Single and multi-dose pharmacology of recombinant and urinary human chorionic gonadotrophin in men.

OBJECTIVE: Human choriogonadotrophin (hCG) treatment of gonadotrophin-deficient infertile men uses hCG of urinary (uhCG) or recombinant (rhCG) origin, but these treatments have not been compared nor are there studies defining rhCG dosing in men. DESIGN: hCG products were studied in randomized cross-over single-dose studies of standard (Study 1, 1500 IU and 62.5 µg, respectively) or high (Study 2, 5000 IU and 250 µg) dose and a multi-dose population pharmacology study of hCG use. PARTICIPANTS: Eight (Study 1) and seven (Study 2) volunteers in cross-over and 52 gonadotrophin-deficient men in the multi-dose study MEASUREMENTS: In cross-over studies, serum testosterone (T), dihydrotestosterone (DHT) and estradiol by liquid chromatography-mass spectrometry (LCMS) and serum hCG, LH, FSH, SHBG and T (observational study) by immunoassays. RESULTS: After standard and high-dose injection, serum hCG and testosterone responses had similar timing and peak concentrations except for a mildly lower early (<48 h) serum testosterone with uhCG. In the multi-dosing study, both hCGs had similar pharmacokinetics (pooled half-life 5.8 days, p < .001), while serum testosterone concentrations were stable after injection and did not differ between hCG products. Bench testing verified that 20% of pens from 4/10 individuals were used inappropriately. CONCLUSIONS: Although hCG pharmacokinetics are not formally bioequivalent, the similar pharmacodynamic effects on serum testosterone indicate that at the doses tested both hCGs provide comparable clinical effects. The starting dose of rhCG for treating gonadotrophin-deficient men should be 62.5 µg (6 clicks) of the rhCG pen.

Controlled dual release of dihydrotestosterone and flutamide from polycaprolactone electrospun scaffolds accelerate burn wound healing.

Wound healing is a complex process involving multiple independent and overlapping sequential physiological mechanisms. In addition to cutaneous injury, a severe burn stimulates physiological derangements that induce a systemic hypermetabolic response resulting in impaired wound healing. Topical application of the anti-androgen drug, flutamide accelerates cutaneous wound healing, whereas paradoxically systemic dihydrotestosterone (DHT) improves burn wound healing. We developed and characterized a PCL scaffold that is capable of controlled release of androgen (DHT) and anti-androgen (F) individually or together. This study aims to investigate whether local modification of androgen actions has an impact on burn injury wound healing. In a full-thickness burn wound healing, mouse model, DHT/F-scaffold showed a significantly faster wound healing compared with F-scaffold or DHT-scaffold. Histology analysis confirmed that DHT/F-scaffold exhibited higher re-epithelization, cell proliferation, angiogenesis, and collagen deposition. Dual release of DHT and F from PCL scaffolds promoted cell proliferation of human keratinocytes and alters the keratinocyte cell cycle. Lastly, no adverse effects on androgen-dependent organs, spleen and liver were observed. In conclusion, we demonstrated DHT plus F load PCL scaffolds accelerated burn wound healing when loading alone did not. These findings point to a complex role of androgens in burn wound healing and open novel therapeutic avenues for treating severe burn patients.

Evaluation of testosterone, estradiol and progesterone immunoassay calibrators by liquid chromatography mass spectrometry.

OBJECTIVES: In clinical practice, steroid measurements are performed mainly by direct, non-extraction immunoassays adapted to high throughput, automated immunoassay platforms and employing secondary calibrators. The accuracy of such steroid immunoassays is limited by cross-reactivity with structurally related steroids and nonspecific matrix interference as well as the metrological traceability of manufacturer supplied calibrators. The accuracy of steroid immunoassay calibrators has been little investigated by independent chemical methods. METHODS: Steroid concentrations of 41 calibrators (4-6 replicates per calibrator) supplied by four manufacturers for use in testosterone (T), estradiol (E2), and progesterone (P4) commercial immunoassays were measured by ultra-pressure liquid chromatography-mass spectrometry (UPLC-MS). RESULTS: Among 14 non-zero T calibrators, six (43 %) deviated significantly from the label concentration with 29 % outside 20 % of it. Among 14 E2 calibrators, eight (57 %) deviated significantly, whereas seven (50 %) were outside 20 % of the label concentration. Among 11 P4 calibrators, eight (73 %) deviated significantly whereas four (36 %) were outside within 20 % of the label concentration. CONCLUSIONS: We conclude that inaccurate calibration of manufacturer's supplied standards may contribute to inaccuracy of commercial direct steroid immunoassays.

Simultaneous measurement of 13 circulating vitamin D3 and D2 mono and dihydroxy metabolites using liquid chromatography mass spectrometry.

OBJECTIVES: Clinical evaluation of vitamin D status is conventionally performed by measuring serum levels of a single vitamin D metabolite, 25-hydroxyvitamin D predominantly by immunoassay methodology. However, this neglects the complex metabolic pathways involved in vitamin D bioactivity, including two canonical forms D3 and D2, bioactive 1,25-dihydroxy metabolites and inactive 24-hydroxy and other metabolites. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can measure multiple analytes in a sample during a single run with high sensitivity and reference level specificity. We therefore aimed to develop and validate a LC-MS/MS method to measure simultaneously 13 circulating vitamin D metabolites and apply it to 103 human serum samples. RESULTS: The LC-MS/MS method using a Cookson-type derivatization reagent phenyl-1,2,4-triazoline-3,5-dione (PTAD) quantifies 13 vitamin D metabolites, including mono and dihydroxy-metabolites, as well as CYP11A1-derived D3 and D2 metabolites in a single run. The lower limit of quantitation was 12.5 pg/mL for 1,25(OH)2D3 with accuracy verified by analysis of National Institute of Standards and Technology (NIST) 972a standards. Quantification of seven metabolites (25(OH)D3, 25(OH)D2, 3-epi-25(OH)D3, 20(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 and 1,20S(OH)2D3) was consistently achieved in human serum samples. CONCLUSIONS: This profiling method can provide new insight into circulating vitamin D metabolite pathways forming the basis for improved understanding of the role of vitamin D in health and disease.

Neuroendocrine androgen action is a key extraovarian mediator in the development of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex hormonal disorder characterized by reproductive, endocrine, and metabolic abnormalities. As the origins of PCOS remain unknown, mechanism-based treatments are not feasible and current management relies on treatment of symptoms. Hyperandrogenism is the most consistent PCOS characteristic; however, it is unclear whether androgen excess, which is treatable, is a cause or a consequence of PCOS. As androgens mediate their actions via the androgen receptor (AR), we combined a mouse model of dihydrotestosterone (DHT)-induced PCOS with global and cell-specific AR-resistant (ARKO) mice to investigate the locus of androgen actions that mediate the development of the PCOS phenotype. Global loss of the AR reveals that AR signaling is required for all DHT-induced features of PCOS. Neuron-specific AR signaling was required for the development of dysfunctional ovulation, classic polycystic ovaries, reduced large antral follicle health, and several metabolic traits including obesity and dyslipidemia. In addition, ovariectomized ARKO hosts with wild-type ovary transplants displayed normal estrous cycles and corpora lutea, despite DHT treatment, implying extraovarian and not intraovarian AR actions are key loci of androgen action in generating the PCOS phenotype. These findings provide strong evidence that neuroendocrine genomic AR signaling is an important extraovarian mediator in the development of PCOS traits. Thus, targeting AR-driven mechanisms that initiate PCOS is a promising strategy for the development of novel treatments for PCOS.

Steroid profiles by liquid chromatography-mass spectrometry of matched serum and single dominant ovarian follicular fluid from women undergoing IVF.

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 3ß, 5α androstanediol (3ß-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 3ß-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.

Elevated expression of the Sertoli cell androgen receptor disrupts male fertility.

Recently, we created a unique gain-of-function mouse model with Sertoli cell-specific transgenic androgen receptor expression (TgSCAR) showing that SCAR activity controls the synchronized postnatal development of somatic Sertoli and Leydig cells and meiotic-postmeiotic germ cells. Moderate TgSCAR (TgSCAR(m)) expression reduced testis size but had no effect on male fertility. Here, we reveal that higher TgSCAR expression (TgSCAR(H)) causes male infertility. Higher SCAR activity, shown by upregulated AR-dependent transcripts (Rhox5, Spinw1), resulted in smaller adult TgSCAR(H) testes (50% of normal) despite normal or elevated circulating and intratesticular testosterone levels. Unlike fertile TgSCAR(m) males, testes of adult TgSCAR(H) males exhibited focal regions of interstitial hypertrophy featuring immature adult Leydig cells and higher intratesticular dihydrotestosterone and 5α-androstane 3α,17ß-diol levels that are normally associated with pubertal development. Mature TgSCAR(H) testes also exhibited markedly reduced Sertoli cell numbers (70%), although meiotic and postmeiotic germ cell/Sertoli cell ratios were twofold higher than normal, suggesting that elevated TgSCAR activity supports excessive spermatogenic development. Concurrent with the higher germ cell load of TgSCAR(H) Sertoli cells were increased levels of apoptotic germ cells in TgSCAR(H) relative to TgSCAR(m) testes. In addition, TgSCAR(H) testes displayed unique morphological degeneration that featured accumulated cellular and spermatozoa clusters in dilated channels of rete testes, consistent with reduced epididymal sperm numbers. Our findings reveal for the first time that excessive Sertoli cell AR activity in mature testes can reach a level that disturbs Sertoli/germ cell homeostasis, impacts focal Leydig cell function, reduces sperm output, and disrupts male fertility.

Measurement of Estradiol in Human Serum by LC-MS/MS Using a Novel Estrogen-Specific Derivatization Reagent.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described that employs a novel derivatization reagent for the measurement of serum estradiol (E2), with simultaneous analysis of underivatized testosterone (T) and dihydrotestosterone (DHT). The main advantage of the new derivatization reagent 1,2-dimethylimidazole-5-sulfonyl chloride is its analyte-specific fragmentation that enables monitoring of confirmatory mass transitions with high sensitivity. The reaction mixture can be analyzed without additional purification steps using a 9.5 min gradient run, and sensitive detection is achieved with a triple quadrupole mass spectrometer using atmospheric pressure photoionization. Method validation was performed with human serum samples, including a comparison with a standard LC-MS/MS method using 120 samples from a clinical study, and analysis of certified E2 serum reference materials BCR-576, BCR-577, and BCR-578. The lower limits of quantification for E2, T, and DHT were 0.5 pg/mL, 25 pg/mL, and 0.10 ng/mL, respectively, from a 200-µL sample. Validation results indicated good accuracy and agreement with established, conventional LC-MS/MS assays, demonstrating suitability for analysis of samples containing E2 in the low pg/mL range, such as serum from men, children, and postmenopausal women.

Testosterone attenuates and the selective estrogen receptor modulator, raloxifene, potentiates amphetamine-induced locomotion in male rats.

Although sex steroids are known to modulate brain dopamine, it is still unclear how testosterone modifies locomotor behaviour controlled, at least in part, by striatal dopamine in adolescent males. Our previous work suggests that increasing testosterone during adolescence may bias midbrain neurons to synthesise more dopamine. We hypothesised that baseline and amphetamine-induced locomotion would differ in adult males depending on testosterone exposure during adolescence. We hypothesised that concomitant stimulation of estrogen receptor signaling, through a selective estrogen receptor modulator (SERM), raloxifene, can counter testosterone effects on locomotion. Male Sprague-Dawley rats at postnatal day 45 were gonadectomised (G) or sham-operated (S) prior to the typical adolescent testosterone increase. Gonadectomised rats were either given testosterone replacement (T) or blank implants (B) for six weeks and sham-operated (i.e. intact or endogenous testosterone group) were given blank implants. Subgroups of sham-operated, gonadectomised and gonadectomised/testosterone-replaced rats were treated with raloxifene (R, 5mg/kg) or vehicle (V), daily for the final four weeks. There were six groups (SBV, GBV, GTV, SBR, GBR, GTR). Saline and amphetamine-induced (1.25mg/kg) locomotion in the open field was measured at PND85. Gonadectomy increased amphetamine-induced locomotion compared to rats with endogenous or with exogenous testosterone. Raloxifene increased amphetamine-induced locomotion in rats with either endogenous or exogenous testosterone. Amphetamine-induced locomotion was negatively correlated with testosterone and this relationship was abolished by raloxifene. Lack of testosterone during adolescence potentiates and testosterone exposure during adolescence attenuates amphetamine-induced locomotion. Treatment with raloxifene appears to potentiate amphetamine-induced locomotion and to have an opposite effect to that of testosterone in male rats.

Glucocorticoid receptor in prostate epithelia is not required for corticosteroid-induced epithelial hyperproliferation in the mouse prostate.

BACKGROUND: Glucocorticoids are used as a last resort treatment for prostate cancer but the cell-specific glucocorticoid receptor (GR) mediated actions and the role of endogenous glucocorticoids in prostate are not understood. METHODS: We evaluated the influence of prostate epithelial GR mediated actions of glucocorticoids in prostate structural development by comparing the intact wild-type (WT) and prostate epithelia selective GR knockout (peGRKO) males at 8, 20, and 35 weeks of age. We also determined the cell-specific role of GR on corticosterone treatment induced prostate abnormalities by treating peGRKO and WT male mice with corticosterone depot pellets or placebo for 4 weeks. RESULTS: GR was not expressed in the epithelial cells of peGRKO prostate unlike WT but was expressed in stromal of both peGRKO and WT mice. Nevertheless, prostate weights, histological appearance, and secretory protein probasin expression in peGRKO were no different from WT. Despite lacking epithelial GR, the peGRKO prostate demonstrated corticosterone treatment induced hyperplasia similar to WT suggesting that stromal rather than epithelial GR mediates the hyperproliferative mouse prostate response to corticosterone. As circulating androgen levels were not affected by corticosterone treatment, this effect is likely to be mediated directly via prostate GR. CONCLUSIONS: Sustained administration of corticosterone induces prostate hyperplasia, which is mediated via GR expressed predominantly in the stroma. Thus GR mediated actions in the prostate may have significant cell-specific effects that could be utilized for more rational therapeutic approaches in prostate cancer treatment. This also illustrates the paracrine hormonal mechanisms in prostate pathophysiology.

Dried blood spot sampling of testosterone microdosing in healthy females.

Capillary dried blood spot (DBS) analysis coupled with multi-analyte steroid liquid chromatography mass spectrometry (LCMS) is attractive for field studies, home-based self-sampling as well as clinical trials by eliminating costly and laborious sample processing involving venipuncture and frozen storage/shipping while providing multiple steroid measurements from a single small sample. We investigated steroid measurements in DBS samples stored for four years at room temperature prior to analysis compared with the original venipuncture serum samples. Healthy women (n=12) provided paired DBS and blood samples over two weeks run-in before seven days treatment with daily transdermal T gel (12.5 mg) and after the end of treatment on days 0, 1, 2, 4, 7 and 14. Compliance with treatment and sampling was high and no adverse effects were reported. Testosterone (T), androstenedione (A4), 17 hydroxyprogesterone (17OHP) and progesterone (P4) were measured in extracted DBS samples as whole blood concentrations with and without adjustment for hematocrit. Using the same LCMS methods, DBS T and A4 measurements had high correlation with minimal bias from prior serum measurements with DBS T displaying the same pattern as serum, with or without hematocrit adjustment. However, serial whole blood measurements of T without hematocrit adjustment provided the best fitting model compared with serum, urine, or hematocrit-adjusted whole blood T measurements. These finding facilitate and simplify DBS methodology for wider field and home-based self-sampling studies of reproductive steroids indicating the need for hematocrit adjustment may be superfluous.

High circulating concentrations of estradiol are anabolic for bone mass and strength in an adult male to female transgender mouse model.

The effects of gender affirming hormone therapy (GAHT) on bone microarchitecture and fracture risk in adult transgender women is unclear. To investigate the concept that skeletal integrity and strength in trans women may be improved by treatment with a higher dose of GAHT than commonly prescribed, we treated adult male mice with a sustained, high dose of estradiol. Adult male mice at 16 weeks of age were administered ~1.3 mg estradiol by silastic implant, implanted intraperitoneally, for 12 weeks. Controls included vehicle treated intact females and males. High-dose estradiol treatment in males stimulated the endocortical deposition of bone at the femoral mid-diaphysis, increasing cortical thickness and bone area. This led to higher stiffness, maximum force, and the work required to fracture the bone compared to male controls, while post-yield displacement was unaffected. Assessment of the material properties of the bone showed an increase in both elastic modulus and ultimate stress in the estradiol treated males. Treatment of male mice with high dose estradiol was also anabolic for trabecular bone, markedly increasing trabecular bone volume, number and thickness in the distal metaphysis which was accompanied by an increase in the histomorphometric markers of bone remodelling, mineralizing surface/bone surface, bone formation rate and osteoclast number. In conclusion, a high dose of estradiol is anabolic for cortical and trabecular bone in a male to female transgender mouse model, increasing both stiffness and strength. These findings suggest that increasing the current dose of GAHT administered to trans women, while considering other potential adverse effects, may be beneficial to preserving their bone microstructure and strength.

Estradiol increases cortical and trabecular bone accrual and bone strength in an adolescent male-to-female mouse model of gender-affirming hormone therapy.

The effects of gender-affirming hormone therapy on the skeletal integrity and fracture risk in transitioning adolescent trans girls are unknown. To address this knowledge gap, we developed a mouse model to simulate male-to-female transition in human adolescents in whom puberty is first arrested by using gonadotrophin-releasing hormone analogs with subsequent estradiol treatment. Puberty was suppressed by orchidectomy in male mice at 5 weeks of age. At 3 weeks post-surgery, male-to-female mice were treated with a high dose of estradiol (~0.85 mg) by intraperitoneal silastic implantation for 12 weeks. Controls included intact and orchidectomized males at 3 weeks post-surgery, vehicle-treated intact males, intact females and orchidectomized males at 12 weeks post-treatment. Compared to male controls, orchidectomized males exhibited decreased peak bone mass accrual and a decreased maximal force the bone could withstand prior to fracture. Estradiol treatment in orchidectomized male-to-female mice compared to mice in all control groups was associated with an increased cortical thickness in the mid-diaphysis, while the periosteal circumference increased to a level that was intermediate between intact male and female controls, resulting in increased maximal force and stiffness. In trabecular bone, estradiol treatment increased newly formed trabeculae arising from the growth plate as well as mineralizing surface/bone surface and bone formation rate, consistent with the anabolic action of estradiol on osteoblast proliferation. These data support the concept that skeletal integrity can be preserved and that long-term fractures may be prevented in trans girls treated with GnRHa and a sufficiently high dose of GAHT. Further study is needed to identify an optimal dose of estradiol that protects the bone without adverse side effects.

Sex steroids and androgen biomarkers in the healthy man study: within-person variability and impact of fasting.

OBJECTIVE: Serum testosterone measurements in clinical practice mostly utilize "direct" (non-extraction) immunoassays which have method-specific bias due to steroid cross-reactivity and nonspecific matrix artifacts. Although more accurate, sensitive, and specific liquid chromatography-mass spectrometry (LCMS) dominates in clinical research, the within-person variability of serum testosterone in healthy men using LCMS measurement is not reported. DESIGN: Longitudinal multi-sampling observational study of men in excellent health over 3 months. METHODS: Elite healthy men (n = 325) over 40 years of age in excellent, asymptomatic health provided 9 blood samples over 3 months with serum testosterone, dihydrotestosterone (DHT), estradiol (E2), and estrone (E1) measured by validated LCMS with conventional biochemical and anthropometric variables. RESULTS: Quantitative estimates of within-person variability within day and between day, week, month, and quarter were stable other than an increase due to fasting. The androgen biomarkers most sensitive to age and testosterone among widely used biochemical and anthropometric variables in middle-aged and older men were identified. CONCLUSIONS: This study provides estimates of variability in serum testosterone and the best androgen biomarkers that may prove useful for future studies of androgen action in male ageing.

Characterizing the neuroendocrine and ovarian defects of androgen receptor-knockout female mice.

Homozygous androgen receptor (AR)-knockout (ARKO) female mice are subfertile due to both intra- and extraovarian (neuroendocrine) defects as defined by ovary transplantation. Using ARKO mice, this study set out to reveal the precise AR-regulated pathways required for optimal androgen-regulated ovulation and fertility. ARKO females exhibit deficient neuroendocrine negative feedback, with a reduced serum luteinizing hormone (LH) response to ovariectomy (OVX) (P < 0.01). Positive feedback is also altered as intact ARKO females, at late proestrus, exhibit an often mistimed endogenous ovulatory LH surge. Furthermore, at late proestrus, intact ARKO females display diminished preovulatory serum estradiol (E2; P < 0.01) and LH (P < 0.05) surge levels and reduced Kiss1 mRNA expression in the anteroventral periventricular nucleus (P < 0.01) compared with controls. However, this reduced ovulatory LH response in intact ARKO females can be rescued by OVX and E2 priming or treatment with endogenous GnRH. These findings reveal that AR regulates the negative feedback response to E2, E2-positive feedback is compromised in ARKO mice, and AR-regulated negative and positive steroidal feedback pathways impact on intrahypothalamic control of the kisspeptin/GnRH/LH cascade. In addition, intraovarian AR-regulated pathways controlling antral to preovulatory follicle dynamics are disrupted because adult ARKO ovaries collected at proestrus have small antral follicles with reduced oocyte/follicle diameter ratios (P < 0.01) and increased proportions of unhealthy large antral follicles (P < 0.05) compared with controls. As a consequence of aberrant follicular growth patterns, proestrus ARKO ovaries also exhibit fewer preovulatory follicle (P < 0.05) and corpora lutea numbers (P < 0.01). However, embryo development to the blastocyst stage is unchanged in ARKO females, and hence, the subfertility is a consequence of reduced ovulations and not altered embryo quality. These findings reveal that the AR has a functional role in neuroendocrine regulation and timing of the ovulatory LH surge as well as antral/preovulatory follicle development.

Unexpected Plasma Gonadal Steroid and Prolactin Levels Across the Mouse Estrous Cycle.

Despite the importance of the mouse in biomedical research, the levels of circulating gonadal steroids across the estrous cycle are not established with any temporal precision. Using liquid chromatography-mass spectrometry, now considered the gold standard for steroid hormone analysis, we aimed to generate a detailed profile of gonadal steroid levels across the estrous cycle of C57BL/6J mice. For reference, luteinizing hormone (LH) and prolactin concentrations were measured in the same samples by sandwich enzyme-linked immunosorbent assay. Terminal blood samples were collected at 8-hour intervals (10 Am, 6 Pm, 2 Am) throughout the 4 stages of the estrous cycle. As expected, the LH surge was detected at 6 Pm on proestrus with a mean (±SEM) concentration of 11 ± 3 ng/mL and occurred coincident with the peak in progesterone levels (22 ± 4 ng/mL). Surprisingly, estradiol concentrations peaked at 10 Am on diestrus (51 ± 8 pg/mL), with levels on proestrus 6 Pm reaching only two-thirds of this value (31 ± 5 pg/mL). We also observed a proestrus peak in prolactin concentrations (132.5 ± 17 ng/mL) that occurred earlier than expected at 2 Am. Estrone and androstenedione levels were often close to the limit of detection (LOD) and showed no consistent changes across the estrous cycle. Testosterone levels were rarely above the LOD (0.01 ng/mL). These observations provide the first detailed assessment of fluctuating gonadal steroid and reproductive hormone levels across the mouse estrous cycle and indicate that species differences exist between mice and other spontaneously ovulating species.

Thyroid Hormone Abuse Among Elite Athletes.

Context: Thyroid hormone (TH) abuse for performance enhancement in sport remains controversial and it is not prohibited in sports under the World Anti-Doping Code. However, the prevalence of TH usage in athletes is not known. Objective: We investigated TH use among Australian athletes undergoing antidoping tests for competition in World Anti-Doping Agency (WADA)-compliant sports by measuring TH in serum and surveying mandatory doping control form (DCF) declarations by athletes of all drugs used in the week prior to the antidoping test. Methods: Serum thyroxine (T4), triiodothyronine (T3), and reverse T3 were measured by liquid chromatography-mass spectrometry and serum thyrotropin, free T4, and free T3 by immunoassays in 498 frozen serum samples from antidoping tests together with a separate set of 509 DCFs. Results: Two athletes had biochemical thyrotoxicosis giving a prevalence of 4 per 1000 athletes (upper 95% confidence limit [CL] 16). Similarly, only 2 of 509 DCFs declared usage of T4 and none for T3, also giving a prevalence of 4 (upper 95% CL 16) per 1000 athletes. These estimates were consistent with DCF analyses from international competitions and lower than the estimated T4 prescription rates in the age-matched Australian population. Conclusion: There is minimal evidence for TH abuse among Australian athletes being tested for competing in WADA-compliant sports.

Circulating Conjugated and Unconjugated Vitamin D Metabolite Measurements by Liquid Chromatography Mass Spectrometry.

CONTEXT: Vitamin D status is conventionally defined by measurement of unconjugated circulating 25-hydroxyvitamin D (25OHD), but it remains uncertain whether this isolated analysis gives sufficient weight to vitamin D's diverse metabolic pathways and bioactivity. Emerging evidence has shown that phase II endocrine metabolites are important excretory or storage forms; however, the clinical significance of circulating phase II vitamin D metabolites remains uncertain. OBJECTIVE: In this study we analyzed the contribution of sulfate and glucuronide vitamin D metabolites relative to unconjugated levels in human serum. METHODS: An optimized enzyme hydrolysis method using recombinant arylsulfatase (Pseudomonas aeruginosa) and beta-glucuronidase (Escherichia coli) was combined with liquid chromatography mass spectrometry (LC-MS/MS) analysis to measure conjugated and unconjugated vitamin D metabolites 25OHD3, 25OHD2, 3-epi-25OHD3, and 24,25(OH)2D3. The method was applied to the analysis of 170 human serum samples from community-dwelling men aged over 70 years, categorized by vitamin D supplementation status, to evaluate the proportions of each conjugated and unconjugated fraction. RESULTS: As a proportion of total circulating vitamin D metabolites, sulfate conjugates (ranging between 18% and 53%) were a higher proportion than glucuronide conjugates (ranging between 2.7% and 11%). The proportion of conjugated 25OHD3 (48 ± 9%) was higher than 25OHD2 conjugates (29.1 ± 10%) across all supplementation groups. Conjugated metabolites correlated with their unconjugated forms for all 4 vitamin D metabolites (r = 0.85 to 0.97). CONCLUSION: Sulfated conjugates form a high proportion of circulating vitamin D metabolites, whereas glucuronide conjugates constitute a smaller fraction. Our findings principally in older men highlight the differences in abundance between metabolites and suggest a combination of both conjugated and unconjugated measurements may provide a more accurate assessment of vitamin D status.

Detection of testosterone microdosing in healthy females.

The ready detectability of synthetic androgens by mass spectrometry (MS)-based antidoping tests has reoriented androgen doping to using testosterone (T), which must be distinguished from its endogenous counterpart making detection of exogenous T harder. We investigated urine and serum steroid and hematological profiling individually and combined to determine the optimal detection model for T administration in women. Twelve healthy females provided six paired blood and urine samples over 2 weeks prior to treatment consisting of 12.5-mg T in a topical transdermal gel applied daily for 7 days. Paired blood and urine samples were then obtained at the end of treatment and Days 1, 2, 4, 7, and 14 days later. Compliance with treatment and sampling was high, and no adverse effects were reported. T treatment significantly increased serum and urine T, serum dihydrotestosterone (DHT), urine 5α-androstane-3α,17ß-diol (5α-diol) epitestosterone (E), and urine T/E ratio with a brief window of detection (2-4 days) as well as total and immature (medium and high fluorescence) reticulocytes that remained elevated over the full 14 posttreatment days. Carbon isotope ratio MS and the OFF score and Abnormal Blood Profile score (ABPS) were not discriminatory. The optimal multivariate model to identify T exposure combined serum T, urine T/E ratio with three hematological variables (% high fluorescence reticulocytes, mean corpuscular hemoglobin, and volume) with the five variables providing 93% correct classification (4% false positive, 10% false negatives). Hence, combining select serum and urine steroid MS variables with reticulocyte measures can achieve a high but imperfect detection of T administration to healthy females.

Recovery of male reproductive endocrine function after ceasing prolonged testosterone undecanoate injections.

CONTEXT: The time course of male reproductive hormone recovery after stopping injectable testosterone undecanoate (TU) treatment is not known. OBJECTIVE: The aim of this study was to investigate the rate, extent, and determinants of reproductive hormone recovery over 12 months after stopping TU injections. MATERIALS AND METHODS: Men (n = 303) with glucose intolerance but without pathologic hypogonadism who completed a 2-year placebo (P)-controlled randomized clinical trial of TU treatment were recruited for further 12 months while remaining blinded to treatment. Sex steroids (testosterone (T), dihydrotestosterone, oestradiol, oestrone) by liquid chromatography-mass sprectometry, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and sex hormone-binding globulin (SHBG) by immunoassays and sexual function questionnaires (Psychosexual Diary Questionnaire, International Index of Erectile Function, and short form survey (SF-12)) were measured at entry (3 months after the last injection) and 6, 12, 18, 24, 40, and 52 weeks later. RESULTS: In the nested cohort of TU-treated men, serum T was initially higher but declined at 12 weeks remaining stable thereafter with serum T and SHBG at 11 and 13%, respectively, lower than P-treated men. Similarly, both questionnaires showed initial carry-over higher scores in T-treated men but after 18 weeks showed no difference between T- and P-treated men. Initially, fully suppressed serum LH and FSH recovered slowly towards the participant's own pre-treatment baseline over 12 months since the last injection. CONCLUSIONS: After stopping 2 years of 1000 mg injectable TU treatment, full reproductive hormone recovery is slow and progressive over 15 months since the last testosterone injection but may take longer than 12 months to be complete. Persistent proportionate reduction in serum SHBG and T reflects lasting exogenous T effects on hepatic SHBG secretion rather than androgen deficiency.