Diuretic effect of Lagopsis supina fraction in saline-loaded rats is mediated through inhibition of aquaporin and renin-angiotensin-aldosterone systems and up-regulation of atriopeptin.

Lagopsis supina (Steph. ex Willd.) lk. -Gal. ex Knorr. has been used as a diuretic agent in China for centuries with limited scientific evidence. This study investigated the diuretic efficacy and underlying mechanism of a macroporous adsorption resin with 30% ethanol elution fraction from L. supina (LSC) in saline-loaded rats and to identify its phytochemicals by ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-qTOF-MS/MS). As a result, 18 phenylpropanoids, 14 flavonoids and 15 others were identified in LSC, among which stachysoside A and acteoside could be the main bio-active constituents responsible for the diuretic effect. In parallel, the daily administration of LSC (16, 32 and 64 mg/kg) markedly promoted urinary excretion after 2 h of treatment. Moreover, LSC had no effect on urinary Na+ and K+ concentrations, as well as on serum Na+-K+-ATPase activity. Meanwhile, LSC significantly decreased the serum levels of angiotensin II (Ang II), anti-diuretic hormone (ADH), aldosterone (ALD), aquaporin (AQP) 1, AQP2 and AQP3, suppressed renal AQP1, AQP2, and AQP3 mRNA expressions, down-regulated AQP1, AQP2 and AQP3 protein levels, and up-regulated serum atriopeptin (ANP) level in a dose-dependent manner. These findings suggest that LSC has acute and prolonged diuretic effects by inhibiting the AQPs, RAAS, and upregulation of atriopeptin in saline-loaded rats, and this finding support LSC as a novel diuretic agent.

Urinary steroid profile in relation to the menstrual cycle.

The interpretation of the steroidal module of the Athlete Biological Passport (ABP) in female athletes is complex due to the large variation of the endogenous urinary steroids. The menstrual cycle seems to be one of the largest confounders of the steroid profile. The duration of the different phases in the menstrual cycle differs between women and is difficult to predict only by counting days after menstruation. Here, we have determined the follicle, ovulation, and luteal phases, by assessing the menstrual hormones in serum samples collected from 17 healthy women with regular menses. Urine samples were collected three times per week during two consecutive cycles to measure the urinary steroid concentrations used in the ABP. The metabolite that was mostly affected by the menstrual phases was epitestosterone (E), where the median concentration was 133% higher in the ovulation phase compared to the follicle phase (p < 0.0001). The women with a large coefficient of variation (CV) in their first cycle also had a large CV in their second cycle and vice versa. The inter-individual difference was extensive with a range of 11%-230% difference between the lowest and the highest T/E ratio during a cycle. In conclusion, E and ratios with E as denominator are problematic biomarkers for doping in female athletes. The timing of the sample collection in the menstrual cycle will have a large influence on the steroid profile. The results of this study highlight the need to find additional biomarkers for T doping in females.

Quantification of steroid hormones in human urine by DLLME and UHPLC-HRMS detection.

A procedure for the quantification of steroid hormones of various classes in human urine (androgens, estrogens, progestins, corticosteroids) has been described consisting of sample preparation by means of dispersive liquid-liquid extraction after enzymatic hydrolysis with ß-glucuronidase from E. Coli followed by ultra-high performance liquid chromatography-high resolution mass spectrometry (quadrupole time-of-flight) detection. Both one-variable-at-a-time and multivariate approaches (full factorial and Box-Behnken designs) were applied to optimize sample preparation conditions. The procedure was validated using synthetic urine in the concentration range of 0.25-500 ng/mL. Then, it was applied to the analysis of real urine samples and the results were compared with those of a common liquid-liquid extraction procedure. The results obtained proved its applicability to the quantification of steroid hormones in human urine with high sensitivity and accuracy.

Simultaneous Determination of Prostaglandin and Hormones in Excreta of Trogopterus xanthipes.

The excreta of Trogopterus xanthipes (also called Wulingzhi in Chinese, WLZ) is a well-known traditional Chinese medicine used for the treatment of irregular menstruation in clinic. Few reports are available on the chemical profiling of WLZ. In this work, qualitative and quantitative analyses of endogenous prostaglandin and hormones in WLZ were performed using UHPLC-orbitrap-MSn. In total, 48 compounds were identified in urine of T. xanthipes. Furthermore, the contents of four target compounds were simultaneously quantitated in 20 batches of samples by UPLC-MS/MS. The quantitative method showed a good linear correlation (R > 0.995) in a wide range for each compound. The method had a high sensitivity with LOD (0.5-1.0 ng/mL) and LOQ (1.0-2.5 ng/mL). The intra- and inter-day precisions were < 9.17 (RSD %), and repeatability and stability were < 6.14 (RSD %). The recovery of the analytes varied between 85.8% and 97.3% at three different concentrations. The present integrated qualitative and quantitative assessment of WLZ provides an evaluation strategy to assess the constituent in traditional Chinese medicine.

Detection of estrous biomarkers in the body exudates of Kangayam cattle (Bos indicus) from interplay of hormones and behavioral expressions.

Behavioral expressions and biochemical composition of body exudates are significantly altered in concert with the endocrine status, which are all clear indicators of physiological conditions of animals. In this study, we sought to infer about the reproductive physiological status of Kangayam cattle (Bos indicus) by analyzing behaviors, endocrine pattern, and body exudates and further to discover estrous biomarkers so as to facilitate timely artificial insemination/mating and to aid in aspects of conservation of the species. Therefore, in this study, we followed Kangayam cows through pre-estrous to post-estrous phases to correlate the endocrine dependence of biochemical constituents in urine and cervical mucus and sought to identify estrous biomarkers. Behavioral estrus was confirmed in 10 cows, from which urine samples were collected and subjected to determination of LH, FSH, estrogens, progesterone, proteins, and lipids. Furthermore, urinary fatty acids and proteins were profiled using gas chromatography and SDS-PAGE, respectively. The volatile compounds in the urine and cervical mucus were identified by gas chromatography-mass spectrometry analysis. The data revealed that LH, FSH, and estrogen levels increased significantly in estrous urine compared with nonestrous urine, whereas progesterone status was vice versa (P < 0.05). The lipid content was also significantly higher in estrous urine than in pre- and post-estrous urines (P < 0.05). There were also cyclical variations of volatiles and fatty acid profiles across phases of the estrous cycle. More acidic compounds were present in estrous urine, rendering it more acidic, than in pre- and post-estrous urines. Interestingly, oleic acid, which was present as a fatty acid in estrous and post-estrous urines, appeared to be a volatile in post-estrous urine and estrous cervical mucus. In addition, octanoic and butanoic acids were specific to both estrous urine and cervical mucus, indicating their possible candidature as estrous biomarkers. SDS-PAGE analysis showed pronounced expression of a 98 kDa protein in post-estrous urine, which in matrix-assisted laser desorption ionization-time of flight mass spectrometry was identified as albumin. Our results demonstrate multiple biomarkers in estrous urine and specific volatiles in cervical mucus that offer scope to develop viable estrus detection kits for Kangayam cows.

Multi-exposures to suspected endocrine disruptors in electronic waste recycling workers: Associations with thyroid and reproductive hormones.

Electronic waste recycling (e-recycling) exposes workers to substances such as flame retardants and metals. Some of them are known or suspected endocrine disruptors that could affect hormonal homeostasis and eventually result in adverse health outcomes. Our aim was to measure biological concentrations of organophosphate ester (OPE) metabolites, polybrominated diphenyl ethers (PBDEs), mercury, lead and cadmium in e-recycling workers, and to explore associations with thyroid and sexual hormones. In a cross-sectional study, end-of-shift blood and urine spot samples were collected from 23 women and 77 men in six e-recycling facilities and one commercial recycling facility. Urinary concentrations of 15 OPE metabolites and mercury, and blood concentrations of 12 PBDE congeners, lead, cadmium, and thyroid (thyroxine [T4], triiodothyronine [T3], thyroid stimulating hormone [TSH]) and sexual (testosterone [T], estradiol, Follicle Stimulating Hormone [FSH], Luteinizing hormone [LH]) hormones were measured. E-recycling workers had higher concentrations of BDE209, all OPE metabolites, and lead than commercial recycling workers. In e-recycling workers, plasma geometric mean concentration of BDE209 was 18 ng/g lipids (geometric standard deviation [GSD]: 2.8) vs.1.7 ng/g lipids (GSD: 2.8) in commercial recycling, and urinary geometric mean concentration of diphenyl phosphate (DPhP), a major metabolite of triphenyl phosphate, was 1.7 ng/ml (GSD: 2.5), vs. 0.95 ng/ml (GSD: 2.0). In men, a two-fold increase in BDE209 was associated with 3.1% (95% Confidence interval: 0.07, 6.1) higher levels of total T4, and a two-fold increase in tert-butyl diphenyl phosphate (tb-DPhP) was associated with 18% (-29, -4.7) lower total T, 18% (-27, -6.9) lower free T and 13% (-25, 0.70) lower free T/estradiol ratio. In women, a two-fold increase in BDE153 was associated with 10% (-17, -3.2) lower free T3. To our knowledge, this is the first study to show associations between OPE metabolites and sex hormones in adults. Although some of our results are not conclusive and need replication, they suggest that prudent avoidance should be applied in risk management of flame retardants.

Determination of hormones in human urine by ultra-high-performance liquid chromatography/triple-quadrupole mass spectrometry.

Steroid hormones play a critical role in maintaining the homeostasis of human metabolism. Urine as a noninvasive sample has been extensively used in clinical diagnosis for hormones homeostasis. In this study, the simultaneous characterization of fourteen hormones in urine was performed based on ultra-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPHLC/ESI(+)-MS/MS) with multiple reaction monitoring in the positive ionization mode. The target hormones were cortisone, cortisol, 11-deoxycortisol, aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, 17-OH-progesterone, pregnenolone, estrone, estradiol, estriol, testosterone and dehydreopiandrosterone. ß-Glucuronidase/sulfatase deconjugation and liquid-liquid extraction (LLE) were conducted for the determination of urinary hormones (free + conjugated forms). The limits of detection (LODs) ranged from 0.2 ng/mL (11-deoxycortisol and testosterone) to 1 ng/mL (cortisone). The extraction recovery of the targeted compounds ranged from 87% to 127%, indicating sufficient extraction efficiency for the LLE process. Intraday precision was below 10% and the accuracy ranged from 84% to 122%. The profiling analysis of hormones in urine samples helps to understand the metabolic state of biological systems and can be employed as a diagnostic tool in diseases developed by endocrine-disrupted systems.

Annual banned-substance review - Analytical approaches in human sports drug testing.

Within the complex construct of today's antidoping work, continuously updated routine doping controls, as well as advancements in sampling and analysis have been of particular relevance and importance. New analytes of existing classes of prohibited substances are frequently included into sports drug testing assays, analytical approaches are optimized to allow for better sensitivities, selectivity, and/or faster turnaround times, and research dedicated to addressing analytical issues concerning scenarios of both (potentially) inadvertent doping and new emerging doping agents is constantly conducted. By way of reviewing and summarizing, this annual banned-substance review evaluates the literature published between October 2018 and September 2019 offering an in-depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2019 Prohibited List.

Development and validation of an LC-MS/MS method for profiling 39 urinary steroids (estrogens, androgens, corticoids, and progestins).

Abnormal production or metabolism of steroid hormones is responsible for the development of endocrine diseases. Thus, accurate quantification of steroid hormones is needed for both research into clinical conditions and diagnostic and monitoring purposes. An improved analytical method for profiling 39 steroids in urine using LC-MS/MS was developed. As a pre-treatment procedure prior to LC-tandem mass spectrometry (LC-MS/MS) analysis, hydrolysis using ß-glucuronidase and solid-phase extraction for purifying the samples were performed. Steroids were separated using Waters ACQUITY BEH C18 column (2.1 × 100 mm, 1.7 µm) and a mobile phase consisting of eluent A (0.01% formic acid and 1 mm ammonium formate in water) and eluent B (0.01% formic acid and 1 mm ammonium formate in methanol) with a gradient program at a flow rate of 0.4 mL/min. Under the optimized method, the linearity of calibration curves was higher than 0.992. The limits of detection at signal-to-noise ratio of 3 were 0.03-90 ng/mL. The developed novel LC-MS/MS method can quantitatively profile 39 steroids in a single analytical run. Steroid profiling based on quantitative results could improve the diagnosis and monitoring of hormone-dependent diseases.

Comprehensive identification of steroid hormones in human urine based on liquid chromatography-high resolution mass spectrometry.

Steroid hormones, structural derivatives of cyclopentanoperhydrophenanthrene, play important roles in modulation of many physiological processes. Comprehensive characterization of steroid hormones is valuable for understanding the process of human life activities and even disease diagnosis. Hitherto systematical characterization of steroid hormones has been rarely investigated. Here, we presented an integrated method for human urine analysis based on ultra-high performance liquid chromatography-high resolution mass spectrometry in data-dependent acquisition mode with the following parallel reaction monitoring mode. To process the data acquired by two scan modes, a comparative study of standards' fragmentation behaviors and diagnostic product ions (DPIs) were firstly conducted to facilitate the characterization of steroid hormones. The fragmentation behaviors, DPIs, elemental composition and double-bond equivalent were then simultaneously utilized for systematical characterization of steroid hormones in human urine. Consequently, fragmentation pathways and DPIs for all types of steroid hormones were comprehensively interpreted. It is interesting to find that dehydration is not restricted in the form of hydroxyl groups loss, elimination of the carbonyl oxygen could also generate dehydrated ions. Ultimately, a total of 80 and 107 steroidal hormones were characterized or tentatively identified in human urine of male and female, respectively. The proposed method is expected to provide valuable insights for chemical characterization in complex matrixes.

Aged Black-and-Gold Howler Monkey Female (Alouatta caraya): A Sign of Reproductive Senescence?

Reproductive senescence patterns have been scarcely studied in Neotropical primates. The few studies available on the hormonal profiles of aging female monkeys indicate that the decline of ovarian function in nonhuman primates may resemble the hormonal events associated with the perimenopause in women. In this study, we explore a reproductive hormone profile of an aged black-and-gold howler monkey female (Alouatta caraya) from a wild population in northeastern Argentina and compare this profile with that of a cycling female in the same population. As part of a larger study, we recorded sociosexual behaviors in adult and subadult females belonging to two groups, and we collected urine (n = 877) to determine the sex hormone profile of each female. These samples were analyzed using enzyme immunoassays for estrone conjugates and pregnanediol-3-glucuronide (PdG). We found differences in mean values of PdG between the younger (cycling) and the older female. These hormone values were lower in the older female, and she did not show any signs of cyclicity for either reproductive hormone. Our results show that the aging female in this wild population shows signs of ovarian senescence, indicated by low, acyclic levels of progesterone metabolites.

Combination of in situ metathesis reaction with a novel "magnetic effervescent tablet-assisted ionic liquid dispersive microextraction" for the determination of endogenous steroids in human fluids.

Herein, a novel magnetic effervescence tablet-assisted microextraction coupled to in situ metathesis reaction of ionic liquid (IS-META-ILDM) is presented for the determination of four endogenous steroids in human urine, pregnant women's blood, and fetal umbilical cord blood. The magnetic effervescent tablets, which were composed of Fe3O4 nanoparticles, sodium carbonate (alkaline source), and tartaric acid (acidic source), were used to disperse the extractant and for convenient magnetic separation. After the effervescent reaction, in situ reaction between NH4PF6 and [C6MIM]BF4 was adopted to change hydrophilic ionic liquid to hydrophobic liquid, which could be separated from the aqueous phase. The newly developed method has three obvious advantages: (1) combination of effervescent dispersion and magnetic nanoparticles' retrieval is cost-effective and the dispersion and collection of the extractant can be completed almost simultaneously; (2) as compared to temperature-controlled ionic liquid dispersive microextraction and cold-induced solidified microextraction, this method avoids a heating and cooling process which significantly reduces the extraction time and energy cost; and (3) the combination of adsorption by magnetic nanoparticles with extraction by in situ metathesis reaction easily produces high recoveries for target analytes. The optimized composition of effervescent tablet and experimental parameters are as follows: 0.64 g mixture of sodium carbonate and tartaric acid, 7 mg of Fe3O4 (20 nm) as magnetic sorbents, 40 µL of [C6MIM]BF4 as the extraction solvent, 0.15 g NH4PF6, and 300 µL of elution solvent. Under the optimized conditions, the newly developed method provided high extraction recoveries (90.0-118.5%) and low LODs (0.14-0.17 µg L-1) in urine and blood samples. In total, this IS-META-ILDM method provided high extraction efficiency, fast and convenient separation, and underutilization of any organic solvent, and thus it has great potential for the determination of trace endogenous steroids in complex human fluids. Graphical abstract The newly developed method has three obvious advantages: combination of effervescent dispersion and magnetic nanoparticles' retrieval is cost-effective and the dispersion and collection of the extractant can be completed almost simultaneously. It avoids a heating and cooling process which significantly reduces the extraction time and energy cost and easily produces high recoveries for target analytes.

Monitoring training load and fatigue in soccer players with physiological markers.

The quantification and monitoring of training load (TL) has been the topic of many scientific works in the last fifteen years. TL monitoring helps coaches to individually prescribe, follow-up, analyse, adjust and programme training sessions. In particular, the aim of the present review was to provide a critical literature report regarding different physiological markers of TL monitoring, particularly in soccer, as the load is specific to individual sports. Therefore, the interests and limitations of heart rate (HR), HR variability (HRV) and biochemical variables (blood, urinary and hormonal variations) were analysed, with a special focus on daily measures (before, during and after training) and monitoring throughout a whole season. It appears that the most relevant markers were the resting HR before training, HR reserve during training, HRV during rest days, blood lactate, and blood and salivary immunological status in follow-ups throughout the season. Urinary markers indicative of the players' hydration status also deserve attention. However, these objective markers should be considered with a subjective marker of TL such as the rating of perceived exertion to give a more precise quantification of TL and its perception. Future research could be directed towards urinary marker analysis and the analysis of specific markers of TL, which could be related to injury occurrence and to performance during competition.

Characterization of hormonal profiles during the luteal phase in regularly menstruating women.

OBJECTIVE: To characterize the variability of hormonal profiles during the luteal phase in normal cycles. DESIGN: Observational study. SETTING: Not applicable. PATIENT(S): Ninety-nine women contributing 266 menstrual cycles. INTERVENTION(S): The women collected first morning urine samples that were analyzed for estrone-3-glucuronide, pregnanediol-3-alpha-glucuronide (PDG), FSH, and LH. The women had serum P tests (twice per cycle) and underwent ultrasonography to identify the day of ovulation. MAIN OUTCOME MEASURE(S): The luteal phase was divided into three parts: the early luteal phase with increasing PDG (luteinization), the midluteal phase with PDG ≥10 µg/mg Cr (progestation), and the late luteal phase (luteolysis) when PDG fell below 10 µg/mg Cr. RESULT(S): Long luteal phases begin with long luteinization processes. The early luteal phase is marked by low PDG and high LH levels. Long luteinization phases were correlated with low E1G and low PDG levels at day 3. The length of the early luteal phase is highly variable between cycles of the same woman. The duration and hormonal levels during the rest of the luteal phase were less correlated with other characteristics of the cycle. CONCLUSION(S): The study showed the presence of a prolonged pituitary activity during the luteinization process, which seems to be modulated by an interaction between P and LH. This supports a luteal phase model with three distinct processes: the first is a modulated luteinization process, whereas the second and the third are relatively less modulated processes of progestation and luteolysis.

Gestation-specific changes in maternal thyroglobulin during pregnancy and lactation in an iodine-sufficient region in China: a longitudinal study.

OBJECTIVES: To describe the changes in thyroglobulin (Tg) based upon gestational and postpartum concentrations in healthy pregnant women from an iodine-sufficient region in China, and to evaluate the use of Tg as a biomarker for iodine-sufficient pregnant women. DESIGN: A longitudinal study of Tg change in normal pregnant women from an iodine-sufficient region. PATIENTS AND MEASUREMENTS: Blood and urine samples were obtained from 133 pregnant women. Urinary iodine concentration (UIC) was measured using an ammonium persulfate method. Serum iodine concentration was required by inductively coupled plasma mass spectrometry (ICP-MS). Serum thyroid-stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3), total thyroxine (TT4), total triiodothyronine (TT3), antithyroid peroxidase antibody (TPOAb), antithyroglobulin antibody (TgAb) and Tg were measured using an electrochemiluminescence immunoassay. RESULTS: Thyroglobulin concentrations were higher in early pregnancy (pregnancy at 8 weeks vs nonpregnancy: 11·42 ng/ml vs 8·8 ng/ml, P < 0·01) and maintained a stable level, and then increased greatly at the 36th week. After delivery, Tg decreased to nonpregnant levels. During pregnancy, maternal Tg was not correlated with thyroid function, UIC or urine iodine-creatinine ratio (UI/Cr). Cord blood Tg was much higher compared to maternal Tg levels at the 36w (57·34 vs 14·86 ng/ml, P < 0·001) and correlated positively with cord FT4 (r = 0·256, P < 0·05), cord TT4 (r = 0·263, P < 0·05) and maternal UI/Cr at 36w (r = -0·214, P < 0·05). CONCLUSIONS: Our work demonstrates that Tg is elevated during pregnancy, and the effect of pregnancy should be taken into consideration when Tg is used as a biomarker for the iodine status. Cord blood Tg is much higher than maternal Tg levels at the 36w and is correlated with maternal iodine status.

Sensitive determination of cholesterol and its metabolic steroid hormones by UHPLC-MS/MS via derivatization coupled with dual ultrasonic-assisted dispersive liquid-liquid microextraction.

RATIONALE: Quantitative analysis of cholesterol and its metabolic steroid hormones plays a vital role in diagnosing endocrine disorders and understanding disease progression, as well as in clinical medicine studies. Because of their extremely low abundance in body fluids, it remains a challenging task to develop a sensitive detection method. METHODS: A hyphenated technique of dual ultrasonic-assisted dispersive liquid-liquid microextraction (dual-UADLLME) coupled with microwave-assisted derivatization (MAD) was proposed for cleansing, enrichment and sensitivity enhancement. 4'-Carboxy-substituted rosamine (CSR) was synthesized and used as derivatization reagent. An ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for determination of cholesterol and its metabolic steroid hormones in the multiple reaction monitoring mode. RESULTS: Parameters of dual-UADLLME, MAD and UHPLC-MS/MS were all optimized. Satisfactory linearity, recovery, repeatability, accuracy and precision, absence of matrix effect and extremely low limits of detection (LODs, 0.08-0.15 pg mL(-1) ) were achieved. CONCLUSIONS: Through the combination of dual-UADLLME and MAD, a determination method for cholesterol and its metabolic steroid hormones in human plasma, serum and urine samples was developed and validated with high sensitivity, selectivity, accuracy and perfect matrix effect results. Copyright © 2016 John Wiley & Sons, Ltd.

Picogram-level quantification of some growth hormones in bovine urine using mixed-solvent bubble-in-drop single drop micro-extraction.

Growth hormones are important biologically active compounds. However, they can cause deleterious effects if not used with care and their use in farmed animals is banned in the European Union. This study presents the development and application of a mixed-solvent "bubble-in-drop single drop micro-extraction" (BID-SDME) method for enrichment of stilbene hormones in bovine urine samples. The hormones are quantified using GC-MS showing good linearity with the coefficient of determination (R(2)) of 0.999 and 0.999 for hexestrol and diethylstilbestrol, respectively, in the concentration range 0.05-10 ng mL(-1). Excellent precision (RSD<10%) and accuracy (using a bovine urine certified reference material) were obtained. The observed detection capability (CCα, or LOD) values were 0.01 ng mL(-1) (hexestrol), 0.03 ng mL(-1) (cis-diethylstilbestrol) and 0.02 ng mL(-1) (trans-diethylstilbestrol), respectively, while the decision limit (CCß, or LOQ) values were 0.03 ng mL(-1), 0.08 ng mL(-1) and 0.07 ng mL(-1), which are comparable to or better than those reported in literature. Importantly, sample handling is significantly simplified by our method and enrichment values are greatly enhanced. The results show that a 3:1 chloroform/toluene mixture gave the highest extraction efficiency with a drop-bubble ratio of 2:1. We highlight the importance of solvent density on the success of the BID-SDME method.

Development of the first urinary reproductive hormone ranges referenced to independently determined ovulation day.

BACKGROUND: Urinary hormone level analysis provides valuable fertility status information; however, previous studies have not referenced levels to the ovulation day, or have used outdated methods. This study aimed to produce reproductive hormone ranges referenced to ovulation day determined by ultrasound. METHODS: Women aged 18-40 years (no reported infertility) collected daily urine samples for one complete menstrual cycle. Urinary luteinising hormone (LH), estrone-3-glucuronide (E3G, an estradiol metabolite), follicle stimulating hormone (FSH) and pregnanediol-3-glucuronide (P3G, a progesterone metabolite) were measured using previously validated assays. Volunteers underwent trans-vaginal ultrasound every 2 days until the dominant ovarian follicle size reached 16 mm, when daily scans were performed until ovulation was observed. Data were analysed to create hormone ranges referenced to the day of objective ovulation as determined by ultrasound. RESULTS: In 40 volunteers, mean age 28.9 years, urinary LH surge always preceded ovulation with a mean of 0.81 days; thus LH is an excellent assay-independent predictor of ovulation. The timing of peak LH was assay-dependent and could be post-ovulatory; therefore should no longer be used to predict/determine ovulation. Urinary P3G rose from baseline after ovulation in all volunteers, peaking a median of 7.5 days following ovulation. Median urinary peak E3G and FSH levels occurred 0.5 days prior to ovulation. A persistent rise in urinary E3G was observed from approximately 3 days pre- until 5 days post-ovulation. CONCLUSIONS: This study provides reproductive hormone ranges referenced to the actual day of ovulation as determined by ultrasound, to facilitate examination of menstrual cycle endocrinology.