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In this study, a one-step method for liquid-liquid extraction has been compared against a two-step procedure for testosterone assays in terms of accuracy, specificity, recovery, lipid removal and baseline noise, using QCs and unknown samples. The difference in accuracy was less than 5% for adult sera, while it was less than 10% for prepubescent sera. To compare specificity, the ion ratio transition of 289 → 97 to 289 → 109 was monitored for all QCs and unknown samples; no interference in the testosterone peak was observed for any tested sample prepared by either the one-step or two-step procedure. The baseline comparison of LC–MS/MS chromatograms of samples indicated that samples prepared by the one-step procedure were of the same quality as those prepared by the two-step procedure; however, recovery in unaltered serum using the one-step procedure was approximately 15% greater across the low to high concentration range. Furthermore, recovery using the one-step procedure was more consistent between stripped and unstripped serum. Lipids were removed efficiently in the two-step procedure as verified by monitoring the typical phospholipid MRM transition of m/z 496 → 184. For every sample processed by the one-step procedure, a one-minute online column wash with 95% methanol was able to remove 95% of the bound lipids, thereby providing a column life-time approximately equivalent to that for the two-step procedure. The presented data indicate that the one-step procedure could replace the two-step procedure while maintaining accuracy, saving time, increasing recovery, and minimizing the potential for errors with the fewer steps required.

INTRODUCTION AND OBJECTIVE:Recurrent urinary tract infections (UTIs) are associated with genitourinary syndrome of menopause (GSM) in postmenopausal women. Androgens, in addition to estrogens, have...

Luciano Martini has been an internationally renowned endocrinologist and a remarkable ambassador of Italian endocrinology through the world. He brought important experimental contributions in support of the new science of intracrinology. After the menopause, the secretion of estrogens stops while serum DHEA attains its lowest values. Estrogens and androgens are produced intra cellularly from the small residual amounts of DHEA. They act within the cells and are released only after inactivation, thus avoiding stimulation of the endometrium and possible actions in other tissues. In three independent 12-week prospective, randomized, double- blind and placebo-controlled clinical studies, it was shown that intravaginal administration of DHEA (prasterone) improves the severity score of the most bothersome symptom of the menopause such as vaginal dryness and dyspareunia. Most importantly, all serum sex steroids remained within normal values, thus explaining the absence of systemic effects. In conclusion, the lack of DHEA availability, not the lack of estrogens, is the main cause of the symptoms of menopause and this notion should guide the therapeutic strategy.

The aim of this study is to confirm the local beneficial effects of intravaginal dehydroepiandrosterone (DHEA, Prasterone) on moderate to severe dyspareunia or pain at sexual activity, the most frequent symptom of vulvovaginal atrophy due to menopause or genitourinary syndrome of menopause (GSM).In a prospective, randomized, double-blind, and placebo-controlled phase III clinical trial, the effect of daily intravaginal 0.50% DHEA (6.5 mg) (Prasterone, EndoCeutics) was examined on four coprimary objectives, namely percentage of parabasal cells, percentage or superficial cells, vaginal pH, and moderate to severe pain at sexual activity (dyspareunia) identified by the women as their most bothersome vulvovaginal atrophy symptom. The intent-to-treat population included 157 and 325 women in the placebo and DHEA-treated groups, respectively.After daily intravaginal administration of 0.50% DHEA for 12 weeks, when compared to baseline by the analysis of covariance test, the percentage of parabasal cells decreased by 27.7% over placebo (P 0.0001), whereas the percentage of superficial cells increased by 8.44% over placebo (P 0.0001), vaginal pH decreased by 0.66 pH unit over placebo (P 0.0001), and pain at sexual activity decreased by 1.42 severity score unit from baseline or 0.36 unit over placebo (P = 0.0002). On the other hand, moderate to severe vaginal dryness present in 84.0% of women improved at 12 weeks by 1.44 severity score unit compared to baseline, or 0.27 unit over placebo (P = 0.004). At gynecological evaluation, vaginal secretions, epithelial integrity, epithelial surface thickness, and color all improved by 86% to 121% over the placebo effect (P 0.0001 for all comparisons with placebo). Serum steroid levels remained well within the normal postmenopausal values according to the involved mechanisms of intracrinology. The only side effect reasonably related to treatment is vaginal discharge due to melting of the vehicle at body temperature and this was reported in about 6% of the participants.The daily intravaginal administration of 0.50% (6.5 mg) DHEA (Prasterone) has shown clinically and highly statistically significant effects on the four coprimary parameters suggested by the US Food and Drug Administration. The strictly local action of Prasterone is in line with the absence of significant drug-related adverse events, thus showing the high benefit-to-risk ratio of this treatment based upon the novel understanding of the physiology of sex steroids in women.

Antiandrogens inhibit the androgen receptor and have an important role in the treatment of prostate cancer. This review provides a historical perspective on the development and clinical benefit of antiandrogens in the treatment of prostate cancer.We searched PubMed® for clinical trials with the search terms antiandrogens and prostate cancer combined with drug names for antiandrogens. This article represents a collaboration of clinical investigators who have made critical scientific contributions leading to the approval of antiandrogens for treating patients with prostate cancer.Antiandrogens differ in chemical structure and exert varying efficacy and safety profiles. The unfavorable therapeutic index of steroidal antiandrogens led to replacement by safer nonsteroidal agents. Flutamide, nilutamide and bicalutamide, which were designed to target the androgen receptor, were developed primarily for use in combination with castration to provide combined androgen blockade. Modest clinical benefits were observed with the combination of first generation antiandrogens and castration vs castration alone. With increased knowledge of androgen receptor structure and its biological functions a new generation of antiandrogens without agonist activity was designed to provide more potent inhibition of the androgen receptor. Randomized clinical trials in patients with metastatic, castration resistant prostate cancer showed significant survival benefits, which led to the approval of enzalutamide in August 2012. Apalutamide was recently approved while darolutamide is not yet approved in the United States. These next generation antiandrogens are being actively tested in earlier disease states such as nonmetastatic prostate cancer. Evolving knowledge of resistance mechanisms to androgen receptor targeted treatments will stimulate research and drug discovery for additional compounds. Further testing in nonmetastatic castration resistant prostate cancer as well as castration sensitive disease states will hopefully augment our ability to treat a broader spectrum of patients with prostate cancer.Antiandrogens have already provided important benefits for prostate cancer treatment. Greater knowledge about the structural and functional biology of the androgen receptor in prostate cancer will facilitate further discovery and development of further improved antiandrogens with enhanced clinical activity in patients with advanced metastatic disease. Testing these new agents earlier in the course of prostate cancer may further improve the survival and quality of life of patients with current local and/or systemic treatment modalities.

Objective:The aim of the study was to determine the range of serum sex-related steroids in normal postmenopausal women and in women of the same age with a diagnosis of vulvovaginal atrophy (VVA).Methods:Validated mass spectrometry-based assays coupled to gas or liquid chromatography were use

Pregnant women with polycystic ovary syndrome (PCOS) are often overweight or obese. To study the effects of maternal androgen excess in obese dams on metabolism, placental function and fetal growth, female C57Bl6J mice were fed a control (CD) or a high fat/high sucrose (HF/HS) diet for 4–10 weeks, and then mated. On gestational day (GD) 15.5–17.5, dams were injected with dihydrotestosterone (CD-DHT, HF/HS-DHT) or a vehicle (CD-Veh, HF/HS-Veh). HF/HS dams had higher fat content, both before mating and on GD18.5, with no difference in glucose homeostasis, whereas the insulin sensitivity was higher in DHT-exposed dams. Compared to the CD groups, the livers from HF/HS dams weighed more on GD18.5, the triglyceride content was higher, and there was a dysregulation of liver enzymes related to lipogenesis and higher mRNA expression of Fitm1. Fetuses from HF/HS-Veh dams had lower liver triglyceride content and mRNA expression of Srebf1c. Maternal DHT exposure, regardless of diet, decreased fetal liver Pparg mRNA expression and increased placental androgen receptor protein expression. Maternal diet-induced obesity, together with androgen excess, affects maternal and fetal liver function as demonstrated by increased triglyceride content and dysfunctional expression of enzymes and transcription factors involved in de novo lipogenesis and fat storage.

Objective The aim of this study was to review the preclinical data showing the role of both estrogens and androgens in the physiology of the vagina, and, most likely, in vulvovaginal atrophy of menopause. Methods Mass spectrometry-based assays (validated according to the FDA guidelines) for the measurement of sex steroids, their precursors, and metabolites were used. In addition to fixation of the vagina for morphological examination, histomorphometry, immunocytochemistry, immunofluorescence, and quantitative reverse transcription polymerase chain reaction were performed. Results The vaginal epithelium of the animals receiving dehydroepiandrosterone (DHEA) was made of large multilayered columnar mucous cells showing distended cytoplasmic vacuoles representative of an androgenic effect. DHEA also stimulates collagen fiber compactness of the lamina propria (second layer)-an effect essentially due to an androgenic effect, whereas stimulation by DHEA of the muscularis in the third vaginal layer is approximately 70% due to the androgenic conversion of DHEA. Stimulation of the surface area of the nerve endings, on the contrary, is exclusively androgenic. Vaginal weight stimulation by DHEA is about 50% androgenic and 50% estrogenic. Conclusions Practically all studies on the influence of steroid hormones in the vagina have focused on luminal epithelial cells. Since all estrogens and androgens in postmenopausal women are made intracellularly and derive from the conversion of circulating DHEA, it is of interest to observe from these preclinical data that DHEA exerts both estrogenic and androgenic activity in the three layers of the vagina, the stimulatory effect on nerve density being 100% androgenic. Taking vaginal weight as a global parameter, the stimulatory effect of DHEA in the rat vagina is about equally estrogenic and androgenic, thus illustrating the importance of androgens in vaginal morphology and function, and the likely importance of androgens in vulvovaginal atrophy of menopause.

OBJECTIVE To quantify the association between vulvovaginal atrophy and depression, major depressive disorder, and anxiety. METHODS Women with vulvovaginal atrophy from the Truven Health MarketScan Commercial and Medicare Supplemental Databases (01/2010-09/2016) with ≥365 days of continuous insurance coverage before and after the first vulvovaginal atrophy/dyspareunia diagnosis (index date) were selected. Women with vulvovaginal atrophy were matched 1:3 to women without (controls) according to age, calendar year, health plan, and region. The study period spanned from 12 months before to 12 months after index date. The ratios of diagnosed depression, major depressive disorder, and anxiety among women with vulvovaginal atrophy and the controls were calculated. Logistic regressions adjusting for proxies of menopause were used to compare prevalence. RESULTS In all, 125,889 women with vulvovaginal atrophy and 376,057 controls were included (mean age 60.7 [45-101]). The prevalence of depression, major depressive disorder, and anxiety was higher among women with vulvovaginal atrophy compared with controls (23.9% vs 18.9%, 6.3% vs 4.7%, 16.6% vs 11.3%), with prevalence ratios of 1.26, 1.33, and 1.47, respectively (all P

We examined associations between intensive lifestyle intervention (ILS) and changes in testosterone and associations with mood among middle-aged men.Secondary analysis of men (n=886) participating in the Diabetes Prevention Program which randomized glucose-intolerant, overweight men to ILS, metformin, or placebo between 1996 and 1999.Changes in testosterone between baseline and 1-year follow-up asnd associations of these changes with mood measures (Beck Depression Inventory [BDI-II], Beck Anxiety Inventory [BAI]).Median baseline testosterone was 10.98nmol/l and 44% (n=385) had testosterone10.41nmol/l or 300ng/dl. Testosterone increases were greater among men randomized to ILS vs. metformin vs. placebo (1.15nmol/l vs. -0.12nmol/l vs. -0.27nmol/l, p0.001). The association between changes in testosterone and mood differed by study arm (p0.001 for interaction); there were no significant associations between changes in testosterone and mood changes among men in the ILS or placebo arms. Among men in the metformin arm, increases in testosterone were significantly associated with decreases in BDI-II (improved depressive symptoms) (β-coefficient -0.2336, p=0.0002) indicating a 0.23 decrease in BDI-II for every 1nmol/l increase in testosterone and decreases in BAI (improved anxiety symptoms) (β-coefficient -0.2147, p=0.0014). Similar patterns were observed for bioavailable testosterone.Among overweight middle-aged men with glucose-intolerance, ILS increased endogenous testosterone slightly but without significant improvements in mood. Metformin did not increase testosterone, but among metformin users, testosterone increases were associated with improvements in mood. Thus, interventions that increase endogenous testosterone may not also improve mood.

Context: Some men who use finasteride for hair loss report persistent sexual and other symptoms after discontinuing finasteride therapy. Objective: To determine whether these persistent symptoms after discontinuation of finasteride use are due to androgen deficiency, decreased peripheral androgen action, or persistent inhibition of steroid 5α-reductase (SRD5A) enzymes. Participants: Finasteride users, who reported persistent sexual symptoms after discontinuing finasteride (group 1); age-matched finasteride users who did not report sexual symptoms (group 2); and healthy men who had never used finasteride (group 3). Outcomes: Sexual function, mood, affect, cognition, hormone levels, body composition, functional magnetic resonance imaging (fMRI) response to sexually and affectively valenced stimuli, nucleotide sequences of androgen receptor (AR), SRD5A1, and SRD5A2; expression levels of androgen-dependent genes in skin. Setting: Academic medical center. Results: Symptomatic finasteride users were similar in body composition, strength, and nucleotide sequences of AR, SRD5A1, and SRD5A2 genes to asymptomatic finasteride users and nonusers. Symptomatic finasteride users had impaired sexual function, higher depression scores, a more negative affectivity balance, and more cognitive complaints than men in groups 2 and 3 but had normal objectively assessed cognitive function. Testosterone, dihydrotestosterone, 5α-androstane-3α,17β-diol-glucuronide, testosterone to dihydrotestosterone and androsterone glucuronide to etiocholanolone glucuronide ratios, and markers of peripheral androgen action and expression levels of AR-dependent genes in skin did not differ among groups. fMRI blood oxygen level-dependent responses to erotic and nonerotic stimuli revealed abnormal function in brain circuitry linked to sexual arousal and major depression. Conclusions: We found no evidence of androgen deficiency, decreased peripheral androgen action, or persistent peripheral inhibition of SRD5A in men with persistent sexual symptoms after finasteride use. Symptomatic finasteride users revealed depressed mood and fMRI findings consistent with those observed in depression.

A series of steroids present in the brain have been named "neurosteroids" following the possibility of their role in the central nervous system impairments such as anxiety disorders, depression, premenstrual dysphoric disorder (PMDD), addiction, or even neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Study of their potential role requires a sensitive and accurate assay of their concentration in the monkey brain, the closest model to the human. We have thus developed a robust, precise and accurate liquid chromatography-tandem mass spectrometry method for the assay of pregnenolone, pregnanolone, epipregnanolone, allopregnanolone, epiallopregnanolone, and androsterone in the cynomolgus monkey brain. The extraction method includes a thorough sample cleanup using protein precipitation and phospholipid removal, followed by hexane liquid-liquid extraction and a Girard T ketone-specific derivatization. This method opens the possibility of investigating the potential implication of these six steroids in the most suitable animal model for neurosteroid-related research.

Human 3α-HSD3 (3α-hydroxysteroid dehydrogenase type 3) plays an essential role in the inactivation of the most potent androgen 5α-DHT (5α-dihydrotestosterone). The present study attempts to obtain the important structure of 3α-HSD3 in complex with 5α-DHT and to investigate the role of 3α-HSD3 in breast cancer cells. We report the crystal structure of human 3α-HSD3·NADP+·A-dione (5α-androstane-3,17-dione)/epi-ADT (epiandrosterone) complex, which was obtained by co-crystallization with 5α-DHT in the presence of NADP+. Although 5α-DHT was introduced during the crystallization, oxidoreduction of 5α-DHT occurred. The locations of A-dione and epi-ADT were identified in the steroid-binding sites of two 3α-HSD3 molecules per crystal asymmetric unit. An overlay showed that A-dione and epi-ADT were oriented upside-down and flipped relative to each other, providing structural clues for 5α-DHT reverse binding in the enzyme with the generation of different products. Moreover, we report the crystal structure of the 3α-HSD3·NADP+·4-dione (4-androstene-3,17-dione) complex. When a specific siRNA (100 nM) was used to suppress 3α-HSD3 expression without interfering with 3α-HSD4, which shares a highly homologous active site, the 5α-DHT concentration increased, whereas MCF7 cell growth was suppressed. The present study provides structural clues for 5α-DHT reverse binding within 3α-HSD3, and demonstrates for the first time that down-regulation of 3α-HSD3 decreases MCF7 breast cancer cell growth.

The objective of the study is to evaluate the acceptability of the intravaginal administration of ovules/suppositories of DHEA (dehydroepiandrosterone, prasterone) for the treatment of vulvovaginal atrophy (VVA) in women with moderate to severe dyspareunia who were administered daily for 12 weeks intravaginal 0.50% (6.5 mg) DHEA or placebo. There were a total of 373 women in the per-protocol population who responded to the questionnaire for both treatment groups. While it was planned that the applicator would be evaluated as suitable if at least 80% of participants have a global score ≤ 2 units, 99% and 100% of participants had a score ≤ 2 units in the placebo and DHEA groups, respectively, for the global score (mean of 5 questions). When asked about like and dislike the technique of drug administration, 284 comments were positive, while 114 women gave no comment. About 92-94% of women indicated that they were very confident to be able use the applicator successfully in the future. The survey shows a high degree of satisfaction and of confidence to use the applicator successfully in the future.

Introduction Previous data have shown that intravaginal dehydroepiandrosterone (DHEA, prasterone) improved all the domains of sexual function, an effect most likely related to the local formation of androgens from DHEA. Aims To confirm in a placebo-controlled, prospective, double-blind and randomized study the benefits of daily intravaginal DHEA for 12 weeks on sexual function using the Female Sexual Function Index (FSFI) questionnaire. Methods Placebo was administered daily to 157 women while 325 women received 0.50% (6.5 mg) DHEA daily for 12 weeks. All women were postmenopausal meeting the criteria of vulvovaginal atrophy (VVA), namely moderate to severe dyspareunia as their most bothersome symptom of VVA in addition to having ≤5% of vaginal superficial cells and vaginal pH > 5.0. The FSFI questionnaire was filled at baseline (screening and day 1), 6 weeks and 12 weeks. Comparison between DHEA and placebo of the changes from baseline to 12 weeks was made using the analysis of covariance test, with treatment group as the main factor and baseline value as the covariate. Main Outcome Measures The six domains and total score of the FSFI questionnaire were evaluated. Results The FSFI domain desire increased over placebo by 0.24 unit (+49.0%, P = 0.0105), arousal by 0.42 unit (+56.8%, P = 0.0022), lubrication by 0.57 unit (+36.1%, P = 0.0005), orgasm by 0.32 unit (+33.0%, P = 0.047), satisfaction by 0.44 unit (+48.3%, P = 0.0012), and pain at sexual activity by 0.62 unit (+39.2%, P = 0.001). The total FSFI score, on the other hand, has shown a superiority of 2.59 units in the DHEA group over placebo or a 41.3% greater change than placebo (P = 0.0006 over placebo). Conclusion The present data show that all the six domains of the FSFI are improved over placebo (from P = 0.047 to 0.0005), thus confirming the previously observed benefits of intravaginal DHEA on female sexual dysfunction by an action exerted exclusively at the level of the vagina, in the absence of biologically significant changes of serum steroids levels.

Sensitive and accurate measurement of androstane-3β,17β-diol and androstane-3α,17β-diol in the circulation is important for clinical research and accurate clinical diagnosis. This report describes a highly sensitive, specific, precise and reliable assay for the simultaneous accurate measurement of serum androstane-3α,17β-diol and androstane-3β,17β-diol in postmenopausal women. The LLOQ of 1 pg/mL has been achieved with nicotinic acid derivatization, which is superior to picolinic acid by a factor of 5 to 10 in terms of signal to noise ratio. The difference is attributed to the higher acidity of picolinic acid which forms a more stable intermediate, thus decreasing derivatization efficiency. Potential interference from androstane-3α, 17α-diol, androstane-3β, 17α-diol, and 5-androstenediol has been well separated from the two target diols. The high level of specificity has been determined by well-developed chromatography and ion ratio monitoring. A good linearity in the range of 1 pg/mL to 200 pg/mL (0.03 pg to 6 pg on column) was obtained for both compounds at R > 0.998. The bias and coefficients of variation of all the QC levels are within the range of 10% while the recovery in both charcoal-stripped and unstripped human serum is around 85%. The matrix effect was evaluated and the results well met the acceptance criteria according to the guidelines of bioanalytical method development and validation. Using this newly developed method, the concentrations of both androstane-3α,17β diol and androstane-3β,17β diol were measured in normal postmenopausal serum, where the concentrations range from 2 pg/mL to 32 pg/mL for androstane-3α,17β diol and from 1 pg/mL to 10 pg/mL for androstane-3β,17β diol, respectively.

The secretion of estrogens by the ovaries stops at menopause. Afterward, dehydroepiandrosterone (DHEA) becomes the only source of both estrogens and androgens during all the postmenopausal years. To maintain very low and biologically inactive concentrations of estrogens and androgens in the circulation, DHEA is transformed intracellularly into cell-specific small amounts of estrogens and androgens (except in the endometrium) which then act and are inactivated locally in the same cells, thus avoiding biologically significant systemic exposure to active sex steroids. The secretion of DHEA, however, mainly of adrenal origin, has already decreased by an average of 60% at the time of menopause and it continues to decrease thereafter with a parallel lowering in available intracellular estrogens and androgens. Consequently, after the arrest of estrogen secretion by the ovaries, the loss of DHEA becomes practically responsible for the symptoms and signs of menopause. Replacing what is missing, namely DHEA, at the right place, at the right time, and in the right amount, seems to be the logical and physiological approach for the treatment of menopausal symptoms and signs, as recently demonstrated for pain at sexual activity (dyspareunia), the most bothersome symptom of vulvovaginal atrophy due to menopause.

The normal blood estrogen concentrations in women treated with physiological amounts of DHEA are not different from the situation observed in about 25% of normal postmenopausal women who have sufficiently high endogenous DHEA activity to avoid the symptoms of menopause: these women are not symptomatic and, consequently, do not need DHEA replacement. The administration of intravaginal DHEA permits to increase DHEA availability locally in the vagina where the symptoms of sex steroid deficiency are present, especially pain at sexual activity and vaginal dryness. The local addition of exogenous DHEA permits to compensate for the absence of a specific stimulator of DHEA secretion when serum DHEA decreases and becomes symptomatic.Starting at menopause, DHEA becomes the exclusive source of all estrogens and androgens in women. It remains, however, that both sex steroids are essential for the normal functioning of most tissues during the remaining life of women. In fact, the marked and continuing decrease in DHEA availability with age is responsible for the menopausal symptoms which should best be corrected by replacing the missing amount of DHEA. It should also be taken into account that women are not only missing estrogens after menopause but are exposed to declining levels of androgens in parallel with the decreasing serum DHEA concentrations starting at the age of 30 years with an average 60% loss already observed at time of menopause.

The control of DHEA action is completely different from that of E2 and testosterone, as engineered by the 500 million years of evolution which have added 30 or more intracrine enzymes controlling DHEA action in the human. In fact, the essential characteristics which differentiate the exposure to E2 and testosterone from the exposure to DHEA, an inactive compound by itself, derive from the major differences between endocrinology and intracrinology, which can be summarized as follows:The arrest of estrogen secretion by the ovaries at menopause causes an abrupt decrease of serum estradiol (E2) below the level of biological activity (established at 9.3 pg E2/ml (95th centile)), thus avoiding stimulation of the endometrium and decreasing the risk of endometrial cancer. As much as the arrest of E2 secretion by the ovaries is essential to protect the uterus, it is important that sex steroids continue to be available in the tissues other than the uterus where estrogens and androgens are needed for normal morphology and functioning. To this end, evolution, through 500 million years, has progressively provided the peripheral tissues with the sophisticated sets of enzymes which make androgens and estrogens from dehydroepiandrosterone (DHEA). The biologically inactive precursor DHEA appeared much later or approximately 20 million years ago with the primates. All elements were then in place for the installment of intracrinology or the cell-specific formation and inactivation of estrogens and androgens in peripheral tissues from the precursor DHEA, a molecule inactive by itself.

Copyright © 2015 by the Endocrine Society.Context: Steroid sex hormones and SHBG may modify metabolism and diabetes risk, with implications for sex-specific diabetes risk and effects of prevention interventions. Objective: This study aimed to evaluate the relationships of steroid sex hormones, SHBG and SHBG single-nucleotide polymorphisms (SNPs) with diabetes risk factors and with progression to diabetes in the Diabetes Prevention Program (DPP). Design and Setting: This was a secondary analysis of a multicenter randomized clinical trial involving 27 U.S. academic institutions. Participants: The study included 2898 DPP participants: 969 men, 948 premenopausal women not taking exogenous sex hormones, 550 postmenopausal women not taking exogenous sex hormones, and 431 postmenopausal women taking exogenous sex hormones. Interventions: Participantswererandomized to receive intensive lifestyle intervention, metformin, or placebo. Main Outcomes: Associations of steroid sex hormones, SHBG, and SHBG SNPs with glycemia and diabetes risk factors, and with incident diabetes over median 3.0 years (maximum, 5.0 y). Results: T and DHT were inversely associated with fasting glucose in men, and estrone sulfate was directly associated with 2-hour post-challenge glucose in men and premenopausal women. SHBG was associated with fasting glucose in premenopausal women not taking exogenous sex hormones, and in postmenopausal women taking exogenous sex hormones, but not in the other groups. Diabetes incidence was directly associated with estrone and estradiol and inversely with T inmen; the association with Twaslost after adjustment for waist circumference. Sex steroids were not associated with diabetes outcomes in women. SHBG and SHBG SNPs did not predict incident diabetes in the DPP population. Conclusions: Estrogens and T predicted diabetes risk in men but not in women. SHBG and its polymorphisms did not predict risk in men or women. Diabetes risk is more potently determined by obesity and glycemia than by sex hormones.

Acupuncture with combined manual and low-frequency electrical stimulation, or electroacupuncture (EA), reduces endocrine and reproductive dysfunction in women with polycystic ovary syndrome (PCOS), likely by modulating sympathetic nerve activity or sex steroid synthesis. To test this hypothesis, we induced PCOS in rats by prepubertal implantation of continuous-release letrozole pellets (200 µg/day) or vehicle. Six weeks later, rats were treated for 5-6 weeks with low-frequency EA 5 days/week, subcutaneous injection of 17β-estradiol (2.0 µg) every fourth day, or a β-adrenergic blocker (propranolol hydrochloride, 0.1 mg/kg) 5 days/week. Letrozole controls were handled without needle insertion or injected with sesame oil every fourth day. Estrous cyclicity, ovarian morphology, sex steroids, gonadotropins, insulin-like growth factor I, bone mineral density, and gene and protein expression in ovarian tissue were measured. Low-frequency EA induced estrous-cycle changes, decreased high levels of circulating luteinizing hormone (LH) and the LH/follicle-stimulating hormone (FSH) ratio, decreased high ovarian gene expression of adiponectin receptor 2, and increased expression of adiponectin receptor 2 protein and phosphorylation of ERK1/2. EA also increased cortical bone mineral density. Propranolol decreased ovarian expression of Foxo3, Srd5a1, and Hif1a. Estradiol decreased circulating LH, induced estrous cycle changes, and decreased ovarian expression of Adipor1, Foxo3, and Pik3r1. Further, total bone mineral density was higher in the letrozole-estradiol group. Thus, EA modulates the circulating gonadotropin levels independently of sex steroids or β-adrenergic action and affects the expression of ovarian adiponectin system.

Quantification of steroidal glucuronide conjugates by the indirect methods of immunoassay and GC-MS/MS may underestimate some conjugates since hydrolysis is needed in sample processing. In the present work, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous direct quantification of androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17β diol 17-glucuronide in postmenopausal women's serum. The quantification limits are 0.1ng/mL for 3α-diol-17G and 4ng/mL for both ADT-G and Etio-G, respectively, with an extraction from 200μL serum while the total run time is less than 6min for all three glucuronides. In this method, solid phase extraction is used for sample preparation. The assay has been validated in compliance with EndoCeutics SOPs and FDA guidelines for bioanalytical method development and validation. The recovery of glucuronides in stripped serum is consistent with that in unstripped serum, where the average difference in stripped and unstripped is less than 10%. A linear regression model fits well the standard curves of all three compounds with R≥0.99 where the weighting factor is 1/X. Interday accuracy and CV for all levels of QCs are within the range of 15% in both stripped and unstripped serum while all calibration curves are within the range of 6% except for LLOQs, which are within the range of 9%. Other parameters have also been assessed such as selectivity, matrix, lipemic and hemolysis effects as well as stabilities in solution and matrix. Incurred sample reanalysis has been performed with a result of over 93% within 20% of the original values. This reliable, sensitive and fast method is ready for large-scale clinical sample assays.

Recently two drugs, namely the antiandrogen MDV-3100 and the inhibitor of 17α-hydroxylase abiraterone have been accepted by the FDA for the treatment of castration-resistant prostate cancer (CRPC) with or without previous chemotherapy, with a prolongation of overall survival of 2.2-4.8 months. While medical (GnRH agonist) or surgical castration reduces the serum levels of testosterone by about 97%, an important concentration of testosterone and dihydrotestosterone remains in the prostate and activates the androgen receptor (AR), thus offering an explanation for the positive data obtained in CRPC. In fact, explanation of the response observed with MDV-3100 or enzalutamide in CRPC is essentially a blockade of the action or formation of intraprostatic androgens. In addition to the inhibition of the action or formation of androgens made locally by the mechanisms of intracrinology, increased AR levels and AR mutations can be involved, especially in very advanced disease. Future developments look at more efficient inhibitors of the action or formation of intraprostatic androgens and starting treatment earlier when blockade of androgens can exert long-term control and even cure prostate cancer treated at a stage before the appearance of metastases. This article is part of a Special Issue entitled 'Essential role of DHEA'.

The role of endogenous androgens for body composition and physical performance in women athletes is still not elucidated.To examine the serum androgen profile in relation to body composition and physical performance in women Olympic athletes and to compare endocrine variables and body composition to controls.Cross-sectional study, conducted between 2011 and 2015 at the Women's Health Research Unit, Karolinska University Hospital, Stockholm.Swedish women Olympic athletes (n=106) and age-matched and body mass index-matched sedentary controls (n=117) were included in the study. Blood sampling was performed in a rested, fasting state for the measurement of serum androgens and their metabolites by liquid chromatography-tandem mass spectrometry. Body composition was determined by dual-energy X-ray absorptiometry (controls n=100, athletes n=65). The athletes performed standardised performance tests (n=59) (squat jump (SJ) and countermovement jump (CMJ).The athletes demonstrated significantly higher levels of the precursor androgens dehydroepiandrosterone (DHEA) and 5-androstene-3β, 17β-diol (5-DIOL) and the metabolite etiocholanolone glucuronide (Etio-G), significantly lower levels of estrone (p0.05, respectively), higher bone mineral density (p0.001) and more lean mass (p0.001) compared with controls. Serum levels of DHEA, 5-DIOL and Etio-G correlated positively to lean mass variables and physical performance in the athletes. DHEA and lean mass legs explained 66% of the variance in SJ, whereas lean mass explained 52% of the variance in CMJ.The present data suggest that endogenous androgens are associated with a more anabolic body composition and enhanced performance in women athletes. These results are of importance for the current discussion regarding hyperandrogenism in women athletes.

Objectives To examine the impact of a weight loss intervention upon follicle stimulating hormone (FSH) levels in postmenopause. Methods Participants were postmenopausal, overweight, glucose-intolerant women not using exogenous estrogen (n = 382) in the Diabetes Prevention Program. Women were randomized to intensive lifestyle change (ILS) with the goals of weight reduction of at least 7% of initial weight and 150 min per week of moderate-intensity exercise, metformin 850 mg twice a day, or placebo administered twice a day. Results Randomization to ILS led to small increases in FSH between baseline and 1-year follow-up vs. placebo (2.3 IU/l vs. −0.81 IU/l, P < 0.01). Increases in FSH were correlated with decreases in weight (r = −0.165, P < 0.01) and estradiol (E2) (r = −0.464, P < 0.0001) after adjustment for age, race/ethnicity, and randomization arm. Changes in FSH were still significantly associated with changes in weight even after adjustment for E2 levels. Metformin users had reductions in weight but non-significant changes in FSH and E2 levels vs. placebo. Conclusions Weight loss leads to small increases in FSH among overweight, postmenopausal women, potentially through pathways mediated by endogenous estrogen as well as other pathways.

Steroids were first analyzed by immunoassay-based methods followed by gas chromatography mass spectrometry (GC-MS or GC-MS/MS) with derivatization techniques since steroids are neutral and do not ionize at a high level using the electrospray ionization technique. We now report a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of seven steroidal compounds, i.e., estradiol (E2), estrone (E1), testosterone (Testo), dihydrotestosterone (DHT), androst-5-ene-3β, 17β-diol (5-diol), dehydroepiandrosterone (DHEA) and androstenedione (4-dione). The system used is a UPLC-MS/MS (Qtrap 6500) system. With this method, the sample preparation is the combination of liquid-liquid extraction and a simple selective derivatization for only E1 and E2. This assay method is simple and practically eliminates potential contamination. Low quantification limits of 1pg/mL, 4pg/mL, 50pg/mL, 10pg/mL, 100pg/mL, 500pg/mL and 100pg/mL have been found, respectively for the steroids mentioned above. Without derivatization, DHT sensitivity can be as low as 4pg/mL with S/N≥5. A full validation has been performed for the seven compounds in compliance with GLP and FDA guidelines for bioanalytical method development and validation. Recovery of all seven compounds in unstripped serum is similar to that in stripped serum: 72.1-84.7% for E2, 83.6-94.5% for E1, 88.2-90.3% for Testo, 82.0-90.6% for DHT, 84.9-92.0% for 5-diol, 88.1-93.8% for DHEA and 86.2-90.3% for 4-dione, respectively. A good linearity is obtained with R>0.99 for each compound within its calibration range. Accuracies of all levels of QC are within the range of 15% for all seven compounds. The between day variation coefficients are 6.1-8.9% for the low limits of quantification of all seven compounds with 0.7-6.1% for higher levels of QCs for all seven compounds. All results of other test parameters similarly meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines. By comparison of GC-MS/MS and LC-MS/MS data for six derivatized and nonderivatized free steroids, the present data show the crucial importance to use validated assays according to the FDA guidelines to increase specificity, precision and reliability of the absolute values associated with MS/MS-based assays. This method has already been applied to series of samples from clinical trials and is ready for wide clinical use.

Introduction To better understand the mechanisms underlying the beneficial effects of the intravaginal administration of dehydroepiandrosterone (DHEA) observed in postmenopausal women on sexual dysfunction. Aims To identify the distribution of the androgen‐synthesizing enzymes as well as androgen receptor (AR) and measure steroid levels in the monkey vagina. Methods The cynomolgus monkey ( M acaca fascicularis ), the closest model to the human, has been used to measure the expression levels of steroidogenic enzymes and androgen receptor by quantitative reverse transcription polymerase chain reaction (n = 4), confirmed by immunohistochemistry, and immunofluorescence (n = 3). DHEA and its androgenic metabolites were quantified by LC‐MS/MS (n = 4). Main Outcome Measures The presence of SRD5A1, SRD5A2, HSD17B3, AR as well as nerve fibers (PGP 9.5) was investigated, and steroid levels were measured. Results AR is widely distributed within the vaginal epithelium and also in the lamina propria with a lower expression in the muscularis layer and blood vessel walls. Androgen‐forming enzymes, on the other hand, are expressed in the vaginal stratified squamous epithelium at a relatively high level where they are uniformly distributed from the basal membrane up to the superficial keratinized cells. The enzymes are at a lower level in blood vessel walls and zona muscularis where nerve fibers are localized. DHEA and its androgen metabolites are present at biologically significant concentrations in the monkey vagina. Conclusion The enzymes responsible for androgen formation as well as AR are at the highest level in the superficial layer of the stratified epithelium and muscularis layers of the vagina. These data provide a potential explanation for the described role of androgens in regulating vaginal lubrication, smooth muscle activity, blood flow, and the neuronal activity potentially involved in the correction of sexual dysfunction. Bertin J, Dury AY, Ouellet J, Pelletier G, and Labrie F. Localization of the androgen‐synthesizing enzymes, androgen receptor, and sex steroids in the vagina: Possible implications for the treatment of postmenopausal sexual dysfunction. J Sex Med 2014;11:1949–1961.

Introduction We have previously observed that intravaginal prasterone (dehydroepiandrosterone, DHEA) improved all domains of female sexual dysfunction (FSD). Aim Investigate the influence of moderate/severe pain at sexual activity (dyspareunia) (MSD) at baseline on FSD following prasterone administration. Methods The effect of daily administration of prasterone (0, 3.25 mg, 6.5 mg or 13 mg) for 12 weeks on FSD in 215 postmenopausal women with or without MSD at baseline was evaluated in a prospective, randomized, double‐blind, and placebo‐controlled phase III clinical trial. Main Outcome Measures Differences were examined on desire, arousal and orgasm. Results Comparable benefits were observed in women not having MSD (n = 56) vs. those having MSD (n = 159). The benefits over placebo in prasterone‐treated women for desire, avoiding intimacy and vaginal dryness as well as for the total sexual domain of the MENQOL (Menopause Specific Quality of Life) questionnaire, ranged between 18.0% and 38.2% with P values of P = 0.01), 118% ( P = 0.001) and 31.1% ( P = 0.03) were observed over placebo, respectively, while similar differences (58.0%, 67.6% and 32.1%) did not reach statistical significance in the MSD− group having up to only 44 prasterone‐treated women compared with 119 in the MSD+ group. Conclusions No MSD at baseline does not apparently affect the effects of intravaginal prasterone on sexual dysfunction. Knowing the absence of significant effects of estrogens on FSD, the present data suggest that vulvovaginal atrophy (VVA) and vulvovaginal sexual dysfunction (VVSD) are two different consequences of sex steroid deficiency at menopause which can respond independently. In addition, the present data seriously question the justification of pain being part of FSD as well as the separation of FSD into separate domains. Labrie F, Archer D, Bouchard C, Fortier M, Cusan L, Gomez J‐L, Girard G, Baron M, Ayotte N, Moreau M, Dube R, Cote I, Labrie C, Lavoie L, Gilbert L, Martel C, and Balser J. Lack of influence of dyspareunia on the beneficial effect of intravaginal prasterone (dehydroepiandrosterone, DHEA) on sexual dysfunction in postmenopausal women. J Sex Med 2014;11:1766–1785.

The discovery of medical castration with GnRH agonists in 1979 rapidly replaced surgical castration and high doses of estrogens for the treatment of prostate cancer. Soon afterwards, it was discovered that androgens were made locally in the prostate from the inactive precursor DHEA of adrenal origin, a mechanism called intracrinology. Taking into account these novel facts, combined androgen blockade (CAB) using a pure antiandrogen combined with castration in order to block the two sources of androgens was first published in 1982. CAB was the first treatment shown in randomized and placebo-controlled trials to prolong life in prostate cancer, even at the metastatic stage. Most importantly, the results recently obtained with the novel pure antiandrogen enzalutamide as well as with abiraterone, an inhibitor of 17α-hydroxylase in castration-resistant prostate cancer, has revitalized the CAB concept. The effects of CAB observed on survival of heavily pretreated patients further demonstrates the importance of the androgens made locally in the prostate and are a strong motivation to apply CAB to efficiently block all sources of androgens earlier at start of treatment and, even better, before metastasis occurs. The future of research in this field thus seems to be centered on the development of more potent blockers of androgens formation and action in order to obtain better results at the metastatic stage and, for the localized stage, reduce the duration of treatment required to achieve complete apoptosis and control of prostate cancer proliferation before it reaches the metastatic or noncurable stage.

In the present study, the impact of the extraction solvent on the accuracy of endogenous progesterone assay in human serum has been investigated using two selective reaction monitoring (SRM) transitions (315>97 & 315>109). Higher levels of noise and more interference were observed when more polar solvents were used for extraction, thus resulting in serious bias of the measured values of progesterone in serum. This is confirmed by monitoring the ion ratio of 315>97-315>109. This issue could not be easily resolved by changes in MS/MS transitions or chromatography conditions. More bias was observed with the SRM transition 315>109 for the polar solvent extraction. Hexane and 1-chlorobutane (polarity index of 0 and 1, respectively) did provide the cleanest samples with a lower noise level in the chromatograms. Moreover, the measured values of progesterone were not changed with different SRM transitions or longer retention time in search of an improved separation. Recovery tests of progesterone have been performed with 1-chlorobutane in matrices with phosphate buffered saline (PBS) 1x, PBS 1×3% bovine serum albumin (BSA), stripped serum/H2O (1:1) and unstripped serum. The recovery (70%∼80%) consistency is observed not only at different levels but also in different matrices. The equivalent recovery between PBS 1x, PBS 1×3% BSA and unstripped serum shows that the impact of progesterone binding to serum proteins on the measurement accuracy can be avoided with this sample preparation procedure. No significant matrix effect on the determination of progesterone was observed with 1-chlorobutane. Within the range of 12.5-2000pg/mL, a good linearity is observed with R>0.99 and weighting factor 1/X. Bias and covariance efficiency of QCs are within 10%. With 1-chlorobutane as the extraction solvent, the concentration of progesterone was measured where the range for postmenopausal serum is 5.74∼91.7pg/mL, which is well below the reported concentrations of 314 pg/mL∼942pg/mL in postmenopausal serum by immunoassay-based techniques, while the range in premenopausal serum is 12.8 pg/mL∼18.6ng/mL.

In order to avoid the risks of non-physiological systemic exposure, serum concentrations of estradiol (E 2 ) and testosterone (as measured by mass spectrometry-based assays) should remain below the 95th centiles measured at 9.3 pg/ml and 0.26 ng/ml for these respective sex steroids in normal postmenopausal women. To document the possibility of achieving this therapeutic objective, we have measured individual 24 h serum E 2 and testosterone concentrations in women with vulvovaginal atrophy (VVA) receiving daily intravaginal administration of a clinically effective dose of 6.5 mg prasterone (dehydroepiandrosterone, DHEA). Serum E 2 and testosterone, as well as DHEA and nine of its other metabolites, were assayed at ten time intervals over 24 h on the first and seventh days of daily vaginal administration of 6.5 mg prasterone. No significant change from baseline of average 24 h serum E 2 or testosterone concentrations was observed. Moreover, average 24 h serum DHEA remained within the normal postmenopausal range. Estrone sulfate and the androgen metabolites androsterone glucuronide and androstane-3α, 17β-diol glucuronide did not change, thus confirming the absence of any biologically relevant systemic exposure to estrogens and androgens, respectively. Serum concentrations of metabolites of both estrogens and androgens remain within the normal postmenopausal range following daily intravaginal administration of 6.5 mg prasterone. As other studies have shown, local formation of sex steroids in peripheral tissues without significant release of E 2 or testosterone in the circulation can be achieved with intravaginal prasterone. Thus, prasterone is a promising physiological and attractive solution to treating VVA symptoms.

Since prostate cancer reaches the advanced and non curable stage in the absence of any specific symptom or sign, it seems reasonable to diagnose this cancer at an early and curable stage. Screening by prostate-specific antigen (PSA) has been the common technology used. The last follow-up of the first two prospective and randomized screening studies for prostate cancer, namely the Quebec and ERSPC (European Randomized Study of Screening for Prostate Cancer) clinical trials started in 1988 and 1991, respectively, have shown reductions of prostate cancer death of 62% (P

Acupuncture has been demonstrated to improve menstrual frequency and to decrease circulating testosterone in women with polycystic ovary syndrome (PCOS). Our aim was to investigate whether acupuncture affects ovulation frequency and to understand the underlying mechanisms of any such effect by analyzing LH and sex steroid secretion in women with PCOS. This prospective, randomized, controlled clinical trial was conducted between June 2009 and September 2010. Thirty-two women with PCOS were randomized to receive either acupuncture with manual and low-frequency electrical stimulation or to meetings with a physical therapist twice a week for 10–13 wk. Main outcome measures were changes in LH secretion patterns from baseline to after 10–13 wk of treatment and ovulation frequency during the treatment period. Secondary outcomes were changes in the secretion of sex steroids, anti-Müllerian hormone, inhibin B, and serum cortisol. Ovulation frequency during treatment was higher in the acupuncture group than in the control group. After 10–13 wk of intervention, circulating levels of estrone, estrone sulfate, estradiol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, testosterone, free testosterone, dihydrotestosterone, androsterone glucuronide, androstane-3α,17β-diol-3-glucuronide, and androstane-3α,17β-diol-17-glucuronide decreased within the acupuncture group and were significantly lower than in the control group for all of these except androstenedione. We conclude that repeated acupuncture treatments resulted in higher ovulation frequency in lean/overweight women with PCOS and were more effective than just meeting with the therapist. Ovarian and adrenal sex steroid serum levels were reduced with no effect on LH secretion.

Studying the mechanisms for the complex pathogenesis of polycystic ovary syndrome (PCOS) requires animal models with endocrine, reproductive, and metabolic features of the syndrome. Hyperandrogenism seems to be a central factor in PCOS, leading to anovulation and insulin resistance. In female rats, continuous administration of letrozole, a nonsteroidal inhibitor of P450 aromatase, at 400 μg/d starting before puberty induces hyperandrogenemia and reproductive abnormalities similar to those in women with PCOS. However, despite high circulating testosterone levels, these rats do not develop metabolic abnormalities, perhaps because of their supraphysiological testosterone concentrations or because estrogen synthesis is completely blocked in insulin-sensitive tissues. To test the hypothesis that continuous administration of lower doses of letrozole starting before puberty would result in both metabolic and reproductive phenotypes of PCOS, we performed a 12-wk dose-response study. At 21 d of age, 46 female Wistar rats were divided into two letrozole groups (100 or 200 μg/d) and a control group (placebo). Both letrozole doses resulted in increased body weight, inguinal fat accumulation, anovulation, larger ovaries with follicular atresia and multiples cysts, endogenous hyperandrogemia, and lower estrogen levels. Moreover, rats that received 200 μg/d had insulin resistance and enlarged adipocytes in inguinal and mesenteric fat depots, increased circulating levels of LH, decreased levels of FSH, and increased ovarian expression of Cyp17a1 mRNA. Thus, continuous administration of letrozole, 200 μg/d, to female rats for 90 d starting before puberty results in a PCOS model with reproductive and metabolic features of the syndrome.

Introduction: Acolbifene (ACOL) is a novel compound completely free of estrogen-like activity in both the human and rat mammary gland and uterus. In previous studies, it was observed that ACOL could have a cytotoxic or tumoricidal rather only tumorostatic activity, with a lack of development of tumor resistance. The objective of the present study was to obtain information on the molecular basis of the tumoricidal properties of acolbifene, as well as on the identity of the genes potentially implicated in the resistance to tamoxifen (TAM). The specificity of action of ACOL was compared to the pure antiestrogen fulvestrant (FUL) as well as to tamoxifen (TAM) and raloxifene (RAL). Methods: The gene expression profile of the ZR-75-1 breast cancer xenografts was studied following treatment with ACOL, RAL, TAM and FUL. Ovariectomized female nude mice (10/group) bearing human ZR-75-1 breast cancer xenografts were supplemented with estrone (E1) (subcutaneous silastic implants) and were injected daily with the vehicle alone or 50 μg of ACOL, RAL, TAM or FUL for 6 months. Mice were killed 24h after the last injection and ZR-75-1 tumors were collected and processed for RNA extraction and microarray analyses (Affymetrix GeneChip U133 Plus 2.0). Results: Long-term exposure of ZR-75-1 xenografts to E1 causes a massive modulation of E1-responsive genes. When the antiestrogens were administered simultaneously with E1, some compound-specific prevention of the effect of E1 was observed. Globally, the efficacy of the compounds was ACOL>FUL or RAL>TAM. ACOL significantly prevented the effect of E1 on 195 responsive genes. Among these, the most enriched gene ontological group in Molecular Function was Receptor Activity, including ER, insulin, retinoid and thrombospondin receptor activity. One of the transcriptional repressors of the estrogen signaling pathway, the NKX31, was down-regulated by E1 and completely blocked by ACOL (fold-change −1.64 and 2.54, respectively). The most enriched biological processes were associated with mammary gland development, positive regulation of glucose metabolic processes and blood vessel morphogenesis. Between the genes implicated in cell death/apoptosis of breast cancer cells, the changes of expression of 57, 51, 48 and 31 genes were significantly neutralized by ACOL, RAL, FUL and TAM, respectively. Sixteen genes implicated in tumor resistance to TAM were significantly blocked by ACOL, including DEGS1, a gene implicated in the inhibition of EGF receptor biosynthesis. The other 15 genes in the group were up-regulated by E1, changes which were significantly blocked by ACOL. Conclusion: The present data offer a possible explanation for the potent tumoricidal action of acolbifene in human breast cancer xenografts, thus offering a new paradigm in the hormonal therapy of breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-06-03.

Objective To determine whether there is a causal effect of oral contraceptive (OC) treatment on general well-being and depressed mood in healthy women. Design Double-blind, randomized, and placebo-controlled trial. Setting University hospital. Patient(s) Three hundred and forty healthy women aged 18–35 years randomized to treatment, of whom 332 completed the data collection at follow-up evaluation. Intervention(s) A combined OC (150 μg levonorgestrel and 30 μg ethinylestradiol) or placebo for 3 months of treatment. Main Outcome Measure(s) Primary outcome measures: global score of Psychological General Well-Being Index (PGWBI) and the Beck Depression Inventory (BDI); secondary outcome measures: six separate dimensions of the PGWBI. Result(s) The OC treatment statistically significantly decreased general well-being compared with placebo −4.12 (95% CI, −7.18 to −1.06). Furthermore, OC decreased the following PGWBI dimensions compared with placebo: positive well-being −3.90 (95% CI, −7.78 to −0.01), self-control −6.63 (95% CI, −11.20 to −2.06), and vitality −6.84 (95% CI, −10.80 to −2.88). The effect of OC on depressive symptoms and on the PGWBI dimension depressed mood were not statistically significant. Conclusion(s) This study demonstrates a statistically significant reduction in general well-being by a first-choice OC in comparison with placebo in healthy women. We found no statistically significant effects on depressive symptoms. A reduction in general well-being should be of clinical importance.

The concentrations of allopregnanolone (Allopreg), pregnenolone (Preg) and androsterone (ADT) are very low in the circulation, especially in postmenopausal women, resulting in a considerable challenge for their accurate measurements in serum or plasma. In this report, a sensitive and reliable LC-MS/MS assay method has been developed using a simple sample preparation and the 1-Amino-4-methylpiperazine (AMP) derivatization procedure. A 5pg/ml (0.1pg on column) of low limit of quantitation has been achieved for Allopreg, Preg and ADT, with a sensitivity comparable to data obtained with the commercial reagent. The major benefit of this reagent is to limit the matrix effect since the excess amount of reagent can be removed during the reaction. Multiple reaction monitoring (MRM) from the derivatization of AMP not only increases the detection of these compounds but also provides a good resolution for Allopreg, Preg and ADT from interferences, especially for Allopreg from its isomers. Within the calibration range of 5pg/ml to 2000pg/ml, a good linearity was obtained with R>0.99 where the weighing factor is 1/X. Bias and coefficients of variance are within 15% for all QC levels. The matrix effect has been evaluated, well meeting the acceptance criteria according to the FDA guidelines. With this method, the concentrations of Allopreg, Preg and ADT in postmenopausal serum are in the range of 6.4-53.6pg/ml, 16.2-68.0pg/ml and 23.9-114.0pg/ml, respectively, while the ranges in premenopausal serum are 8.2-701.5pg/ml, 31.2-135.2pg/ml and 47.8-310.0pg/ml, respectively.

Objective Serum concentrations of estradiol (E2) and testosterone (testo) measured by mass spectrometry-based assays should remain below the 95th centile measured at 9.3 pg/mL for E2 and 0.26 ng/mL for testo in normal postmenopausal women in order to avoid the risk of non-physiological systemic exposure to elevated serum concentrations of these two sex steroids. Methods Serum E2 and testo, as well as dehydroepiandrosterone (DHEA) and nine of its other metabolites, were measured at 10 time intervals over 24 h on the first and seventh days of daily intravaginal administration of 0.50% (6.5 mg) DHEA by validated mass spectrometry-based assays. Results No biologically significant change in the individual serum concentrations of E2, testo or DHEA was observed. Most importantly, estrone sulfate (E1-S) and the glucuronidated androgen metabolites also remained within normal values, thus confirming the absence of biologically significant systemic exposure in line with intracrinology. Using data from the literature, comparison is made with serum E2 above normal postmenopausal values following administration of 10-μg E2 tablets. Conclusion While the clinical program on vulvovaginal atrophy has shown the efficacy and safety of intravaginal 6.5 mg of DHEA (prasterone), the present data illustrate in detail the serum levels of the individual sex steroids and their metabolites derived from DHEA. The data obtained are in line with the physiology of intracrinology and confirm an action limited to the vagina as the serum concentrations of all sex steroids are maintained within the normal values of menopause, thus protecting the uterus and most likely other tissues.

The objective is to review how the cell-specific amounts of intracellular androgens are all made in women from circulating dehydroepiandrosterone (DHEA) in each peripheral tissue, independently from the rest of the body. Following 500 million years of evolution, approximately three dozen cell-specific intracrine enzymes have been engineered in human peripheral tissues whereby the inactive sex steroid precursor DHEA mainly of adrenal origin is transformed into the appropriate minute intracellular amounts of androgens. These intracellular androgens are inactivated in the same cells, with no biologically significant release of active androgens in the circulation. The best estimate is that approximately 50% as much androgens are synthesized in women, compared to men of the same age. The problem with DHEA, however, the exclusive source of androgens in women of all ages, is that DHEA secretion has already decreased by an average of 60% at time of menopause and continues to decrease thereafter. The human-specific and highly sophisticated mechanisms of intracrinology permit each cell to control androgen availability according to its own needs independently from the remaining of the body. Such a mechanism is completely different from classical endocrinology well understood in men where testosterone of testicular origin is transported through the blood and has indiscriminate access to the androgen receptor (AR) in all AR-containing cells of the body. In men, both the endocrine and intracrine mechanisms are in operation while, in women, only the intracrine mechanisms responsible for intracellular formation from DHEA provide androgens.

To examine maternal insulin resistance in relationship with maternal and fetal androgen levels as well as with term placenta mRNA and protein abundance of steroidogenic enzymes implicated in androgen dynamics.The study included 20 women with gestational diabetes mellitus and 27 controls tested using a 120 min., 75 g oral glucose tolerance test. Maternal and fetal plasma concentrations of total testosterone, dihydrotestosterone (DHT) and dehydroepiandrosterone (DHEA) were measured by high-performance gas chromatography and chemical ionization mass spectrometry at 26.1 ± 3.7 weeks of pregnancy.Glycemic response to oral glucose over 120 min. as well as Matsuda insulin sensitivity and HOMA insulin resistance (HOMA-IR) indices were significantly associated with maternal testosterone levels (r = 0.31, r = -0.37 and r = 0.35 respectively, p ≤ 0.05 for all). Among male offspring, a positive association between maternal and fetal testosterone levels was observed (r = 0.43, p ≤ 0.05). Testosterone levels were higher in the cord blood of newborns from insulin-resistant mothers compared to newborns from insulin-sensitive mothers (0.48 ± 0.36 nmol/L vs. 0.29 ± 0.18 nmol/L p ≤ 0.05). No difference was observed in mRNA abundance or protein expression of placental steroidogenic enzymes according to the degree of maternal insulin resistance.Our results demonstrate a possible association between fetal and maternal androgen concentrations in relationship with insulin resistance.

Menopause has been chosen by evolution as the convergence of three factors, namely cessation of ovarian function (reproduction and estrogen secretion), high circulating dehydroepiandrosterone (DHEA), and intracrine enzymes able to convert DHEA into active sex steroids in peripheral tissues. The arrest of estrogen secretion by the ovaries at menopause causes a decrease of circulating estradiol below the threshold of biological activity, thus eliminating stimulation of the endometrium and risk of endometrial cancer. As much as the arrest of secretion of estradiol by the ovaries is essential to protect the uterus, it is of major importance that sex steroids continue to be made available in most other tissues which need estrogens and/or androgens for their normal functioning. Evolution, through 500 million years, has progressively provided the peripheral tissues with the enzymes able to make androgens and estrogens while high levels of DHEA, the precursor of all sex steroids, have appeared much later with the primates approximately 20 million years ago. All elements were thus in place for the functioning of intracrinology or the cell-specific formation of estrogens and androgens in peripheral tissues from the inactive precursor DHEA, with no significant release of active sex steroids in the circulation, thus eliminating the risks of adverse effects in the other tissues, especially the uterus. The presence of subthreshold levels of circulating estradiol combined with the formation of sex steroids from DHEA in specific peripheral tissues (intracrinology) makes menopause a positive characteristic supporting many years of good-quality postmenopausal life, useful for taking care of children and grandchildren. DHEA, however, decreases with age and is present at very different concentrations between different women, with the consequence that approximately 75% of postmenopausal women have too low circulating DHEA levels and suffer from symptoms/signs of hormone deficiency.