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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 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 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.

Fate and occurrence of fourteen androgens, four estrogens, five glucocorticoids and five progestagens were investigated in three swine farms and three dairy cattle farms with different farming scales and wastes disposal systems in China. Twenty-one, 22, and 12 of total 28 steroids were detected in feces samples with concentrations ranging from below method limit of quantitation (

Hormonal changes associated with reproductive activity in the unique pollen and nectar-feeding marsupial Honey possum, Tarsipes rostratus, have been monitored by the measurement of sex steroids excreted via the faeces. From a radio-metabolism study, 63% of administered [(14)C]oestradiol was excreted in the faeces and 37% via the urine. Peak levels in the faeces were reached 6h after injection and by a mean 12h, 95% of steroid was eliminated. The principal metabolic products of progesterone that were identified by chromatographic analysis were the isomers 5alpha- and 5beta-pregnan-3beta-ol-20-one with only trace amounts of progesterone and the isomers 5beta-pregnan-3beta,20beta- and 20alpha-diols. Extended excretory profiles for faecal progestagens (PM) and oestradiol-17beta (E(2)) are reported for the first time in a marsupial. The profiles from 4 females held in indoor cages with an artificial photoperiod suggest that long days inhibit reproductive activity in this species, as is the case in a number of other marsupials. One female appeared to resume cycling after a 5-month period and the time between peak levels of both E(2) and PM suggest that the length of the oestrous cycle in the Honey possum is approximately 25 days. The PM profile suggests that the corpora lutea secrete low levels of progesterone for approximately the first 19 days after ovulation, followed by increased rates of excretion during the final 6 days.

One source of both bias and "noise" in fecal steroid analysis is temporal change in steroid concentrations resulting from duration or conditions of fecal sample storage. However, no consensus currently exists regarding correct procedures or precautions necessary for fecal sample storage, and conditions vary widely within field endocrinology literature. This study considered the effects of short-term, weeks-long, storage conditions on quantifiable fecal testosterone (fT), glucocorticoids (fGC), estrogens (fE), and progestagen (fP) metabolite concentrations in wild baboons (Papio cynocephalus). Quadruplicate subsamples of fecal samples (n=29) collected at Amboseli National Park and its environs were subjected to four different storage conditions prior to lyophilization, in order to determine the effects of storage on subsequent steroid concentrations, as assessed by 125I radioimmunoassays. As expected, the best alternative to the "initial condition" of lyophilization at three days after collection was to freeze fecal samples at -20 degrees C for two weeks prior to lyophilization. This storage method resulted in no significant change from initial steroid concentrations for fE, fT, or fP, although fGC showed a slight but significant decline. Storage for two weeks in a charcoal refrigerator caused a mean increase in all four steroid concentrations. However, the results from this storage condition were robust in terms of practical questions asked of the data: fE and fP values still reflected pregnant versus non-pregnant states in baboon females; a fGC profile constructed by age class resembled that created from the samples from the initial condition, although slightly inflated across age classes; and there were only moderate changes in relative fT concentrations across adult males. Knowledge of the effects of storage upon each steroid analyzed within one's study is a necessary component in determining the optimal compromise for storage protocol in a particular research project.

Measurements of reproductive hormones (progestins and estrogens) excreted in feces were used to discriminate between 25 conceptive and 76 nonconceptive (including undetected early abortion) cycles of free-ranging yellow baboons at Mikumi National Park, Tanzania. Conceptive cycles had significantly higher luteal-phase progestin and estrogen concentrations than nonconceptive cycles as early as Day 4 postovulation. However, mean early luteal-phase progestin concentrations in conceptive cycles were lower when conception occurred during ecologically optimal vs. suboptimal times, and among females of high compared to low dominance rank. Mean estrogen concentrations in conceptive cycles showed the opposite dominance rank pattern: mean luteal-phase estrogen concentrations were higher in conceptive cycles of high-compared to low-ranking females. None of these relations existed for nonconceptive cycles. These data suggest that successful implantation is facilitated by relatively high early luteal-phase progestin and estrogen concentrations. However, long-term environmental cues predicting the probability of offspring survival appear to influence the amount of progesterone required for successful implantation; progesterone concentrations necessary to facilitate successful implantation are higher during suboptimal seasons or among females of low dominance rank-cues that also suggest that offspring survival conditions are relatively poor. This may act as a reproductive filter, restricting conception to females whose immediate condition (e.g., low social stress and good physical health) enables them to compensate physiologically and behaviorally for effects associated with these relatively harsh offspring survival conditions.

Oocytes of pre-ovulatory follicles were collected by laparoscopy for in-vitro fertilization in 118 women. Patients were treated either during the natural cycle, or after induction of ovulation with clomiphene citrate, or with human menopausal gonadotrophin (HMG), or clomiphene citrate combined with HMG. The oestrogens (oestrone + oestradiol) and total aromatizable androgens (androstenedione, testosterone and dehydroepiandrosterone and its sulphate) were assayed in follicular fluids using an enzymatic method. The levels of progesterone, 17 alpha OH-progesterone, thromboxane, and prostaglandins (PGE2 and PGF2 alpha) were determined by radioimmunoassay. The best follicular fluid indicator of oocyte fertilization in vitro was the A/E ratio, which was less than 1 when the oocyte was fertilized in vitro and led to a pregnancy. The lowest value for the A/E ratio was obtained with spontaneous ovulation protocols. Regardless of oocyte development, the progesterone level was always greater than 2000 ng/ml, and the PGE2/PGF2 alpha ratio was greater than 1 in all stimulated cycles. Our investigations show that a combination of clomiphene citrate and HMG provides the best stimulation, on the basis of follicular fluid analysis and the outcome of fertilized oocytes.