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.

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.

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.

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.

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.

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.

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.

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.

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.

Depot Pure GnRH Antagonist for Long-term Treatment of Ovarian Hyperthecosis Monitored by Multisteroid LCMS Profiling.

Ovarian hyperthecosis (OHT), severe hyperandrogenism after menopause in the absence of ovarian or adrenal tumors, is usually treated by surgical excision. We report a 58-year-old woman presenting with severe hyperandrogenism (serum testosterone 15.7-31.0 nmol/L, normal female <1.8 nmol/L) with menopausal gonadotropins and virilization but no adrenal or ovarian lesions. Multisteroid profiling by liquid chromatography mass spectrometry (LCMS) of adrenal and ovarian vein samples identified strong gradients in the left ovarian vein (10- to 30-fold vs peripheral blood in 17OHP4, 17 hydroxyprogesterone, 17 hydroxypregnenolone, androstenedione, testosterone, dehydroepiandrosterone) but the right ovarian vein could not be cannulated with the same findings in a second ovarian vein cannulation. OHT diagnosis was confirmed by an injection of a depot pure gonadotropin-releasing hormone (GnRH) antagonist (80 mg Degarelix, Ferring) producing a rapid (<24 hour) and complete suppression of ovarian steroidogenesis as well as serum luteinizing hormone and follicle-stimulating hormone lasting at least 8 weeks, with reduction in virilization but injection site reaction and flushing and vaginal spotting ameliorated by an estradiol patch. Serum testosterone remained suppressed at 313 days after the first dose despite recovery of menopausal gonadotropins by day 278 days. This illustrates use of multisteroid LCMS profiling for confirmation of the OHT diagnosis by ovarian and adrenal vein sampling and monitoring of treatment by peripheral blood sampling. Injection of a depot pure GnRH antagonist produced rapid and long-term complete suppression of ovarian steroidogenesis maintained over 10 months. Hence a depot pure GnRH antagonist can not only rapidly confirm the OHT diagnosis but also induce long-term remission of severe hyperandrogenism without surgery.

Reformulation of PULSAR for Analysis of Pulsatile LH Secretion and a Revised Model of Estrogen-Negative Feedback in Mice.

The recent use of the tail-tip bleeding approach in mice has enabled researchers to generate detailed pulse and surge profiles of luteinizing hormone (LH) secretion in mice. However, the analysis of pulsatile LH secretion is piecemeal across the field with each laboratory using their own methodology. We have reformulated the once-popular PULSAR algorithm of Merriam and Wachter to operate on contemporary computer systems and provide downloadable and online pulse analysis platforms. As it is now possible to record the activity of the gonadotropin-releasing hormone pulse generator in freely behaving mice, we have been able to unambiguously define LH pulses in intact and gonadectomized male and female mice. These data sets were used to determine the appropriate PULSAR parameter sets for analyzing pulsatile LH secretion in the mouse. This was then used to establish an accurate model of estrogen negative feedback in the mouse. Intact and ovariectomized mice given Silastic capsules containing 1, 2, and 4 µg 17-ß-estradiol/20 g body weight were tail-tip bled at 6-min intervals, and the resultant LH profiles were analyzed with PULSAR. Only the 4 µg 17-ß-estradiol capsule treatment was found to return LH pulse amplitude and frequency to that of intact diestrous mice. Ultrasensitive mass spectrometry analysis showed that the 4 µg 17-ß-estradiol capsule generated circulating estradiol levels equivalent to that of diestrous mice. It is hoped that the reformulation of PULSAR and generation of a realistic model of estrogen-negative feedback will provide a platform for the more uniform assessment of pulsatile hormone secretion in mice.

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.

Direct measurement of pregnanediol 3-glucuronide (PDG) in dried urine spots by liquid chromatography-mass spectrometry to detect ovulation.

BACKGROUND: Non-invasive self-testing using an objective chemical method to detect ovulation is valuable for women planning conception, practising contraception or undergoing infertility investigations or treatment. METHODS: Based on luteal phase secretion of progesterone (P4) and excretion of its major metabolite, pregnanediol glucuronide (PDG), we developed a novel direct liquid chromatography-mass spectrometry (LCMS) method to measure PDG and other steroid glucuronides in urine and in dried urine spots (DUS) without deconjugation or derivatization. Urine PDG by LCMS and immunoassay (P3G) and P4 by immunoassay with and without adjustment for creatinine were evaluated in daily first void urine samples from 10 women through a single menstrual cycle in which ovulation was confirmed by serial transvaginal ultrasound. RESULTS: Urine PDG with and without creatinine adjustment was stable during the follicular phase with the expected striking rise in the luteal phase peaking at 5 days after ovulation. Using a single spot urine sample (100 µL) or a DUS (<20 µL urine) and an optimal threshold to distinguish pre- from post-ovulatory samples, in ROC analysis urine PDG adjusted for creatinine accurately identified ovulation in 92 % of samples was comparable with P3G immunoassay and superior to urine P4 with or without adjustment for creatinine. Extending the analysis to two or three consecutive daily samples reduced the false negative rate from 8% to 2.6 % for two and 1.9 % for three urine samples. CONCLUSIONS: This method holds promise as a non-invasive self-test method for women to determine by an objective chemical method their ovulatory status.

Cortisol-dehydroepiandrosterone ratios are inversely associated with hippocampal and prefrontal brain volume in schizophrenia.

While high levels of glucocorticoids are generally neuro-damaging, a related adrenal steroid, dehydroepiandrosterone (DHEA), has anti-glucocorticoid and neuroprotective properties. Previous work has shown increased circulating levels of DHEA and abnormal cortisol/DHEA ratios in people with schizophrenia, however reports are limited and their relationship to neuropathology is unclear. We performed the largest study to date to compare levels of serum DHEA and cortisol/DHEA ratios in people with schizophrenia and healthy controls, and investigated the extent to which cortisol/DHEA ratios predict brain volume. Serum cortisol and DHEA were assayed in 94 people with schizophrenia and 81 healthy controls. T1-weighted high-resolution anatomical scans were obtained using a 3 T Achieva scanner on a subset of 59 people with schizophrenia and 60 healthy controls. Imaging data were preprocessed and analyzed using SPM12. People with schizophrenia had significantly increased serum DHEA levels (p = 0.002), decreased cortisol/DHEA ratios (p = 0.02) and no difference in cortisol levels compared to healthy controls. Cortisol/DHEA ratios were inversely correlated with hippocampal (r = -0.33 p = 0.01) and dorsolateral prefrontal cortex (r = -0.30, p = 0.02) volumes in patients. Our findings suggest that the cortisol/DHEA ratio may be a molecular blood signature of hippocampal and cortical damage. These results further implicate the role of DHEA and hypothalamic-pituitary-adrenal axis dysfunction in the pathophysiology of schizophrenia.

Ultrasensitive Serum Estradiol Measurement by Liquid Chromatography-Mass Spectrometry in Postmenopausal Women and Mice.

Accurate measurement of very low circulating estradiol (E2) (<5 pg/ml) in postmenopausal women and in mice is essential to investigating sex steroid action in target tissues. However, direct immunoassays are too inaccurate and conventional mass spectrometry-based measurement too insensitive at these serum E2 levels. We report application of an ultrasensitive method using a novel estrogen-selective derivatization in liquid chromatography-mass spectrometry to measure serum E2, with a detection limit of 0.25 pg/ml in small (0.2 ml) serum volumes that can quantify serum E2 in 98% and serum E1 in 100% of healthy postmenopausal women. Aromatase inhibitor (AI) treatment of postmenopausal women with breast cancer further reduces serum E2 by 85% and serum estrone (E1) by 80%. The wide scatter of circulating E2 in AI-treated women suggests that the degree of sustained E2 depletion, now quantifiable, may be an efficacy or safety biomarker of adjuvant AI treatment. This ultrasensitive method can also measure serum E2 in most (65%) female but not in any male mice. Further studies are warranted using this and comparable ultrasensitive liquid chromatography-mass spectrometry estrogen measurements to investigate the relationship of circulating E2 (and E1) in male, postmenopausal female, and childhood health where accurate quantification of serum estrogens was not previously feasible. This will focus on the direct impact of estrogens as well as the indirect effects of androgen aromatization on reproductive, bone, and brain tissues and, notably, the efficacy and safety of AIs in adjuvant breast cancer treatment.

Defining the impact of dietary macronutrient balance on PCOS traits.

Lifestyle, mainly dietary, interventions are first-line treatment for women with polycystic ovary syndrome (PCOS), but the optimal diet remains undefined. We combined a hyperandrogenized PCOS mouse model with a systematic macronutrient approach, to elucidate the impact of dietary macronutrients on the development of PCOS. We identify that an optimum dietary macronutrient balance of a low protein, medium carbohydrate and fat diet can ameliorate key PCOS reproductive traits. However, PCOS mice display a hindered ability for their metabolic system to respond to diet variations, and varying macronutrient balance did not have a beneficial effect on the development of metabolic PCOS traits. We reveal that PCOS traits in a hyperandrogenic PCOS mouse model are ameliorated selectively by diet, with reproductive traits displaying greater sensitivity than metabolic traits to dietary macronutrient balance. Hence, providing evidence to support the development of evidence-based dietary interventions as a promising strategy for the treatment of PCOS, especially reproductive traits.

Estrone Is a Strong Predictor of Circulating Estradiol in Women Age 70 Years and Older.

IMPORTANCE: After menopause, estradiol (E2) is predominately an intracrine hormone circulating in very low serum concentrations. OBJECTIVE: The objective of this work is to examine determinants of E2 concentrations in women beyond age 70 years. DESIGN AND SETTING: A cross-sectional, community-based study was conducted. PARTICIPANTS: A total of 5325 women participated, with a mean age of 75.1 years (±â€…4.2 years) and not using any sex steroid, antiandrogen/estrogen, glucocorticoid, or antiglycemic therapy. MAIN OUTCOME MEASURES: Sex steroids were measured by liquid chromatography-tandem mass spectrometry. Values below the limit of detection (LOD; E2 11 pmol/L [3 pg/mL] were assigned a value of LOD/√2 to estimate total E2. RESULTS: E2 and estrone (E1) were below the LOD in 66.1% and 0.9% of women, respectively. The median (interdecile ranges) for E1 and detectable E2 were 181.2 pmol/L (range, 88.7-347.6 pmol/L) and 22.0 pmol/L (range, 11.0-58.7 pmol/L). Women with undetectable E2 vs detectable E2 were older (median age 74.1 years vs 73.8, P = .02), leaner (median body mass index [BMI] 26.8 kg/m2 vs 28.5, P < .001), and had lower E1, testosterone and DHEA concentrations (P < .001). A linear regression model, including age, BMI, E1, and testosterone, explained 20.9% of the variation in total E2, but explained only an additional 1.2% of variation over E1 alone. E1 and testosterone made significant contributions (r2 = 0.162, P < .001) in a model for the subset of women with detectable E2. CONCLUSIONS: Our findings support E1 as a principal circulating estrogen and demonstrate a robust association between E1 and E2 concentrations in postmenopausal women. Taken together with prior evidence for associations between E1 and health outcomes, E1 should be included in studies examining associations between estrogen levels and health outcomes in postmenopausal women.

Androgen signaling pathways driving reproductive and metabolic phenotypes in a PCOS mouse model.

As the mechanistic basis of polycystic ovary syndrome (PCOS) remains unknown, current management relies on symptomatic treatment. Hyperandrogenism is a major PCOS characteristic and evidence supports it playing a key role in PCOS pathogenesis. Classically, androgens can act directly through the androgen receptor (AR) or, indirectly, following aromatization, via the estrogen receptor (ER). We investigated the mechanism of androgenic actions driving PCOS by comparing the capacity of non-aromatizable dihydrotestosterone (DHT) and aromatizable testosterone to induce PCOS traits in WT and Ar-knockout (ARKO) mice. DHT and testosterone induced the reproductive PCOS-like features of acyclicity and anovulation in WT females. In ARKO mice, DHT did not cause reproductive dysfunction; however, testosterone treatment induced irregular cycles and ovulatory disruption. These findings indicate that direct AR actions and indirect, likely ER, actions of androgens are important mediators of PCOS reproductive traits. DHT, but not testosterone, induced an increase in body weight, body fat, serum cholesterol and adipocyte hypertrophy in WT mice, but neither androgen induced these metabolic features in ARKO mice. These data infer that direct AR-driven mechanisms are key in driving the development of PCOS metabolic traits. Overall, these findings demonstrate that differing PCOS traits can be mediated via different steroid signaling pathways and indicate that a phenotype-based treatment approach would ensure effective targeting of the underlying mechanisms.