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PCOS is common in adolescents and includes excess testosterone concentrations. The clinical importance of 11-oxyandrogens in PCOS is unclear. We sought to determine if fasted morning 11-oxyandrogens: 1) better identify PCOS diagnosis compared to testosterone, 2) relate to clinical comorbidities of PCOS, 3) are altered with combined oral contraceptive pill (COCP) or metformin therapy. Data from 186 adolescent females aged 12-21 years, mostly obese, enrolled in one of 6 studies in our division were included: 115 untreated PCOS (13 lean, BMI 22.5±2.2 kg/m2; 102 overweight/obese, BMI 35.4±5.3), 9 obese PCOS treated with COCP (BMI 33.7±5.7), 6 obese PCOS treated with metformin (BMI 36.4±5.3) and 70 normally cycling controls (19 lean, BMI 19.9±1.4; 52 overweight/obese BMI 35.5±10.4). Three 11-oxyandrogens (11-hydroxyandrostenedione (11OHA), 11-hydroxytestosterone (11OHT), and 11-ketotestosterone (11KT) and total testosterone were analyzed via liquid chromatography tandem mass spectroscopy (LabCorp/Esoterix, Calabasas, CA). Data between 1) untreated PCOS and controls and 2) untreated PCOS and the 2 treatment groups were compared. ROC analysis was performed to evaluate accuracy of diagnosis of PCOS and Pearson’s correlation coefficient was calculated for 11-oxyandrogens and clinical measures. Untreated PCOS girls had higher 11OHA (129±77 ng/dL PCOS vs 97±52 control, p=0.003) and 11OHT (13.8±7.9 ng/dL PCOS vs 10.5±6.7 control, p=0.005) compared to controls, with no difference in 11KT (30.7±17.3 ng/dL PCOS vs 26.6±16.6 control, p=0.208). Only 11OHA remained significant after controlling for obesity via multiple linear regression. However, neither of these metabolites better predicted PCOS status compared to testosterone (ROC analysis: 11OHA AUC=0.620, p=0.006; 11OHT AUC=0.638, p=0.002; total testosterone AUC=0.840, p&lt;0.001, free androgen index AUC 0.860, p&lt;0.001). Among girls with PCOS, all three 11-oxyandrogens correlated with hirsutism severity as graded by Ferriman Gallwey score. COCP treatment decreased 11KT concentrations compared to untreated PCOS (32±17 untreated vs 17±8 ng/dL COCP; p=0.039) as well as total testosterone (46.9±21 untreated vs 28.1±12.9 ng/dL COCP; p&lt;0.006). However, metformin treatment had no effect 11-oxyandrogens, although total testosterone was lower (46.9±21 untreated vs 26.5±5.9 metformin; p=0.01). Whereas 11-oxyandrogens would not aid in the diagnosis of PCOS, they relate to excess hair growth and 11KT decreases with COCP therapy. Further work is needed to determine the relationship with metabolic outcomes and the clinical utility of measuring these androgens in PCOS.

ENDO 2021

11-Oxyandrogens in Youth with Polycystic Ovary Syndrome

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ENDO 2020

ENDO is the place where leading experts, researchers, and the most respected clinicians in the field come together to share the latest findings in hormone science and discuss breakthroughs in clinical care.

PCOS is common in adolescents and includes excess testosterone concentrations. The clinical importance of 11-oxyandrogens in PCOS is unclear. We sought to determine if fasted morning 11-oxyandrogens: 1) better identify PCOS diagnosis compared to testosterone, 2) relate to clinical comorbidities of PCOS, 3) are altered with combined oral contraceptive pill (COCP) or metformin therapy. Data from 186 adolescent females aged 12-21 years, mostly obese, enrolled in one of 6 studies in our division were included: 115 untreated PCOS (13 lean, BMI 22.5±2.2 kg/m2; 102 overweight/obese, BMI 35.4±5.3), 9 obese PCOS treated with COCP (BMI 33.7±5.7), 6 obese PCOS treated with metformin (BMI 36.4±5.3) and 70 normally cycling controls (19 lean, BMI 19.9±1.4; 52 overweight/obese BMI 35.5±10.4). Three 11-oxyandrogens (11-hydroxyandrostenedione (11OHA), 11-hydroxytestosterone (11OHT), and 11-ketotestosterone (11KT) and total testosterone were analyzed via liquid chromatography tandem mass spectroscopy (LabCorp/Esoterix, Calabasas, CA). Data between 1) untreated PCOS and controls and 2) untreated PCOS and the 2 treatment groups were compared. ROC analysis was performed to evaluate accuracy of diagnosis of PCOS and Pearson’s correlation coefficient was calculated for 11-oxyandrogens and clinical measures. Untreated PCOS girls had higher 11OHA (129±77 ng/dL PCOS vs 97±52 control, p=0.003) and 11OHT (13.8±7.9 ng/dL PCOS vs 10.5±6.7 control, p=0.005) compared to controls, with no difference in 11KT (30.7±17.3 ng/dL PCOS vs 26.6±16.6 control, p=0.208). Only 11OHA remained significant after controlling for obesity via multiple linear regression. However, neither of these metabolites better predicted PCOS status compared to testosterone (ROC analysis: 11OHA AUC=0.620, p=0.006; 11OHT AUC=0.638, p=0.002; total testosterone AUC=0.840, p<0.001, free androgen index AUC 0.860, p<0.001). Among girls with PCOS, all three 11-oxyandrogens correlated with hirsutism severity as graded by Ferriman Gallwey score. COCP treatment decreased 11KT concentrations compared to untreated PCOS (32±17 untreated vs 17±8 ng/dL COCP; p=0.039) as well as total testosterone (46.9±21 untreated vs 28.1±12.9 ng/dL COCP; p<0.006). However, metformin treatment had no effect 11-oxyandrogens, although total testosterone was lower (46.9±21 untreated vs 26.5±5.9 metformin; p=0.01). Whereas 11-oxyandrogens would not aid in the diagnosis of PCOS, they relate to excess hair growth and 11KT decreases with COCP therapy. Further work is needed to determine the relationship with metabolic outcomes and the clinical utility of measuring these androgens in PCOS.

technical paper

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Bone geometry can be described in terms of periosteal and endocortical growth and is partly determined by sex steroids. Periosteal and endocortical apposition are thought to be regulated by testosterone and estrogen, respectively. Gender-affirming hormone (GAH) treatment with sex steroids in transgender people might affect bone geometry. However, in adult transgender people no change in bone geometry during GAH was observed. In this study, we investigated changes in bone geometry among transgender adolescents using a gonadotropin-releasing hormone agonist (GnRHa) and GAH prior to achieving peak bone mass. Transgender adolescents treated with GnRHa and subsequent GAH at our center before the age of 18 years were eligible for inclusion. Participants were grouped based on their Tanner stage at the start of GnRHa treatment and divided into early, mid, and late puberty groups. Hip Strength Analysis software calculating subperiosteal width (SPW) and endocortical diameter (ED) was applied to dual-energy X-ray absorptiometry scans performed at start of GnRHa and GAH treatments, and after ≥ 2 years of GAH treatment. Mixed model analyses were performed to study differences over time. Data were visually compared with reference values of the general population retrieved from the literature. A total of 322 participants were included, of whom 106 trans women and 216 trans men. In both trans women and trans men participants resembled the reference curve for SPW and ED of the experienced gender, but only when GnRHa was started during early puberty. Those who started during mid- and late puberty remained within the reference curve of the gender assigned at birth. A possible explanation might be sought in the phenomenon of programming, which conceptualizes that stimuli during critical windows of development can have major consequences throughout one’s lifespan. Therefore, this study adds insights into sex-specific bone geometry development during puberty of transgender adolescents treated with GnRHa, as well as the general population.

Development of Hip Bone Geometry During Gender-Affirming Hormone Therapy in Transgender Adolescents Resembles that of Experienced Gender When Pubertal Suspension Is Started Early in Puberty

Introduction and Purpose: Polycystic Ovary Syndrome (PCOS) is recognized as the most common endocrine disorder in women of reproductive age. Notably, PCOS women with hyperandrogenism have a pronounced increased risk for cardio-metabolic comorbidities compared with healthy individuals. Bile acids are endocrine signaling molecules that modulate hepatic lipid, glucose, and energy metabolism by aiding in absorption of lipids. Alteration of bile acid homeostasis affects overall metabolic homeostasis and contributes to pathogenesis of an array of metabolic diseases, although the molecular mechanisms of this have not been studied in PCOS. Methods: Four-week old C57BL/6N female mice were implanted subcutaneously with dihydrotestosterone (DHT, 8.0 mg) or vehicle silastic tubes (n=8/grp). Weekly body weight, food intake, and body composition was assessed. Fasting serum was obtained and the oral glucose tolerance test (OGTT) was performed in the last week of treatment. Animals were euthanized on treatment day 90 and livers were harvested. Expression levels of mRNA were assessed using RT-qPCR. Results: DHT treated females had significantly higher liver mass (1,387 ± 51 vs 1,197 ± 29 g, p<0.05), increased lean mass (21.25 ± 0.27 vs 19.58 ± 0.23 g, p<0.05) and increased fat mass (4.83 ± 0.47 vs 3.59 ± 0.36 g, p<0.05) compared to the vehicle counterparts. These hyperandrogenemic females additionally showed altered glucose homeostasis, having increased fasting glucose (201.10 ± 11.11 vs 152.80 ± 9.23 mg/dL, p<0.05) and an increased area under the curve (209.2± 11.0 vs 160.8± 3.5 mg.min/dL, p<0.05) following OGTT. Hepatic expression of both classic (Cyp8b1, 1.4 ± 0.1-fold, p<0.05) and alternative (Cyp7b1, 2.0 ± 0.3-fold, p<0.05) bile acid synthesis cytochrome P450 enzyme genes were significantly upregulated in DHT treated animals. Additionally, expression of sulfotransferase Sult2a2 was completely abolished in DHT treated animals compared with vehicle animals, indicating the possibility of androgen regulation of the sulfonation of bile acids marked for elimination. Liver expression of both the bile acid receptor G-protein coupled bile acid receptor 1 and the androgen receptor were both significantly downregulated (Gpbar1: 0.68 ± 0.08-fold, AR: 0.46 ± 0.04-fold, p<0.05) in DHT treated animals. Conclusions: Bile acid synthesis, transport, and elimination are tightly controlled processes in the liver to maintain a constant bile acid pool and limit reabsorption. Together, our results highlight the potential role of androgens in DHT-treated female mice in the dysregulation of bile acid homeostasis and its potential contribution to influence metabolic dysfunction. (Supported by NIH grants NIGMS P20GM-121334 to LLYC and DGR, and NIH NIDDK R21DK-113500 to DGR and the Mississippi Center of Excellence in Perinatal Research.)?? 

Hepatic Dysregulation of Bile Acid Homeostasis in Hyperandrogenemic Female Mouse Model of Polycystic Ovary Syndrome

Introduction: During the 22-week core LINC 2 study, the oral 11β-hydroxylase inhibitor osilodrostat normalized mean urinary free cortisol (mUFC) in 79% (15/19) of patients with Cushing’s disease. This report describes long-term LINC 2 efficacy and safety results following an optional extension. Methods: Patients receiving clinical benefit at week 22 could enter the extension (that ran until Oct 22, 2019), continuing the same osilodrostat dose; dose adjustments were permitted based on efficacy and safety. Response rate (mUFC ≤ULN [controlled] or mUFC >ULN but ≥50% decrease from baseline [BL; partially controlled]) was assessed over time. Efficacy/safety were assessed for all patients from core BL until study end. Results: Of 19 enrolled patients (female:male 14:5; mean [SD] age 36.8 years [8.4]), 16 entered the optional extension and 8 of them remained on treatment until study end. Median (range) osilodrostat exposure was 282 weeks (2-351). Mean mUFC decreased from BL (9.9 x ULN) to ≤ULN by week 4 and remained stable throughout the study. All 19 patients achieved mUFC ≤ULN at least once during the study. At each assessment up to month 70 of the extension phase, 50-88% of ongoing patients were controlled, and up to 18% were partially controlled. Mean percentage change in clinical signs from BL (mean [SD]) to last assessment were: fasting plasma glucose, -10.8% (22.1) (from BL: 105.6 mg/dL [49.2]); HbA1c, -2.1% (9.0) (from BL: 5.7% [0.7]); systolic BP, -3.3% (12.6) (from BL: 132.6 mmHg [11.6]); diastolic BP, -2.0% (10.4) (from BL: 85.0 mmHg [6.5]); BMI, -5.9% (8.8) (from BL: 30.7 kg/m2 [7.0]). Overall, 9 patients discontinued treatment (n=2 core and n=7 extension), mostly because of AEs or no longer requiring treatment (n=3 each). The most common AEs during the entire treatment period were nausea (n=10), adrenal insufficiency, and headache (both n=9). AEs related to hypocortisolism and adrenal hormone precursor accumulation occurred in 11 (mostly adrenal insufficiency, n=9) and 12 patients (mostly hypertension, n=4), respectively; most were grade 1/2 and managed with dose adjustment/interruption and/or concomitant medication. Mean (SD) plasma ACTH increased from 1.8 x ULN (0.9) at BL to 7.1 x ULN (12.3) at week 22 and 6.9 x ULN (12.6) at last assessment. Mean (SD) 11-deoxycortisol increased from 1.2 x ULN (1.3) at BL to 13.6 x ULN (12.2) at week 22 and 3.6 x ULN (4.2) at last assessment. In females, mean (SD) testosterone increased from 0.8 x ULN (0.4) at BL to 2.4 x ULN (2.1) at week 22 and 1.0 x ULN (0.9) at last assessment. Two patients, both female, reported an AE of hirsutism.?Conclusions: Rapid reductions in mUFC were sustained for up to 6 years of osilodrostat treatment and were accompanied by improvements in clinical signs of hypercortisolism. Osilodrostat was well tolerated, with no new safety signals during long-term treatment.?? 

Long-Term Control of Urinary Free Cortisol with Osilodrostat in Patients with Cushing's Disease: Final Results from the LINC 2 Study

Depot injection formulations of peptide somatostatin receptor ligands (SRLs) are routinely used to treat acromegaly and neuroendocrine tumors (NETs). Paltusotine (CRN00808), an orally administered small molecule nonpeptide selective somatostatin receptor 2 (SST2) agonist has been shown to maintain GH and IGF-1 levels in acromegaly patients previously on depot SRLs (ACROBAT Edge NCT03789656). In this study, a capsule formulation was used, which did not exhibit dose proportional pharmacokinetics (PK) at doses >40 mg, required a 2-hour post-dose fast in overnight fasted patients, and had the potential for reduced bioavailability when taken with proton-pump inhibitors (PPI). A spray-dried dispersion (SDD) tablet formulation was developed with improved solubility in the physiological pH range and its performance was evaluated in healthy volunteers. Male and female healthy volunteers who met inclusion/exclusion criteria were enrolled in a single-center Phase 1 study (ANZCTR registration ACTRN12619001562167). A Cohort of 12 subjects was administered a single dose of paltusotine in a four-period cross-over design. Periods 1 and 2 assessed the effect of lansoprazole (a PPI) on PK of 20 mg dose of paltusotine SDD tablets. In Period 3, 20 mg dose of paltusotine SDD tablets was co-administered with a high fat, high-calorie meal. In Period 4, a 60 mg dose of paltusotine SDD tablets was administered to assess dose proportionality. In a separate cohort of subjects (n=12; also, a 4-period cross-over design), the relative bioavailability of capsules and SDD tablets was assessed, and the effect of food administration 0.5, 1, and 2 hour post-dose was evaluated. Subsequently, in another cohort of 12 subjects (a 3-period cross-over design), dose proportionality of the SDD tablets was evaluated at 40 mg and 80 mg dose with a 1-hour post-dose fast. A 4 hours post-dose fast was also assessed for the 80 mg dose. Pharmacokinetics and safety of paltusotine were evaluated. Paltusotine was generally well tolerated in this study. SDD tablets exhibited dose proportional increase in total systemic exposure (AUC) up to a dose of 80 mg. Healthy volunteers pretreated with the PPI, lansoprazole (15 mg bid for 3 days), and co-administered with paltusotine SDD tablets exhibited a small decrease (approximately 25%) in systemic exposure to paltusotine compared with the same subjects that had washed out from the PPI-pretreatment. SDD tablets exhibited significant reduction in systemic exposure when co-administered with a high-fat, high-calorie meal. However, the SDD formulation was less sensitive to timing of post-dose food administration compared to the capsule formulation. These data suggest that the SDD tablet formulation of paltusotine improves flexibility in dose administration, can be co-administered with PPIs and other agents that increase stomach pH, and reduces the post-dose fasting requirement.

Pharmacokinetics and Safety of an Improved Oral Formulation of Paltusotine, a Selective, Nonpeptide Somatostatin Receptor 2 (SST2) Agonist for the Treatment of Acromegaly

Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.?? 

Canine Pituitary Organoids as 3D in vitro model for Cushing Disease

Background: Cushing’s disease (CD) is caused by high levels of blood cortisol resulting from excess secretion of ACTH from a corticotroph adenoma in the anterior pituitary gland. Clinical features include hypertension, diabetes, osteoporosis, and depression. If untreated CD has an increased mortality of five-fold owing to cardiovascular comorbidities, stroke or raised vulnerability to infection. Transsphenoidal surgery is considered the first-line treatment but remission is achieved in only 65% of cases and the relapse rate is high. Furthermore, medical treatments are often accompanied by unpleasant side-effects. Antisense therapy is a technique for suppressing gene expression at the level of translation using antisense oligonucleotides (ASOs) against the mRNA of interest. Aims: To investigate antisense therapy as a treatment for CD by targeting ASOs against ACTH-encoding POMC mRNA thereby reducing secretion of the hormone. To transfect mouse AtT20 cells (cells that secrete high levels of ACTH) with ASOs against POMC at varying doses to determine which is the most effective at reducing ACTH secretion. Methods: AtT-20 cells that secrete high levels of ACTH were used as the model system. ASOs were designed to specifically target exon 3 of the POMC gene. Transfection of AtT-20 was carried out using Lipofectamine. FACS was used to determine transfection efficiency. ACTH levels secreted by AtT-20 cells were determined by immunoassay. Statistical analysis was done using ANOVA with P values < 0.05 considered significant. Results: ASOs that targeted POMC exon 3 (ASO-2 and ASO-3) were transfected into AtT-20 cells at 10 and 100 nM. Control ASOs were ASO-1 (matched to POMC sense strand) and ASO-4 (a scrambled version of ASO-3). Experiments included untreated AtT-20 cells and AtT-20 cells treated with transfection reagent or ASOs alone. The results of six experiments indicated that ACTH secretion from AtT-20 cells was reduced after transfection with ASO-2 and ASO-3 at 100 nM (ANOVA, P = < 0.05) and 10 nM (ANOVA, P < 0.05) when compared with untreated AtT20 cells. ASO-1 and ASO-4 had no effect on ACTH secretion by AtT-20 cells (ANOVA, P > 0.05). Conclusions: Initial experiments have shown that ASOs against POMC can reduce ACTH secretion from AtT-20 cells and may be useful as a novel therapy for CD.?? 

Antisense Oligonucleotides as a Novel Therapy for Cushing's Disease

Closing the Loop With Pump and Sensor Communication in Diabetes Mellitus: How to Digest Data and Develop Treatment Recommendations During a Clinic Visit

11-Oxyandrogens in Youth with Polycystic Ovary Syndrome

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Development of Hip Bone Geometry During Gender-Affirming Hormone Therapy in Transgender Adolescents Resembles that of Experienced Gender When Pubertal Suspension Is Started Early in Puberty

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