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Detection of doping with steroids that are also endogenous in the horse can be challenging, and a variety of approaches to distinguish exogenous administration from their natural presence are employed. Knowledge of endogenous concentrations of various steroids in different genders of horses (intact male, castrated male and female) and factors that could naturally affect them is beneficial for establishing ways for detection of their use. The current internationally adopted approaches include concentration-based thresholds in urine and plasma, steroid ratios in urine and targeting the administered intact steroid esters in plasma and hair. However, these have their limitations, and therefore, other strategies, such as additional biomarkers and steroid profiling based on longitudinal testing and multivariate analysis, have been investigated and could potentially improve detection of the use of endogenous steroids in horses. This paper aims to provide a comprehensive overview of the steroids (androgens, oestrogens and progestogens) that have been reported to be endogenous to horses in literature, their concentration ranges in different genders and factors potentially affecting them as well as current and possible future approaches to detect their use., (© 2023 John Wiley & Sons, Ltd.)

A film composed of agarose and graphene (G) and magnetic nanoparticles (G-MNPs) is proposed as a sorbent for the extraction and determination of medroxyprogesterone (MED), levonorgestrel (LEV), norethisterone (NOR) and progesterone (PRO) in natural water samples. Both the preparation of the film and the extraction procedure were optimized. The optimal extraction parameters were as follows: isopropyl alcohol as activation solvent, sample pH value of 3.0, extraction time of 30 min, 1.00 mL of acetonitrile as eluent, elution time of 5 min and sample volume of 100.00 mL. HPLC with photodiode array detector was used for the separation and determination. The method presented a linear range between 2.50 and 75.0 μg L -1 for all analytes, and the LODs were between 1.40 and 1.80 μg L -1 . The method was applied to natural water samples, obtaining satisfactory recovery values (75-111 %). In conclusion, for the immobilization of the G-MNPs, agarose was used, which is a non-toxic, renewable and biodegradable material. The G-MNPs-agarose film was reused up to 70 times, without losing its extraction capacity significantly and presenting excellent sorbent properties, which allow the extraction and preconcentration of the progestogens under study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The occurrence, environmental risks and contribution of organic UV filters to detected (anti-)progestogenic activities were examined in samples of wastewater treatment plant influents and effluents, various surface waters and fish from the Czech Republic. Of the 20 targeted UV filters, 15 were detected in the WWTP influent samples, 11 in the effluents, and 13 in the surface water samples. Benzophenone-3, benzophenone-4, and phenyl benzimidazole sulfonic acid (PBSA) were found in all water samples. Octocrylene, UV-327 and 4-methylbenzylidene camphor exceeded the risk quotient of 1 at some sites. In the anti-progestogenic CALUX assay, 10 out of the 20 targeted UV filters were active. Anti-progestogenic activities reaching up to 7.7 ng/L, 3.8 ng/L, and 4.5 ng/L mifepristone equivalents were detected in influents, effluents, and surface waters, respectively. UV filters were responsible for up to 37 % of anti-progestogenic activities in influents. Anti-progestogenic activities were also measured in fish tissues from the control pond and Podroužek (pond with the highest number of detected UV filters) and ranged from 2.2 to 9.5 and 1.9 to 8.6 ng/g dw mifepristone equivalents, respectively. However, only benzophenone was found in fish, but it does not display anti-progestogenic activity and thus could not explain the observed activities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Concerns are growing about adverse effects of progestins on biota, even at ultra-trace concentrations. The enrichment factor (EF) from extraction of analytes in environmental samples that is needed for sample pre-concentration can affect not only performance of the analytical method but also the matrix effect. Therefore, the present study aimed to assess the influence of high sample EF on performance of the high-performance liquid chromatography with atmospheric pressure chemical ionization and photoionization coupled with high-resolution mass spectrometry (HPLC-APCI/APPI-HRMS) method for analysis of progestins in waste water treatment plant (WWTP) effluents and surface waters and analysis of (anti-)progestogenic activities measured by (anti-)PR-CALUX bioassays. The results showed that HPLC-APCI/APPI-HRMS coupled with solid-phase extraction and a high EF (33,333 L water /L extract ) enabled the detection of more compounds compared to samples with lower sample EF (10,000 L water /L extract ). The matrix effect did not increase proportionally compared to lower EFs (10,000 and 16,666 L water /L extract ), and lower limits of quantification were achieved in WWTP effluents and surface waters. The results of bioassays have shown that relative EF of 25 L water /L bioassay appears high enough to detect progestogenic activity in treated waste water. Our study is one of the first to provide insights into sample pre-concentration in analysis of progestins and progestogenicity in aquatic environments., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: The aim of this study was to investigate the effect of solid fuel use on serum sex hormone levels. Furthermore, the effects of improved kitchen ventilation and duration of cooking time on the relationship between solid fuel use and serum sex hormone levels will be further explored., Methods: In this cross-sectional study, 5386 individuals were recruited. Gender and menopausal status modified associations between solid fuel type and serum sex hormone levels was investigated through generalized linear models and further analyzed by improving kitchen ventilation and length of cooking time on the relationship between solid fuel use and serum sex hormone levels. To identify the causal association, mendelian randomization of two-sample was performed., Results: In observational analyses, for ln-17-hydroxyprogesterone, ln-testosterone, and ln-androstenedione among premenopausal women, the estimated β and 95 % CI of sex hormone levels for the effect of solid fuel users was -0.337 (-0.657, -0.017), -0.233 (-0.47, 0.005), and - 0.240 (-0.452, -0.028) respectively, and - 0.150 (-0.296, -0.004) in ln-progesterone among postmenopausal women. It was found that combining solid fuels with long cooking periods or no ventilation more effectively reduced testosterone and androstenedione in premenopausal women. We further found the adverse effects of using solid fuel on progesterone, testosterone, and androstenedione levels were enhanced with the increases of PM 1 , PM 2.5 , PM 10 , and NO 2 . Corresponding genetic, the causal risk effect of solid fuel were - 0.056 (-0.513, 0.4) and 0.026 (-3.495, 3.547) for testosterone levels and sex hormone binding globulin, respectively., Conclusion: Using gas or solid fuel was negatively related to sex hormone levels. A combination of using solid fuels, cooking for a long time, or cooking without ventilation had a stronger effect on sex hormone levels. However, genetic evidence did not support causality for the associations. WHAT IS ALREADY KNOWN ON THIS TOPIC?: The mechanisms underlying these associations household air pollution (HAP) from incomplete combustion of such fuels and occurrence of chronic diseases remained obscure. Recent years, extensive evidences from animal as well as human researches have suggested that progestogen and androgen hormones are involved in the development of diabetes, hypertension, and cardiovascular disease, which indicated that changes in serum progestogen and androgen hormones levels might play a role in these pathological mechanisms. However, limited evidence exists examining the effect of HAP from solid fuel use on serum sex hormone levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The residues of progestins in milk are dangerous to consumers, but an immunoassay capable of multi-determining progestins in milk has not been reported thus far. In this study, the ligand binding domain of the human progesterone receptor was expressed and its intermolecular interactions with the commonly used steroid hormones were studied. The docking results showed that the receptor fragment only recognized progestins and did not recognize other steroid hormones. Then, it was used as recognition material to develop a pseudo-direct competitive enzyme-linked immunosorbent assay for multi-determination of five progestins in milk. Because biotinylated horseradish peroxidase was combined with streptavidinated horseradish peroxidase to enhance the signal, the sensitivities for the five progestins (IC 50 of 0.029-0.097 ng/mL) were improved 96-143-fold in comparison to the use of the conventional horseradish peroxidase signal system (IC 50 of 3.0-12.5 ng/mL). This method showed negligible cross-reactivities to other steroid hormones, consistent with the docking results. This was the first paper developing a progesterone-receptor-based method for detection of progestins, and this method exhibited generally better performance than all of the previously reported immunoassays for progestins.

The presence of endocrine-disrupting chemicals (EDCs) in aquatic environments such as water, sediment, and sludge received more and more attention. However, the bioaccumulate properties of EDCs, particularly progestins and androgens, in various tissues of different wild freshwater fish species, as well as their effects on human health, have not been fully studied. The muscle, liver, and gills of three wild fish species obtained from the East Dongting Lake in southern China were examined for the presence of 19 EDCs (4 progestins, 5 androgens, 6 estrogens, and 4 phenols). Seventeen analytes were detected in all fish samples, and the concentrations of progestins, androgens, estrogens, and phenols ranged from ND-78.80 ng/g (wet weight, ww), ND-50.40 ng/g ww, ND-3573.82 ng/g ww, and ND-88.17 ng/g ww, respectively. The bioaccumulation of some EDCs in wild fish from East Dongting Lake was species-specific. Additionally, AND, EES, P4, and E2 were discovered in the liver at higher levels than in the muscle, suggesting that livers had a larger ability for enriching these EDCs than the muscle. Furthermore, the relationships between the fish sizes and the EDC concentrations indicated that total weight and length had a negligible impact on the bioaccumulation of EDCs in various fish species. Most importantly, the effects of EDCs on human health as a result of fish consumption were assessed. Although the estimated daily intakes (EDIs) of most EDCs were much lower compared with the corresponding acceptable daily intakes (ADIs) via consuming fish collected in this study, the EDI of EE2 in Silurus asotus was higher than the ADI of E2, indicating that Silurus asotus from East Dongting Lake should be eaten in moderation by local residents., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Gestagens, a class of veterinary drugs also called progestogens, are synthetic hormones used to increase feed efficiency and rate of gain in heifers. The Canadian Food Inspection Agency analyzes progestogens melengestrol acetate (MGA), megestrol acetate, and chlormadinone acetate using liquid chromatography-mass spectrometry (LC-MS). Our conventional gestagen method for kidney fat has many time-consuming steps, including solid-phase extraction. A sample preparation procedure having fewer clean-up steps was developed for routine diagnostic analysis of kidney fat and provided similar results faster, and at lower cost. A confirmatory liver method for gestagens, developed using salt-assisted extraction, employed minimal clean-up steps that resulted in high chemical background at the desired lower limit of quantification (LLOQ). Differential ion mobility spectrometry, specifically high-field asymmetric waveform ion mobility spectrometry (FAIMS), was used to filter chemical background in the gas phase. The effect of the ionization probe position on FAIMS parameters, including sensitivity, is described. With LC-FAIMS-MS, chemical background for each gestagen was virtually eliminated, resulting in a quantitative liver method having the desired 0.6 ng/g LLOQ and estimated limits of detection (LODs) up to 140 times lower than LC-MS. Incurred MGA samples, analyzed using kidney fat and liver methods from the same animal, show levels within the quantitative ranges of both methods.

Many reports on anti-progestogenic activities in aquatic environments have been published in the past decade. These are monitored mainly by in vitro reporter gene bioassays based upon the human progesterone receptor (PR). However, results obtained by some human in vitro bioassays may not be relevant for aquatic animals, especially fish. The present work aimed to detect fish (anti-)PR activity in waste- and receiving surface waters. In parallel, human (anti-)PR activity was analysed to determine if there was any connection between human and fish (anti-)PR activities. Finally, (anti-)PR activities were linked to the occurrence of progestins in water samples. Human PR agonistic activity was detected in all wastewater and most receiving surface water samples. Nevertheless, zebrafish PR (zfPR) agonistic activity was found in only two influent wastewater samples (max. 117 ng/L 17α,20β-dihydroxy-4-pregnen-3-one [DHP] equivalents). Analysed synthetic progestins and progesterone accounted for 14 % to 161 % of detected human PR (hPR) agonistic activity in water samples. Progesterone also contributed significantly to zfPR agonistic activity (up to 10 %) in raw wastewater. The anti-hPR activity was detected also in most wastewater and some surface water samples, but synthetic progestins did not trigger anti-zfPR activity in excess of LOQ values. In addition, altrenogest, dienogest, and ulipristal acetate were tested for their potency to zfPR for the first time. The activity analyses of both pure substances and environmental samples showed that human and zebrafish progesterone receptors are differentially activated. Therefore, results based on human PR in vitro bioassays could not predict fish PR activities in the environment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hana Kocour Kroupova reports financial support was provided by Czech Science Foundation. Patrick Balaguer reports financial support was provided by National Agency for Food Environmental and Occupational Health and Safety., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Over recent decades, steroidal estrogens have become an emerging and very serious issue as they pose a serious threat to living organisms, soil, plants, and water resources in general. Estrogens have therefore been the subject of considerable scientific attention in order to develop new methodologies for its determination, being able of detecting them at very low concentrations. Those procedures minimize or eliminate the consumption of organic solvents and reagents that may be incompatible with the environment. In this respect, we developed a sensitive, selective method for the simultaneous determination of thirteen natural and synthetic hormones present at the nanogram-per-liter level in various types of water by using continuous solid-phase extraction in combination with gas chromatography and mass spectrometry (GC-MS). The target analytes were preferentially sorbed on an Oasis HLB sorbent column (80 mg) and eluted with acetone (600 µL) for derivatization with a mixture of 70 µL of N,O-bis(trimethylsilyl) trifluoroacetamide and trimethylchlorosilane and 35 µL of petroleum ether in a household microwave oven at 200 W for 4 min. Under optimum conditions, the ensuing method exhibited good linearity (r ≥ 0.998), good precision (RSD ≤ 7%), high recoveries (92-103%), and low detection limits (0.01-0.3 ng L -1 ). The method outperforms existing alternatives in robustness, sensitivity, throughput, flexibility-it allows both estrogens, progestogens, and androgens to be determined simultaneously-and compliance with the principles of Green Chemistry. It was successfully used to analyze various types of water samples (mineral, tap, well, pond, swimming pool, river, and waste) that were found to contain four estrogens (estrone, 17β-estradiol, 17α-ethinylestradiol, and hexestrol), two progestogens (testosterone, dihydrotestosterone), and one progestogen (progesterone) at concentrations ranging from 3.0 to 110 ng L -1 ., (© 2022. The Author(s).)

Hormones play critical roles in facilitating pregnancy progression and the onset of parturition. Several classes of environmental contaminants, including fine particulate matter (PM 2.5 ) and ambient temperature, have been shown to alter hormone biosynthesis or activity. However, epidemiologic research has not considered PM 2.5 in relation to a broader range of steroid hormones, particularly in pregnant women. Using metabolomics data collected within 20-40 weeks of gestation in an ethnically diverse pregnancy cohort study, we identified 42 steroid hormones that we grouped into five classes (pregnenolone, androgens, estrogens, progestin, and corticosteroids) based on their biosynthesis type. We found that exposure to PM 2.5 during the pre-conception and early prenatal periods was associated with higher maternal androgen concentrations in late pregnancy. We also detected a positive association between early pregnancy PM 2.5 exposure and maternal pregnenolone levels and a marginal positive association between early pregnancy PM 2.5 exposure and progestin levels. When considering each hormone metabolite individually, we found positive associations between early pregnancy PM 2.5 exposure and five steroids, two of which survived multiple comparison testing: 11beta-hydroxyandrosterone glucuronide (a pregnenolone steroid) and adrosteroneglucuronide (a progestin steroid). None of the steroid classes were statistically significant associated with ambient temperature. In sex-stratified analyses, we did not detect any sex differences in our associations. This is the first study showing that exposure to fine particulate matter during the pre-conception and early prenatal periods can lead to altered steroid adaptation during the state of pregnancy, which has been shown to have potential consequences on maternal and child health., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Long-term exposure to ambient ozone (O 3 ) and residential greenness independently relate to altered hormones levels in urban settings and developed countries. However, independent and their joint associations with progestogen and androgen were sparsely studied in rural regions., Materials and Methods: A total of 6211 individuals were recruited in this study. Random forest model was applied to predict the daily average concentrations of O 3 using the satellites data. Residential greenness was reflected by the normalized difference vegetation index (NDVI). Liquid chromatography-tandem mass spectrometry was used to measure serum progestogen and androgen concentrations. Gender and menopausal status modified associations of long-term exposure to O 3 and residential greenness with hormones levels were analyzed by generalized linear models., Results: Long-term exposure to O 3 was negatively related to 17-hydroxyprogesterone, testosterone, and androstenedione in both men and women (premenopausal and postmenopausal); the estimated β and 95% CI of ln-progesterone in response to per 10 μg/m 3 increment in O 3 concentration was -0.560 (-0.965, -0.155) in postmenopausal women. Association of long-term exposure to O 3 with serum androgen levels in premenopausal and postmenopausal women were alleviated by residing in places with higher greenness. Additionally, a prominent effect of long-term exposure to O 3 related to decreased serum progestogen and androgen levels was found in participants with middle- or high-level of physical activity or lower education level., Conclusions: The results suggested that long-term exposure to high levels of O 3 related to decreased serum androgen levels was attenuated by living in high greenness places in women regardless of menopause status. Future studies are needed to confirm the positive health effects of residential greenness on the potential detrimental effects due to exposure to O 3 ., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This study investigated the overall occurrence and spatiotemporal variation of 19 progestogens in 608 samples collected from 17 wastewater treatment plants (WWTPs) distributed across China during four seasons. The aqueous removal efficiencies of progestogens were calculated and the efficacies of process segments, secondary and advanced processes, and process units in the removal of progestogens were explored. The results indicated that progestogens were widely detected in investigating WWTPs, with the progesterone, dydrogesterone, dienogest, ethisterone, and norethindrone were always dominant in the influent, secondary effluent, final effluent, and excess sludge. Seasonally, the influent exhibited more variability than the other matrices, that 10 progestogens concentrations varied significantly during the four seasons. Spatially, the influent concentrations of progestogens were generally higher in northern WWTPs than that in southern WWTPs during spring and summer. Eight progestogens were stably removed by the WWTPs across seasons, and most progestogens varied considerably in removal in different WWTPs. The conventional process segment was the dominant contributor to progestogen removal. The anaerobic-anoxic-oxic process and a combined process consisting of densadeg and cloth media filter and ultraviolet disinfection showed the highest removal of progestogens among various secondary and advanced treatment processes, respectively. Mass balance analysis showed that most progestogens were effectively eliminated in the aerobic unit, with biodegradation being the primary removal pathway. This study presents the first picture of the spatiotemporal dynamics of the distribution of progestogens in WWTPs of China and provides valuable information for better understanding of the occurrence and removal of progestogens in WWTPs., (Copyright © 2022. Published by Elsevier Ltd.)

A method for rapid screening and quantification of progesterone and progestins in milks by ultrahigh-performance liquid chromatography coupled with quadrupole-high field Orbitrap high-resolution mass spectrometry (UHPLC QE HF HRMS) was established. Milks samples were extracted by acetonitrile + hexane (80 + 20), purified by prime HLB SPE and analyzed by UHPLC QE HF HRMS. The detection limit of progesterone and 21 progestins in milk is between 0.05 µg/kg -0.3 μg /kg, the correlation coefficient of progesterone and progestins in the corresponding concentration range is more than 0.99, recoveries for milk samples are between 80.7% and 108.3% with the relative deviation is less than 15%.The method fulfils the requirements of veterinary drug residue detection validation of EU and China, and successfully applied to detecting the μg/kg level of progesterone and monitoring residual of progestins in real milk., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The quality of surface waters is threatened by pollution with low concentrations of bioactive chemicals, among which those interfering with steroid hormone systems. Induced by reports of anti-progestogenic activity in surface waters, a two-year four-weekly survey of (anti-)progestogenic activity was performed at three surface water locations in the Netherlands that serve as abstraction points for the production of drinking water. As certain endogenous and synthetic progestogenic compounds are also potent (anti-)androgens, these activities were also investigated. Anti-progestogenic and anti-androgenic activities were detected in the majority of the monitoring samples, sometimes in concentrations exceeding effect-based trigger values, indicating the need for further research. To characterize the compounds responsible for the activities, a high resolution Effect-Directed Analysis (hr-EDA) panel was combined with PR and AR CALUX bioassays, performed in agonistic and antagonistic modes. The influent and effluent of a domestic wastewater treatment plant (WWTP) were included as effluent is a possible emission source of active compounds. As drivers for androgenic and progestogenic activities several native and synthetic steroid hormones were identified in the WWTP samples, namely androstenedione, testosterone, DHT, levonorgestrel and cyproterone acetate. The pesticides metolachlor and cyazofamid were identified as contributors to both the anti-progestogenic and anti-androgenic activities in surface water. In addition, epiconazole contributed to the anti-progestogenic activities in the rivers Rhine and Enclosed Meuse. This study showed the strength of hr-EDA for the identification of bioactive compounds in environmental samples and shed light on the drivers of (anti-)progestogenic and (anti-)androgenic activities in the aquatic environment., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A robust and confirmative method was established for the determination of six acetylgestagen residues, namely, flurogestone acetate (FGA), megestrol (MA), melengestrol acetate (MGA), chlormadinone acetate (CMA), medroxyprogesterone (MPA), and hydroxyprogesterone acetate (HPA) in animal-derived matrix samples by utilizing enhanced matrix removal lipid (EMR-lipid) clean-up in combination with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The analytes were extracted with acetonitrile, purified with a EMR-lipid cartridge, and separated with a reversed-phase C18 column. The limit of quantification (S/N ≥ 10) for CMA, FGA, HPA, MA, and MGA in all matrices was 0.5 ng/g, and for MPA, it was 1.0 ng/g; the limit of detection (S/N ≥ 3) for CMA, FGA, HPA, MA, and MGA in all matrices was 0.1 ng/g, and for MPA, it was 0.2 ng/g. The recoveries were between 61.0% and 114.8%, and the relative standard deviations (RSDs) were below 12%. The method was calibrated in a matrix-assisted standard solution in various linear ranges for the analytes and matrices, and the correlation coefficients (R 2 ) exceeded 0.99 for all the matrices., Competing Interests: Declaration of Competing Interest We hereby confirm that this manuscript is our original work and has not been published nor has it been submitted simultaneously elsewhere. We further confirm that all authors have checked the manuscript and have agreed to the submission., (Copyright © 2021. Published by Elsevier B.V.)

Fabric phase sorptive extraction (FPSE) is a recently introduced sample preparation technique that has attracted substantial interest of the scientific community dealing with bioanalysis. This technique is based on a permeable and flexible substrate made of fabric, coated with a sol-gel organic-inorganic sorbent. Among the benefits of FPSE are its tunable selectivity, adjustable porosity, minimized sample preparation workflow, substantially reduced organic solvent consumption, rapid extraction kinetics and superior extraction efficiency, many of which are well-known criteria for Green Analytical Chemistry. As such, FPSE has established itself as a leading green sample preparation technology of 21st century. In this review, we discuss the principal steps for the development of an FPSE method, the main method optimization strategies, as well as the applications of FPSE in bioanalysis for the extraction of a wide range of analytes (e.g., estrogens, benzodiazepines, androgens and progestogens, penicillins, anti-inflammatory drugs, parabens etc.).

The occurrence of biologically potent sex hormones in agricultural soils is of growing concern due to their ability to disrupt the endocrine systems of aquatic organisms after being transported to surface waters via runoff. This study, therefore, examined the large-scale occurrence of 34 natural and synthetic sex hormones (13 progestins, 16 androgens, and 5 estrogens) in soils from 7 provinces and 1 municipality in China. The target sex hormones were detected in 99.3% of the soil samples, indicating their widespread occurrence in most agricultural areas. Additionally, seven synthetic progestins were detected in soils for the first time. The total concentration of the 34 sex hormones (Σsex hormones) in the sampled soils ranged from below the method detection limit to 23.7 ng/g (mean of 4.72 ± 4.07 ng/g), with androgens and progestins being the most dominant hormone groups. Significant correlations were observed among the concentrations of Σestrogens, Σandrogens, and Σprogestins (r = 0.117-0.433, p < 0.001), suggesting similar sources of sex hormones. The mean concentration of Σsex hormones varied considerably across the selected provinces/municipality. Notably, the annual slaughter of poultry and swine (R 2  = 0.75-0.88), female population (R 2  = 0.57-0.58), and soil organic carbon content (R 2  = 0.20-0.55) in each province were significantly correlated with the concentrations or mean concentrations of Σsex hormones, Σestrogens, or Σprogestins. This finding implies that these parameters contributed to the occurrence and distribution of sex hormones in the studied soils. Finally, risk quotients for some sex hormones exceeded 0.01, indicating medium or high risks to agroecosystems. This study highlights the importance of designing an optimal manure fertilization strategy in order to mitigate the risks posed by sex hormones in agroecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A label-free electrochemical progesterone (P4) aptasensor was successfully developed by covalently immobilizing NH 2 -functionalized P4-specific aptamer on the electrode surface. The NiO-Au hybrid nanofibers were synthesized by the electrospinning technique. GQDs-NiO-AuNFs nanocomposite was prepared by dispersing of electrospun NiO-AuNFs in the as-synthesized graphene quantum dots (GQDs) solution and stirring for 24 h. Novel GQDs-NiO-AuNFs nano-architecture in combination with functionalized multiwalled carbon nanotubes (f-MWCNTs) were further utilized to modify screen printed carbon electrode (SPCE) in order to construct an effective immobilization matrix with plenty of carboxylic functional groups. The stepwise assembly process of the designed aptasensor was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The aptamer-progesterone complex formation led to a hindered electron transfer reaction on the sensing interface, which decreased the redox probe peak current. Based on of this, progesterone could be quantitatively detected by monitoring the decrease of differential pulse voltammetric (DPV) responses of [Fe(CN) 6 ] 3-/4- peak current with increasing the progesterone concentration. Under optimized experimental parameters, the aptasensor exhibited a dynamic concentration range from 0.01 to 1000 nM and a detection limit of 1.86 pM. The proposed aptasensor was successfully employed for the determination of progesterone in human serum samples and pharmaceutical formulations., Competing Interests: Declaration of Competing Interest The authors confirm that this article content has no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Obtaining longitudinal endocrinological data from free-ranging animals remains challenging. Steroid hormones can be extracted sequentially from non-invasively sampled biologically inert keratinous tissues, such as feathers, nails, hair and whiskers. However, uncertainty regarding the type and levels of steroids incorporated into such tissues complicates their utility in wildlife studies. Here, we developed a novel, comprehensive method to analyze fourteen C 19 and fourteen C 21 steroids deposited chronologically along the length of seal whiskers in a single, 6-minute chromatographic step, using ultra-performance convergence chromatography-tandem mass spectrometry. The limits of detection and quantification ranged from 0.01 to 2 ng/mL and from 0.1 to 10 ng/mL, respectively. The accuracy and precision were within acceptable limits for steroids at concentrations ≥2 ng/mL. The recovery (mean = 107.5% at 200 ng/mL), matrix effect and process efficiency of steroids evaluated, using blanked whisker matrix samples, were acceptable. The method was applied to the analysis of steroid hormone levels in adult female whisker segments obtained from southern elephant seals (Mirounga leonina), n = 10, and two fur seal species, Antarctic fur seals (Arctocephalus gazella; n = 5) and subantarctic fur seals (Arctocephalus tropicalis; n = 5), sampled between 2012 and 2017. In the whisker subsamples analyzed (n = 71), the median concentration of steroid hormones detected above the LOQ ranged from 2.0 to 273.7 pg/mg. This was the first extraction of multiple C 19 and C 21 steroids, including their C11-oxy metabolites, from the whiskers of mammals. Measuring hormones sequentially along the whisker lengths can contribute to our understanding of the impact of stress associated with environmental/climate changes that affect the health, survival of organisms, as well as to delineate the reproductive cycles of free-living mammals with cryptic life stages., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The occurrence of endocrine disrupting chemicals (EDCs) in swimming pool waters has been scarcely investigated. In this study, the concentrations of 20 EDCs (4 phenols, 6 estrogens, 4 progestogens, 5 androgens, and 1 pharmaceutical) in 40 outdoor and indoor swimming pools in Changsha, China were investigated. Out of them, two phenols (bisphenol A and 4-tert-octylphenol), three estrogens (17β-estradiol, 17ɑ-ethinlestradiol (EE2), and hexestrol), one pharmaceutical (caffeine), and two progestogens (progesterone and levonorgestrel) were detected in the collected samples. The androgens were not detected. Bisphenol A and caffeine were the dominant EDCs at concentrations of ND-23.22 ng/L and ND-39.08 ng/L, respectively. The levels of caffeine were significantly higher in indoor swimming pools (11.15 ng/L in average) than those in outdoor pools (1.90 ng/L in average) (p < 0.05), owing to the less sun's UV radiation and less use of sunscreens containing caffeine. The progestogens (progesterone and levonorgestrel) and estrogens (17β-estradiol and hexestrol) were only detected in outdoor swimming pools. The detection frequencies and concentrations of bisphenol A and caffeine in downtown pools were significantly higher than those in outskirt pools. Besides, the correlations between the concentrations of EDCs and water quality parameters evaluated by the Spearman correlation analysis implied that residual chlorine had strong oxidant capable to bisphenol A and suggested that caffeine could be a potential indicator of organic contamination in swimming pool water. Finally, a quantitative risk assessment revealed that non-athletic child and athletic adult female were vulnerable subpopulations. The EDI total of EE2 for athletic child, non-athletic female, non-athletic male, and non-athletic child were higher than ADI EE2 adopted by Australia and the EDI total of EE2 for athletic female and athletic male were higher than ADI EE2 adopted by the United States., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The North American cheetah population serves as a reservoir for the species, and acts as a research population to help understand the unique biology of the species. Little is known about the intrauterine physiology of the cheetah, including embryo differentiation, implantation, and the development of the placenta. After mating, cheetah females frequently experience (30-65% of matings) a non-pregnant luteal phase where progestogen metabolite levels match those found in pregnant females for the first ~55 days of gestation, but parturition does not occur. Immunoglobulin J chain (IgJ) is a molecule that is involved in the activation of the secretory immune response and has been found to be indicative of pregnancy in the cheetah using fecal monitoring. In this study, western blotting was employed to track IgJ abundance in pooled weekly fecal samples following natural breeding or exogenous stimulation to ovulate, and IgJ levels were compared between individuals undergoing a pregnant (n = 12) and non-pregnant (n = 19) luteal phase. It was revealed that IgJ abundance was increased in pregnant females compared to non-pregnant females at week 4 and week 8 post-breeding, indicating the potential modulation of maternal immunity in response to sensitive events such as implantation and the increased secretory activity of the placenta. IgJ levels also tended to be higher early after breeding in females that were bred naturally with intact males compared to exogenously stimulated females with no exposure to seminal plasma, potentially indicating a response to the act of intromission or the stress of breeding, or possibly demonstrating an immune response resulting in the promotion of maternal tolerance to seminal antigens present upon embryonic implantation. Monitoring fecal IgJ may be a potential method to determine gestational status in the cheetah and will aid future conservation efforts of the species., Competing Interests: The authors have declared that no competing interests exist.

In many mammalian species, corpus luteum derived progesterone (P4) is the main functional gestagen during the estrous cycle and pregnancy. P4 can be metabolized into various metabolites, of which some are biologically active. While some metabolites target the classical nuclear progesterone receptor (PR), neurosteroids bind the receptors of type A γ-aminobutyric acid (GABA A -r) in the brain. According to the position of reduction within the molecule, metabolites of P4 can be characterized into C20-reduced progestogens (20α-dihydroprogesterone (20α-DHP) and 20β-dihydroprogesterone (20β-DHP)), C3-reduced progestogens (3α-dihydroprogesterone (3α-DHP) and 3β-dihydroprogesterone (3β-DHP)), 5α-reduced progestogens (5α-dihydroprogesterone (5α-DHP), allopregnanolone and isopregnanolone) and 5β-reduced progestogens (5β-dihydroprogesterone (5β-DHP), pregnanolone and epipregnanolone). We questioned whether the reduced progestogens are present in bovine plasma during the estrous cycle and whether their profiles differed from the profile of the common precursor P4 around the time of luteolysis. The analytes were monitored in plasma samples using liquid chromatography mass spectrometry (LC-MS). While progestogens lagged behind the drop of P4 at luteolysis, they followed the profile of P4 during the estrous cycle. The abundance of P4 was predominant followed by allopregnanolone, pregnanolone, epipregnanolone and 20β-DHP. Further studies will need to focus particularly on the period around luteolysis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Women are 2 to 9 times more likely to experience an anterior cruciate ligament (ACL) injury than men. Various hormones including relaxin, progesterone, and estrogen influence ACL strength. Oral contraceptives (OCs) alter these hormone levels; however, studies have yet to comprehensively compare different OCs' effects on the ACL., Hypothesis: OCs with increased progestin-to-estrogen ratios will (1) increase ACL collagen expression, (2) decrease ACL matrix metalloproteinase expression, and (3) increase ACL strength., Study Design: Controlled laboratory study., Methods: Untreated female rats were compared with rats treated with 1 of 5 clinically used OCs: norethindrone (NE) only, NE plus ethinylestradiol (EE), etynodiol diacetate (ED) plus EE, norgestimate (NG) plus EE, and drospirenone (DS) plus EE. Doses were scaled from human doses to account for differences in bioavailability and body weight, and OCs were administered daily via oral gavage for 4 rat estrous cycles (20 days). A total of 36 rats were then sacrificed (6 rats/group). ACLs underwent biomechanical testing to assess ACL strength, stiffness, and maximum load before failure. ACL specimens were also isolated for quantitative real-time polymerase chain reaction analysis to assess collagen, matrix metalloproteinase, and relaxin receptor-1 expression., Results: While the primary structural property of interest (ACL maximum load before failure) was not significantly improved by OC treatment, the main material property of interest (ACL strength) in rats treated with NE only, DS + EE, ED + EE, and NE + EE was significantly increased compared with untreated controls ( P = .001, P = .004, P = .004, and P = .04, respectively). The order from strongest to weakest ACLs, which was also the same order as the highest to lowest progestin-to-estrogen ratios, was groups treated with NE only, DS + EE, ED + EE, NE + EE, and lastly NG + EE. Higher ratio formulations also increased the expression of type I collagen ( P = .02) and decreased the expression of matrix metalloproteinase-1 ( P = .04)., Conclusion: OC formulations with higher progestin-to-estrogen ratios may be more protective for the ACL than formulations with lower ratios., Clinical Relevance: OC formulations with high progestin-to-estrogen ratios may benefit female athletes by reducing their ACL injury risk by decreasing the effects of relaxin on the ACL.

Effect-based monitoring is increasingly applied to detect and-in conjunction with chemical analysis-to identify endocrine-disrupting compounds (EDCs) in the environment. Although this approach of effect-directed analysis has been successfully demonstrated for estrogenicity and androgenicity, data on progestogens and glucocorticoids driving endocrine disruption are quite limited. We investigated progestogenic and glucocorticoid activities in Danube River water receiving untreated wastewater from Novi Sad, Serbia. After a 2-step fractionation, all fractions were tested with reporter gene bioassays for agonistic and antagonistic hormonal responses at progestogenic and glucocorticoid hormone receptors as well as with target and nontarget analytical screening of active fractions by liquid chromatography-high-resolution mass spectrometry. Due to masking by cytotoxic mixture components, the effects could not be detected in the raw water extract but were unraveled only after fractionation. Target chemical screening of the fraction that was active in the progesterone receptor (PR) assay revealed that progesterone and megestrol acetate were predominant drivers of PR-mediated activity along with medroxyprogesterone, dihydrotestosterone, androsterone, and epiandrosterone. Hydrocortisone was detected at sub-ng/L concentration in the active fraction in the glucocorticoid receptor (GR) assay but could not explain a significant fraction of the observed GR activity. The present study indicates that effect-based monitoring is a powerful tool to detect EDCs in the aquatic environment but that fractionation may be required to avoid masking effects of mixture components. Future effect-directed analysis studies are required to better understand the occurrence of EDCs and masking compounds in different lipophilicity windows, to finally reduce fractionation requirements for monitoring to a smart clean-up. Environ Toxicol Chem 2019;39:189-199. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC., (© 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.)

Progestagens discharged from municipal wastewater treatment plants (WWTPs) have increasingly gained attention due to their potential risks to the aquatic organisms. However, limited information is available on the occurrence and removal of various progestagens in WWTPs in different cities of China. This work investigated the occurrence and removal of 11 progestagens in 21 WWTPs from 19 Chinese cities. Results showed that progestagens are widely distributed in the investigated WWTPs, with higher influent concentrations of total progestagens in northern WWTPs. The concentration of progestagens in WWTP influent were closely correlated with influent quality, service population and daily service volume of the WWTPs. Additionally, progesterone (PGT) and dydrogesterone (DDT) were two predominant progestagens in influent, effluent and excess sludge. Up to 5 of 11 progestagens showed high aqueous removal efficiencies (median removal efficiency >90%), whereas megestrol acetate (MTA), chlormadinone acetate (CMA), drospirenone (DSP) and levonorgestrel (LNG) had a removal efficiency of below 50%. Specially, the behaviors of progestagens along the anaerobic-anoxic-oxic of a WWTP were further explored and the aerobic tank is the main contributor to the removal of progestagens. Finally, in the effluent of these 21 WWTPs, daily mass loadings of the total progestagens ranged from 0.51 to 10.4 g d -1 . Notably, LNG exhibited high potential risk to the fish base on risk quotient., (Copyright © 2019. Published by Elsevier B.V.)

Animal manure contains various organic contaminants such as steroids. The fate of these steroids during composting is still unknown. Here we investigated the fate of androgens, progestogens, and glucocorticoids during animal manure composting and evaluated their residues in compost-applied soils. The results showed the presence of 16 steroid hormones in the initial compost with concentrations ranging from 3.26 ng/g dw (Cortisol) to 2520 ng/g dw (5α-dihydroprogesterone). The concentrations of almost all detected hormones increased on the 2nd day of composting, and some of them increased several or even dozens of times. Steroids such as hydroxyprogesterone caproate, melengestrol acetate, and methyltestosterone were not found in the initial compost but later detected during the composting process. After 171 days of composting, only 40.4% of detected steroid hormones was removed, and the total concentration of detected steroids was still as high as 3210 ng/g dw. The removal rates of some target compounds were negative, especially for the natural androgens androsta-1,4-diene-3,17-dione and the synthetic androgen 17β-boldenone whose concentrations significantly increased by the end of composting, indicating conversion from their conjugates or transformation from other steroids. The steroid hormones were mainly eliminated in the first three weeks; prolonged composting time did not obviously promote further removal. The variations in steroid concentration were related to the changes in compost properties such as pH and temperature during the composting process. The dissipation of steroid hormones was also linked to the changes of microbial communities in the compost to some extent. Twelve steroids were detected in the compost-treated soils of a kailyard, while 26 steroid hormones were detected in the roots of Chinese cabbages grown on the soil. The results suggest that the application of manure compost product can lead to soil contamination and plant uptake., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the present study, both bioanalytical and instrumental tools were employed to examine the endocrine-disruptive potentials of water samples, cyanobloom samples, and sediment samples collected from in the northern region of Taihu Lake (China) during cyanobloom season. Results from cell-based bioassays suggested the occurrence of estrogenic, anti-estrogenic, anti-androgenic, and anti-glucocorticogenic activities, while no androgenic and glucocorticogenic activities were observed in the collected samples. Using an UPLC-MS/MS system, 29 endocrine disrupting compounds including seven estrogens, seven androgens, six progestogens, and five adrenocortical hormones and four industrial pollutants were simultaneously detected. 17, 20 and 12 chemicals were detected at least in one of the water samples, cyanobloom samples and sediment samples, respectively. Since both agonistic and antagonistic endocrine-disruptive activities were detected in the present study, commonly used receptor-based in vitro bioassays resulted in net effects, suggesting that the hormone receptor agonistic potentials might be underestimated with this practice. The EDCs detected in cyanobloom samples also highlight the necessity to consider the phytoplankton matrix for understanding the mass fluxes of endocrine disruptors in eutrophic freshwaters and to consider it in monitoring strategies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Research Question: Can IVF outcomes be predicted from the steroid profile generated by liquid chromatography-mass spectrometry (LC-MS/MS) from follicular fluid collected from a single dominant follicle and serum after ovarian stimulation., Design: Prospective observational cohort study in which serum and follicular fluid were collected from women and used to generate steroid profiles by LC-MS/MS. A total of 93 consecutive women enrolled for IVF treatment were recruited at the Fertility Unit, Royal Prince Alfred Women and Babies Hospital, Sydney between September 2014 and July 2015. Baseline and serum levels at oocyte retrieval, as well as follicular fluid samples from the largest single antral follicle, were collected. All samples underwent steroid analysis within a single batch to measure progesterone (P4), oestradiol (E2), oestrone (E1), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), and 3 α, 5α androstanediol (3α-diol) and 3β, 5α androstanediol (3β-diol)., Results: P4, E2, E1, A4, T, DHEA and A4 were detectable in all baseline serum levels, at oocyte retrieval and in follicular fluid samples, whereas DHT, 3α-diol and 3β-diol were only detectable in a minority of samples. The most consistent predictor of pre-transfer (number of follicles >14mm in diameter, oocytes retrieved or fertilized, day-5 blastocysts) outcomes was baseline serum anti-Müllerian hormone. In follicular fluid, E2 was a negative predictor of the number of oocytes retrieved and the number of day-5 blastocysts but no follicular fluid steroids predicted pregnancy outcome., Conclusions: None of the nine steroids measured in follicular fluid predicted pregnancy outcome in women undergoing IVF., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

The present study investigated the occurrence and removal of 10 steroid hormones (4 androgens, 3 progestagens and 3 estrogens) in two WSP systems, Mafisa and Mzumbe in Morogoro, Tanzania. All 10 steroid hormones were detected in the influent of both WSP systems in the dry as well as in the rainy season. The concentrations of steroids in influent wastewater ranged from 0.1 ng/L for 17-OH-pregnenolone to 445 ng/L for estrone and from below limit of detection for 17-OH-pregnenolone to 45 ng/L for estrone in effluent. During dry season, the overall mean ± standard deviation removal efficiency for the 10 steroids were 70 ± 21% for Mzumbe WSP and 97 ± 3% for Mafisa WSP. During the rainy season the overall mean removal efficiency for all the steroid hormones were 52 ± 32% for Mzumbe WSP and 94 ± 8% for Mafisa WSP. Risk was characterized by calculating the risk quotients (RQs) for fish and humans. 46% of the total RQs calculated were above one, indicating high risk. Low RQs were estimated for androgens and progestagens but the estrogen concentrations measured in the WSP systems and Morogoro River indicated a high risk for fish. However, estrogens appeared not to pose an appreciable risk to human health from water intake and fish consumption. The results indicated that WSP systems are quite effective in removing steroid hormones from wastewater. Thus, low technology systems such as WSP systems are suitable techniques in low income counties due to relatively low costs of building, operating and maintaining these systems., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A multiclass screening method to detect fifty-three forbidden substances by liquid-chromatography coupled to hybrid high-resolution mass spectrometry (LC-Q-Orbitrap) was developed and validated in bovine bile and urine. Eight classes of compounds were included in the method's scope (β-agonists, corticosteroids, nitroimidazoles, progestins, resorcylic acid lactones (RALs), sedatives, steroids and stilbenes) plus chloramphenicol and dapsone. After hydrolysis, the sample was divided in two aliquots, which followed two parallel purification steps. The reunified extracts were injected and two chromatographic runs performed in positive and negative ionization mode, respectively. The validation data (60 different samples per matrix) proved that the method was fit for purpose with detection capabilities lower than 1 μg L -1 in both matrices. The combined application of accurate mass acquisition and two-stage mass spectrometry (parallel reaction monitoring) was crucial to achieve suitable selectivity, which is the most critical parameter mainly for urines. Finally, the long-standing problem of the high rate of false positive results for RALs, due to the natural ingestion of mycotoxin, zearalenone, was taken on including all their labelled standards. That allowed a very satisfactory management of this screening test., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Emission of compounds with biological activities from waste water treatment plant (WWTP) effluents into surface waters is a topic of concern for ecology and drinking water quality. We investigated the occurrence of hormone-like activities in waste water sample extracts from four Dutch WWTPs and pursued to identify compounds responsible for them. To this aim, in vitro reporter gene bioassays for androgenic, anti-androgenic, estrogenic, glucocorticoid and progestogenic activity and a UPLC-tQ-MS target analysis method for 25 steroid hormones used in high volumes in pharmacy were applied. Principal component analysis of the data was performed to further characterize the detected activities and compounds. All five types of activities tested were observed in the WWTP samples. Androgenic and estrogenic activities were almost completely removed during WW treatment, anti-androgenic activity was only found in treated WW. Glucocorticoid and progestogenic activities persisted throughout the treatment. The androgenic activity in both influent could predominantly be attributed to the presence of androstenedione and testosterone. Anti-androgenic activity was explained by the presence of cyproterone acetate. The glucocorticoid activity in influent was fully explained by prednicarbate, triamcinolone acetonide, dexamethasone and amcinonide. In effluent however, detected hormones could only explain 10-32% of the activity, indicating the presence of unknown glucocorticoids or their metabolites in effluent. Progesterone and levonorgestrel could explain the observed progestogenic activity. The principle component analysis confirmed the way in which hormones fit in the spectrum of other emerging contaminants concerning occurrence and fate in WWTPs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Vast numbers of xenobiotics are known still to be present in treated municipal wastewater treatment plant (WWTP) effluents. Some of these possess endocrine-disrupting potency and pose risks for exposed aquatic animals. We searched for 17 potential environmental contaminants having affinity to the progesterone receptor. Relative potency values of these progesterone receptor-active chemicals were obtained. On the basis of relative potencies and measured environmental concentrations, the contribution of progestins to measured progestagenic activities was evaluated. Wastewaters (influent and effluent) and surrounding surface waters (upstream and downstream) at six municipal WWTPs were screened using instrumental chemical analysis and in vitro reporter gene bioassay. We showed the presence of target compounds and (anti-)progestagenic activities in municipal wastewater and surface water. Nine and seven progestins were identified in influent and effluent wastewaters, respectively. Only two compounds, progesterone and medroxyprogesterone were found in surface waters. Progestagenic agonistic activities in influents were partially masked by strong anti-progestagenic activities that were detected in all influents and ranged from 2.63 to 83 ng/L of mifepristone equivalents (EQs). Progestagenic activities were detected in all effluents and ranged from 0.06 to 0.47 ng/L of reference compound ORG 2058 EQs (a synthetic progestin equivalents), thus indicating incomplete removal of progestins during wastewater treatment processing. This activity poses a continuing risk for the aquatic environment. By contrast, anti-progestagenic activities showed better removal efficiency in WWTPs compared to progestagenic agonistic activities. Anti-progestagenic activities were found in only three of six effluents and ranged from 0.26 to 2.1 ng/L mifepristone EQs. We explained most of the progestagenic activity in municipal WWTP effluents by the presence of synthetic progestins and progesterone, which contributed 65-96% of such activity in samples where no antagonistic activity was found. The progestins medroxyprogesterone acetate, megestrol acetate and progesterone contributed most to the progestagenic activity detected in municipal effluents. Anti-progestagenic activities were found in some municipal effluents, but no causative agents were revealed because two analysed selective progesterone receptor modulators (SPRMs) with anti-progestagenic activities, mifepristone and ulipristal acetate, were not present in the effluents., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Trace levels of airborne hormones during the pharmaceutical compounding process are analyzed by air monitoring and surface sampling. This study is discussed in two parts: the first part discusses the analysis of particulates suspended in the air by using video particle counting; the second part discusses the analysis of particulates settled on the surface by using surface wipe sampling. One solution to the problem of airborne hormones is to incorporate the hormones into a vehicle like a semisolid cream with high surface tension such that there are no powders to be used during the pharmaceutical compounding process. The analytical method for this study was performed., (Copyright© by International Journal of Pharmaceutical Compounding, Inc.)

The aim of this study was to develop a reliable analytical method for the measurement of 17 selected progestogens in waste water and surface water. Automated whole water solid phase extraction (SPE) was used for sample concentration. Liquid chromatography tandem atmospheric pressure chemical ionization/atmospheric pressure photoionization with hybrid quadrupole/orbital trap mass spectrometry operated in high resolution product scan mode (LC-APCI/APPI-HRPS) was applied for the analyses. The whole-method recoveries ranged from 60% to 140% for all analytes at two different spike levels (5 and 50ng/L) in the studied matrices. The method is very sensitive with LOQs ranging from 0.02 to 0.87ng/L. The developed method was used for the determination of progestogens in real samples of waste water from three waste water treatment plants (WWTPs) and in surface water from the corresponding recipients. Progesterone was detected in all samples with concentrations in the range of 0.82 to 1.1ng/L in surface water and 0.11 to 110ng/L in waste water samples. Three synthetic progestogens, namely, megestrol acetate, medroxyprogesterone acetate, and dienogest, were detected most frequently in effluents; therefore, further attention should be paid to the monitoring of these compounds. To the best of our knowledge, this study is the first to present analysis of altrenogest, etonogestrel, dienogest, nomegestrol acetate and ulipristal acetate in waste water and surface water using a solid-phase extraction method., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A broad number of natural and synthetic progestins are widely used in human and veterinary therapies. Although progestins exhibit adverse effects in aquatic organisms, information about environmental occurrence and fate have been limited to several compounds, hampering the accuracy of risk assessments of the compounds. In this study, a selective and sensitive analytical method was established to simultaneously determine 19 natural and 42 synthetic progestins in environmental waters, and the synthetic progestins included 19-nortestosterone, 17α-hydroxyprogesterone and progesterone derivatives. All of the target compounds were effectively separated using an HSS T3 column, and the recoveries for effluent and river samples were 80-115% and 75-105%, respectively. The detection limits for the 61 analytes were in the range of 0.05-0.60 ng/L and 0.03-0.40 ng/L for the effluent and river samples, respectively. The developed method is applied to analyze the target progestogens in sewage effluent and river water samples from Beijing. The detected concentrations of natural progesterone metabolites (3α-hydroxy-5β-tetrahydroprogesterone) were up to 63 times higher than those of the parent compound. Of the three groups of synthetic progestins, the progesterone derivatives were detected for the first time and had the highest concentrations followed by the 19-nortestosterone and 17α-hydroxyprogesterone derivatives. In contrast to previous studies, the predominant derivative compounds of 19-nortestosterone were found to be 19-nortestosterone, gestodene and mifepristone, and those of 17α-hydroxyprogesterone were 6-epi-medroxy progesterone 17-acetate and melengestrol acetate. The toxicities and environmental risk of these emerging progestins deserves more attention in the future., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A highly sensitive and robust method was developed for routine analysis of two progestin metabolites, 17α-hydroxypregnanolone (17OH-Δ5P) and pregnanediol (PD), and 31 other natural and synthetic steroids and related metabolites (estrogens, androgens, corticosteroids, progestins) in river water, as well as influents and effluents of municipal wastewater treatment plants (WWTP) using HPLC-MS/MS combined with solid-phase extraction. For the various matrixes considered, the optimized method showed satisfactory performance with recoveries of 70-120% for most of target steroids. The method detection limits (MDLs) ranged from 0.01 to 3ng/L for river water, 0.02 to 10ng/L for WWTP effluents, and 0.1 to 40ng/L for influents with good linearity and reproducibility. The developed method was successfully applied for the analysis of steroids in rivers and WWTP influent and effluents. WWTP influents concentrations of 17OH-Δ5P and PD were 51-256ng/L and up to 400ng/L, respectively, along with androstenedione (concentration range: 38-220ng/L), testosterone (11-26ng/L), estrone (2.3-37ng/L), 17β-estradiol (N.D.-8.7ng/L), 17α-hydroxyprogesterone (N.D.-66ng/L), medroxyprogesterone acetate (N.D.-5.3ng/L), and progesterone (2.0-22ng/L), while only androstenedione (ADD), estrone (E1), and estriol (E3) were detected in effluent with concentrations ranging up to 1.7ng/L, 0.90ng/L and 0.8ng/L, respectively. In river water samples, only ADD and E1 were detected with concentrations up to 1.0ng/L and 0.91ng/L. Our procedure represents the first method for analyzing 17OH-Δ5P and PD in environmental samples along with a large series of steroids., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Body mass index is a known breast cancer risk factor due to, among other mechanisms, adipose-derived hormones. We developed a method for steroid hormone profiling in adipose tissue to evaluate healthy tissue around the tumor and define new biomarkers for cancer development. A semi-automated sample preparation method based on gel permeation chromatography and subsequent derivatization with trimethylsilyl (TMS) is presented. Progestagens and androgens were determined by GC-EI-MS/MS (LOQ 0.5 to 10 ng/g lipids). For estrogen measurement, a highly sensitive GC-APCI-MS/MS method was developed to reach the required lower limits of detection (0.05 to 0.1 ng/g lipids in matrix, 100-200 fg on column for pure standards). The combination of the two methods allows the screening of 27 androgens and progestagens and 4 estrogens from a single sample. Good accuracies and repeatabilities were achieved for each compound class at their respective limit of detection. The method was applied to determine steroid hormone profiles in adipose tissue of 51 patients, collected both at proximity and distant to the tumor. Out of the 31 tested steroid hormones, 14 compounds were detected in human samples. Pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone (DHEA), and androstendione accounted together for 80% of the observed steroid hormone profiles, whereas the estrogens accounted for only 1%. These profiles did not differ based on sampling location, except for ß-estradiol; steroid hormone conversions from androgens to estrogens that potentially take place in adipose or tumoral tissue might not be detectable due a factor 100 difference in concentration of for example DHEA and ß-estradiol. Graphical Abstract Schematic overview of the determination of steroid hormones and metabolites in adipose tissue in proximity and distal to the tumor.

Approximately 80% of cheetahs living in typical zoological collections never reproduce. In more than 60% of breedings, the female is confirmed to ovulate, but parturition fails to occur. It is unknown if these non-pregnant intervals of elevated progesterone (deemed luteal phases) are conception failures or a pregnancy terminating in embryonic/fetal loss. There have been recent advances in metabolic profiling and proteome analyses in many species with mass spectrometry used to identify 'biomarkers' and mechanisms indicative of specific physiological states (including pregnancy). Here, we hypothesized that protein expression in voided cheetah feces varied depending on pregnancy status. We: 1) identified the expansive protein profile present in fecal material of females; and 2) isolated proteins that may be candidates playing a role in early pregnancy establishment and diagnosis. Five hundred and seventy unique proteins were discovered among samples from pregnant (n = 8), non-pregnant, luteal phase (n = 5), and non-ovulatory control (n = 5) cheetahs. Four protein candidates were isolated that were significantly up-regulated and two were down-regulated in samples from pregnant compared to non-pregnant or control counterparts. One up-regulated candidate, immunoglobulin J chain (IGJ; an important component of the secretory immune system) was detected using a commercially available antibody via immunoblotting. Findings revealed that increased IGJ abundance could be used to detect pregnancy successfully in >80% of 23 assessed females within 4 weeks after mating. The discovery of a novel fecal pregnancy marker improves the ability to determine reproductive, especially gestational, status in cheetahs managed in an ex situ insurance and source population.

Non-invasive fecal hormone metabolite monitoring was used to characterize the estrous cycle, pregnancy, and seasonal anestrus of the critically endangered addra gazelle (Nanger dama ruficollis). With less than 250 animals remaining in the wild and ∼168 individuals managed in captivity, it is crucial to maintain sustainable populations. Progestogen and estrogen profiles were obtained from analysis of fecal samples collected approximately every other day, within varying intervals, over the course of 7 years (n = 8 adult females). Average estrous cycle length was 19.5 ± 0.4 days (range, 14-26 days), with a luteal phase length of 14.6 ± 1.2 days (range, 10-16 days), and an inter-luteal period of 5.2 ± 1.4 days (range, 2-7 days). Mean gestation length for six pregnancies was 200.7 ± 0.4 days (range, 200-202 days). Fecal progestogens increased at 12 weeks of gestation and remained elevated until parturition. Addra gazelle females exhibited a period of seasonal anestrus with consistently low progestogen concentrations and no cyclic activity from about September to March. Analysis of reproductive and climate records demonstrated a peak in U.S. births that coincided with maximal rainfall in the native habitat of the addra gazelle. Results show that estrous cycle, luteal phase, and inter-luteal phase lengths in addra are similar to those observed in other gazelle species, however, to our knowledge, this is the first study to demonstrate seasonal anestrus in the Nanger genus., (Copyright © 2017. Published by Elsevier Inc.)

The presence of endocrine disrupting chemicals in the aquatic environment poses a risk for ecosystem health. Consequently there is a need for sensitive tools, such as in vitro bioassays, to monitor endocrine activity in environmental waters. The aim of the current study was to assess whether current in vitro bioassays are suitable to detect endocrine activity in a range of water types. The reviewed assays included androgenic (n=11), progestagenic (n=6), glucocorticoid (n=5), thyroid (n=5) and estrogenic (n=8) activity in both agonist and antagonist mode. Existing in vitro bioassay data were re-evaluated to determine assay sensitivity, with the calculated method detection limit compared with measured hormonal activity in treated wastewater, surface water and drinking water to quantify whether the studied assays were sufficiently sensitive for environmental samples. With typical sample enrichment, current in vitro bioassays are sufficiently sensitive to detect androgenic activity in treated wastewater and surface water, with anti-androgenic activity able to be detected in most environmental waters. Similarly, with sufficient enrichment, the studied mammalian assays are able to detect estrogenic activity even in drinking water samples. Fewer studies have focused on progestagenic and glucocorticoid activity, but some of the reviewed bioassays are suitable for detecting activity in treated wastewater and surface water. Even less is known about (anti)thyroid activity, but the available data suggests that the more sensitive reviewed bioassays are still unlikely to detect this type of activity in environmental waters. The findings of this review can help provide guidance on in vitro bioassay selection and required sample enrichment for optimised detection of endocrine activity in environmental waters., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This work describes the development and validation of a method for the simultaneous determination of 13 estrogens and progestogens in honey by high performance liquid chromatography-tandem mass spectrometry. The hormones were preconcentrated by solid phase extraction. Pretreatment variables were optimized for a better compatibility with electrospray ionization interfaced mass spectrometry. The analytes were analyzed in multiple-reaction monitoring mode with two pairs of precursor product ion transitions. The proposed method was validated with method detection limits of 0.01-0.33ng/g and good linearities (r 2 >0.9901) throughout the studied concentration range. The recoveries of analytes at the spiking levels (5ng/g and 25ng/g) ranged from 71.2% to 99.7%, with relative standard deviations below 20%. The method was used to determine the target compounds in honey samples (orange blossom, clover and multiflower) obtained from supermarkets. Two samples of honey were found to contain trace amounts of estrone (

The present study investigated the occurrence of 13 parent and conjugated estrogens and progestagens in surface water of the Santa Ana River. With the exception of the synthetic hormones 17α-ethynylestradiol and mestranol, other compounds were detected at least twice at 10 representative sites, with the ubiquitous estrone (E1) and 17β-estradiol-3-sulfate as the dominant compounds quantified (0.24-6.37 ng/L and 0.49-9.25 ng/L, respectively). Sites near dairy farms exhibited high levels of conjugates, whereas those close to a sewage treatment plant (STP) effluent outlet displayed relatively high concentrations of E1. Principle component analysis coupled with multiple linear regression revealed dairy farms and the STP as the 2 significant contamination sources, accounting for 69.9% and 31.1% of the total hormone burden, respectively. Risk assessment results suggested E1 and 17β-estradiol (E2) as the 2 hormones with the largest risks to aquatic organisms, and which combined, contributed >90% of the total estrogenicity. Most of the sites investigated showed that E1 and E2 posed a medium risk (0.1 < risk quotient < 1), whereas each induced a high risk (risk quotient >1) at sites severely impacted by the STP and dairy farms. These results suggest that river health would benefit from effective treatment of waste at the STP and dairy farms prior to discharge. Environ Toxicol Chem 2016;35:2657-2664. © 2016 SETAC., (© 2016 SETAC.)

The secretory patterns of progestagens and estrogens were characterized throughout 28 normal pregnancies until two month post-partum in eleven killer whales. HPLC analysis of serum from different reproductive stages (luteal phase, EARLY, MID, and LATE pregnancy) identified three major immunoreactive progestagen peaks; progesterone (P4), 5α-pregnane-3,20-dione (5α-DHP) and pregnanediol, with 5α-DHP approximately half of that for P4 in the luteal phase, and EARLY, but approximately 2/3 of P4 during MID and LATE pregnancy. At birth, 5α-DHP was the only significant (>10% immunoreactivity) immunoreactive progestagen detected in placental (umbilical cord) serum. Maternal recognition of pregnancy appears to occur between day 21 and 28 post-ovulation when a significant deviation in progestagen concentrations between conceptive and non-conceptive cycles was detected. Progestagen concentrations during pregnancy displayed a bimodal pattern with significant peaks (P<0.05) in EARLY (indexed month post-conception [IMPC] 2, 3, 4) and MID (IMPC 9, 10) before decreasing (P<0.05) over an 11day interval to luteal phase concentrations on the day of parturition. Among estrogens, estriol was secreted in the highest concentrations but only estrone (free and conjugated) and estradiol increased (P<0.001) during pregnancy, with peaks observed during the final month of gestation, and an influence (P<0.05) of fetal sex on estradiol production was detected. Collective findings indicate that P4 derived from the corpus luteum is the major biologically active progestagen during the luteal phase and pregnancy, and that 5α-DHP production, possibly from both luteal and placental sources, increases during the second half of pregnancy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Synthetic hormones have been widely reported in treated sewage effluents, and consequently receiving aquatic environments. Ethinylestradiol (EE2) is a potent synthetic estrogen commonly used in conjunction with levonorgestrel in oral contraceptive pills. Both EE2 and levonorgestrel have been identified in the aquatic environment, but although there is a significant amount of literature on EE2, there is much less information on levonorgestrel. Using Australian prescription data as well as excretion and predicted wastewater removal rates, the concentrations of EE2 and levonorgestrel in Australian wastewater were calculated at 0.1 ng/L to 0.5 ng/L and 0.2 ng/L to 0.6 ng/L, respectively. Both compounds were analyzed in treated wastewater and surface water grab samples from 3 Southeast Queensland, Australia sites. The predicted no-effect concentration (PNEC) for EE2 of 0.1 ng/L was exceeded at most sites, with EE2 concentrations up to 2 ng/L in treated effluent, albeit quickly diluted to 0.1 ng/L to 0.2 ng/L in the receiving environment. A provisional PNEC for levonorgestrel of 0.1 ng/L derived in the present study was slightly lower than predicted effluent concentrations of 0.2 ng/L to 0.6 ng/L, indicating a potential risk of endocrine-related effects in exposed aquatic species. The detection limit for levonorgestrel in the present study was 2.5 ng/L, and all samples were below detection limit. The present study's results suggest that improvements in analytical capabilities for levonorgestrel are warranted to more accurately quantify the risk of this compound in the receiving environment. Environ Toxicol Chem 2016;35:1378-1385. © 2015 SETAC., (© 2015 SETAC.)

Synthetic and natural steroid hormones have attracted some attention in recent years as endocrine active substances (EAS) that interact or interfere with the endocrine system. Endogenous hormones occur naturally in food of animal origin, among which bovine milk represents an important source. This study was conducted to determine the occurrence of steroid hormones (oestrogens, androgens, progestogens and glucocorticoids) in cow's milk samples from three farms in Switzerland. An isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of 12 hormones in milk. Some hormonal levels from individual cows showed large variations. The average levels of the hormones analysed (17α-estradiol = 31 ng kg(-)(1), 17β-estradiol = 6 ng kg(-)(1), estrone = 159 ng kg(-)(1), 4-androstenedione = 684 ng kg(-)(1), progesterone = 15486 ng kg(-)(1), 17-hydroxyprogesterone = 214 ng kg(-)(1), cortisone = 112 ng kg(-)(1), and cortisol = 235 ng kg(-)(1)) were comparable with literature data. Estriol, testosterone and androstenediols were not detected at their respective limit of quantification. No significant differences of hormonal content among milk from cows at different lactation/calving numbers were evidenced, except for progesterone and 4-androstenedione. Due to confounding parameters linked to the physiological stage of the animal, like pregnancy and gestational stage (pregnancy trimester), the causal correlation between the variation of the levels for these two hormones and the lactation/calving number could not be unambiguously demonstrated.

Androgens and progestogens are two important groups of endocrine disrupting compounds (EDCs) which are implicated to produce severe detrimental impact over aquatic biota, even at very low concentrations of ngL(-1). For this reason, one of the major challenges to analytical chemists is the development of sensitive and selective extraction processes which allow the rapid and green determination of these emerging pollutants at low concentrations in environmental samples. Fabric phase sorptive extraction is a new, highly sensitive, efficient and solvent minimized technique which combine the advantages of sol-gel derived microextraction sorbents and the rich surface chemistry of cellulose fabric substrate. This process has several advantages such as minimum usage of organic solvents, short extraction times, small sample volumes and high analyte preconcentration factors. In this study, an extraction method based on sorptive fabric phase coupled to ultra-high-performance liquid chromatography tandem mass spectrometry detection (FPSE-UHPLC-MS/MS) has been developed for the determination of four progestogens and six androgens in environmental and biological samples. All the parameters involved in the extraction, such as sample volume, extraction and desorption times, desorption solvent volume and sample pH values have been optimized. The developed method provides satisfactory limits of detection (between 1.7 and 264ngL(-1)), good recoveries and low relative standard deviations (below 10% in tap and osmosis water and below 20% in wastewater and urine). Subsequently, the method was used to analyse tap water, wastewater treated with different processing technologies and urine samples. The concentrations of the detected hormones ranged from 28.3 to 227.3 ngL(-1) in water samples and from 1.1 to 3.7μgL(-1) in urine samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The Himalayan musk deer (Moschus chrysogaster), an endangered species, is facing threat of extinction globally due to severe hunting for its musk, and efforts are under way in India to breed them in captivity. However, no information is available on the reproductive cycles of the species. In this study, we aimed to standardize an enzyme immunoassay (EIA) procedure for monitoring pregnancy using fecal samples. We collected fecal samples for 12 months from five captive females maintained at the Musk Deer Research Centre, Bageshwar, Uttarakhand, India. Three of these females were observed mating and gave birth, whereas two were seen mating but did not give birth. The gestation periods for the three females were 183, 185, and 199 days, respectively. High-pressure liquid chromatography revealed the presence of immunoreactive pregnanediol-3-glucuronide (PdG), progesterone, and 5α-pregnan-3α-ol-20-one (5-alpha-pregnane) metabolites in the fecal samples. We used EIAs against progesterone, PdG, and 5-alpha-pregnane to monitor pregnancy. We found PdG EIA to be a highly accurate and sensitive assay compared with the other two assays in detecting pregnancy. We conclude that PdG EIA can be used to diagnose and monitor pregnancy in Himalayan musk deer using fecal steroid analysis, at an early stage of 3 months after mating. This study would help in conservation breeding of musk deer in captivity and in monitoring the reproductive status of the species in the wild., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Basic reproductive information in female jaguars (Panthera onca) is lacking, thus longitudinal fecal samples from seven females were analyzed via enzyme immunoassay to measure estradiol and progestin metabolites throughout the year. Mean estrus length of 194 estrus periods measured hormonally was 6.5±0.3d, mean peak fecal estradiol concentration was 138.7±5.7ng/g; and in one female, estrus resumption occurred approximately 15d post-partum. Ovulation, as indicted by sustained elevated progestin concentrations (>20d), was successfully induced one time by treatment with exogenous hormones in one female and by physical vaginal stimulation in two females a combined total of three times. Elevated fecal progestin was observed outside exogenous stimulation on five occasions, suggesting ovulation occurred spontaneously. Mean length of physically induced and spontaneous pseudopregnancies was 24.7±4.2d and 29.6±2.6d, respectively, and mean length of pregnancy (n=2) was 98.0±0.0d. Mean peak progestin concentration for spontaneous and induced pseudopregnancies, and pregnancy was 7.4±1.4μg/g, 6.4±1.2μg/g, and 13.7±1.0μg/g, respectively. This data suggests jaguars are polyestrous and generally induced ovulators, with a moderate incidence of spontaneous ovulation. Additionally, two protocols to successfully stimulate ovarian activity in jaguars are described., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Routine faecal steroid monitoring has been used to aid the management of five captive Asian elephant (Elephas maximus) females at Chester Zoo, UK, since 2007. Progestagen analysis initially revealed synchronised oestrous cycles among all females. However, a 14- to 20-week period of temporary acyclicity subsequently occurred in three females, following several management changes (increased training, foot-care and intermittent matriarch removal for health reasons) and the initiation of pregnancy in another female. The aim of this study was to retrospectively investigate whether these management changes were related to increased adrenal activity and disruption of ovarian activity, or whether social factors may have been involved in the temporary cessation of cyclicity. Faecal samples collected every other day were analysed to investigate whether glucocorticoid metabolites were related to reproductive status (pregnant, cycling, acyclic) or management (training, foot-care, matriarch presence). Routine training and foot-care were not associated with adrenal activity; however, intensive foot-care to treat an abscess in one female was associated with increased glucocorticoid concentration. Matriarch presence influenced adrenal activity in three females, being lower when the matriarch was separated from the group at night compared to being always present. However, in the females that exhibited temporary acyclicity, there was no consistent relationship between glucocorticoids and cyclicity state. Although the results of this study do not fully explain this occurrence, the highly synchronised nature of oestrous cycles within this group, and the concurrent acyclicity in three females, raises the question of whether social factors could have been involved in the temporary disruption of ovarian activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)