe; double-codified gold nanoparticles (DC-AuNPs) modified with both goat anti-rabbit antibody and SH-dsDNA-biotin as a signal amplifier; and avidin-FITC was added to link to the SH-dsDNA-biotin as a tracer. The competitive reaction of the anti-E2 antibody immobilized on magnetic microparticles with estradiol in the sample solution and with the goat anti-rabbit antibody on double-codified gold nanoparticles results in a complex involving the DC-AuNPs and MMPs. Under optimized conditions, the linear range of E2 is from 1.0 × 10(-5) to 1.0 ng mL(-1), and the detection limit of the assay could reach up to 6.37 × 10(-6) ng mL(-1). It was applied to determine E2 in human urine, with mean percent recoveries in the range of 96.5%-107.4%, and relative standard deviations were below 8.1%.

Analysis of glucuronide and sulfate steroids in urine by ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry.

Profiling conjugated urinary steroids to detect anabolic-steroid misuse is recognized as an efficient analytical strategy in both chemical-food-safety and anti-doping fields. The relevance and robustness of such profiling rely on the analysis of glucuronide and sulfate steroids, which is expected to have properties including accuracy, specificity, sensitivity, and, if possible, rapidity. In this context, the ability of ultra-high-performance supercritical-fluid chromatography (UHPSFC) hyphenated tandem mass spectrometry (MS-MS) to provide reliable and accurate phase II analysis of steroids was assessed. Four stationary phases with sub-2 µm particles (BEH, BEH 2-ethyl-pyridine, HSS C18 SB, and CSH fluorophenyl) were screened for their capacity to separate several conjugated steroid isomers. Analytical conditions including stationary phase, modifier composition and percentage, back pressure, column temperature, and composition and flow rate of make-up solvent were investigated to improve the separation and/or the sensitivity. Thus, an analytical procedure enabling the analysis of eight glucuronide and 12 sulfate steroids by two different methods in 12 and 15 min, respectively, was optimized. The two procedures were evaluated, and UHPSFC-MS-MS analysis revealed its ability to provide sensitive (limits of quantification: 0.1 ng mL(-1) and 0.5 ng mL(-1) for sulfate and glucuronide steroids, respectively) and reliable quantitative performance (R(2) > 0.995, RSD < 20%, and bias < 30%) through the use of suitable labeled internal standards. Comparison with UHPLC-MS-MS was performed, and UHPSFC-MS-MS obtained better performance in terms of sensitivity. Finally, as a proof of concept, this so-called green technology was used in a chemical-food-safety context to profile steroid conjugates in urine samples from bovines treated with estradiol. Graphical Abstract Glucuronide and sulfate steroids analysis in urine by ultra-high performance supercritical fluid chromatography hyphenated tandem mass spectrometry.

Seasonal variation in urinary and salivary reproductive hormone levels in Amazonian manatees (Trichechus inunguis).

The Amazonian manatee (Trichechus inunguis) is a threatened aquatic mammal endemic to the Amazon basin. The aim of this study was to evaluate the urinary and salivary reproductive hormone levels of captive Amazonian manatees collected during two seasons of the year. Salivary samples from four males and urinary and salivary samples from three females were collected during two seasons (March-June and September-November) over two consecutive years. Salivary testosterone in males was measured by radioimmunoassay and reproductive hormones in females (salivary progesterone and oestradiol and urinary progestogens, oestrogens and luteinising hormone) were measured by enzyme immunoassay. The data were analysed in a 2×2 factorial design, where the factors were year and season. There was no effect of year or season for salivary testosterone. All female hormones showed a seasonal effect (higher hormone levels during March-June than September-November) or an interaction between year and season (P<0.05). These results strongly indicate the existence of reproductive seasonality in Amazonian manatees; however, apparently only females exhibit reproductive quiescence during the non-breeding season. Further long-term studies are necessary to elucidate which environmental parameters are related to reproductive seasonality in T. inunguis and how this species responds physiologically to those stimuli.