Androstenedione Concentrations Research Articles

Effect of age on androgens pattern in cyclic mares

Androgens are produced in both sexes. In females produced by the adrenal gland and the ovaries they play a crucial role in regulating ovarian function, estrogen synthesis and follicular growth. Age leads to a reduction in androgen concentrations, although, at present, these mechanisms are not elucidated in mares. The objective of this study was to evaluate the concentrations of testosterone (T), androstenedione (A4) and dehydroepiandrosterone (DHEA) in mares of different ages. Blood samples were drawn from seventy cyclic Spanish Purebred mares belonging to five age groups: 3–5 years, 6–9 years, 10–13 years, 14–16 years and > 16 years. The concentrations of T, A4 and DHEA were determined by EIA, validated specifically for horses. Mares aged 3–5, 6–9 and 10–13 years had higher T concentrations (P < 0.05) than mares aged >16 years, and mares aged 6–9 years had also higher concentrations than those 14–16 years old (P < 0.05). A4 concentrations were lower (P < 0.05) in mares >16 years old when compared with those of other age groups. DHEA concentrations were lower (P < 0.05) in mares 14–16 years and > 16 years old when compared with those of other age groups. DHEA was positively correlated with T (r = 0.61; P < 0.05) and A4 (r = 0.51; P < 0.05). Age induces reduction in androgens' synthesis in physiologically cyclic Spanish Purebred mares. These physiological variations must be duly considered for a correct and objective interpretation of the analytical data.

Sex-steroid hormones and risk of postmenopausal estrogen receptor-positive breast cancer: a case-cohort analysis.

Sex-steroid hormones are associated with postmenopausal breast cancer but potential confounding from other biological pathways is rarely considered. We estimated risk ratios for sex-steroid hormone biomarkers in relation to postmenopausal estrogen receptor (ER)-positive breast cancer, while accounting for biomarkers from insulin/insulin-like growth factor-signaling and inflammatory pathways. This analysis included 1208 women from a case-cohort study of postmenopausal breast cancer within the Melbourne Collaborative Cohort Study. Weighted Poisson regression with a robust variance estimator was used to estimate risk ratios (RRs) and 95% confidence intervals (CIs) of postmenopausal ER-positive breast cancer, per doubling plasma concentration of progesterone, estrogens, androgens, and sex-hormone binding globulin (SHBG). Analyses included sociodemographic and lifestyle confounders, and other biomarkers identified as potential confounders. Increased risks of postmenopausal ER-positive breast cancer were observed per doubling plasma concentration of progesterone (RR: 1.22, 95% CI 1.03 to 1.44), androstenedione (RR 1.20, 95% CI 0.99 to 1.45), dehydroepiandrosterone (RR: 1.15, 95% CI 1.00 to 1.34), total testosterone (RR: 1.11, 95% CI 0.96 to 1.29), free testosterone (RR: 1.12, 95% CI 0.98 to 1.28), estrone (RR 1.21, 95% CI 0.99 to 1.48), total estradiol (RR 1.19, 95% CI 1.02 to 1.39) and free estradiol (RR 1.22, 95% CI 1.05 to 1.41). A possible decreased risk was observed for SHBG (RR 0.83, 95% CI 0.66 to 1.05). Progesterone, estrogens and androgens likely increase postmenopausal ER-positive breast cancer risk, whereas SHBG may decrease risk. These findings strengthen the causal evidence surrounding the sex-hormone-driven nature of postmenopausal breast cancer.

Circulating endogenous sex steroids and risk of differentiated thyroid carcinoma in men and women.

Thyroid cancer (TC) is substantially more common in women than in men, pointing to a possible role of sex steroid hormones. We investigated the association between circulating sex steroid hormones, sex hormone binding globulin (SHBG) and the risk of differentiated TC in men and women within the European Prospective Investigation into Cancer and nutrition (EPIC) cohort. During follow-up, we identified 333 first primary incident cases of differentiated TC (152 in pre/peri-menopausal women, 111 in post-menopausal women, and 70 in men) and 706 cancer-free controls. Women taking exogenous hormones at blood donation were excluded. Plasma concentrations of testosterone, androstenedione, dehydroepiandrosterone, estradiol, estrone and progesterone (in pre-menopausal women only) were performed using liquid chromatography/mass spectrometry method. SHBG concentrations were measured by immunoassay. Odds ratios (ORs) were estimated using conditional logistic regression models adjusted for possible confounders. No significant associations were observed in men and postmenopausal women, while a borderline significant increase in differentiated TC risk was observed with increasing testosterone (adjusted OR T3 vs T1: 1.68, 95% CI: 0.96-2.92, ptrend = .06) and androstenedione concentrations in pre/perimenopausal women (adjusted OR T3 vs T1: 1.78, 95% CI: 0.96-3.30, ptrend = .06, respectively). A borderline decrease in risk was observed for the highest progesterone/estradiol ratio (adjusted OR T3 vs T1: 0.54, 95% CI: 0.28-1.05, ptrend = .07). Overall, our results do not support a major role of circulating sex steroids in the etiology of differentiated TC in post-menopausal women and men but may suggest an involvement of altered sex steroid production in pre-menopausal women.

Serum steroid and thyroid hormone concentrations in healthy domestic male and female alpacas.

Alpacas are high quality fiber producing animals, kept for production purpose and as pets. Endocrine imbalances from adrenal glands, gonads, or thyroid gland may result in coat abnormalities in domestic animals and affect reproduction. Contrary to many domesticated animals, information on hormone concentrations in alpacas is scarce. The purpose of this study was to provide steroid and thyroid hormone values in domestic alpacas. Blood was collected from healthy male (35 intact, 2 castrated) and female (48 non-pregnant, 3 pregnant) alpacas from local farms in Tennessee. Adrenal, reproductive, and thyroid hormones were analyzed. There were no significant differences in median concentrations of progesterone, estradiol, thyroxine (T4), and triiodothyronine (T3) between intact male and female non-pregnant alpacas. Median concentrations of testosterone, androstenedione, 17-hydroxyprogesterone, and cortisol were significantly higher in intact male alpacas compared to female non-pregnant alpacas. This information provides adrenal, gonadal, and thyroid hormone concentrations in alpacas to help with diagnosis of endocrine disorders.

Plasma Androstenedione Concentration Can Discriminate Frail versus Non-Frail Men with Prostate Cancer under Androgen Deprivation Therapy.

Androgen deprivation therapy (ADT) is a mainstay of prostate cancer in both adjuvant and palliative settings. Since androgens are crucial for functional status and psychological functions, we evaluated whether blood testosterone, androstenedione, or DHEA concentrations were associated with functional status and psychological alterations in patients with localised (PCa) or metastatic prostate cancer (mPCa) receiving ADT with analogues of luteinising hormone-releasing hormone (LHRH). The five Fried criteria were considered to identify frailty syndrome. In addition, complementary evaluations were carried out to measure other variables of interest. Sleep quality was assessed using the Athens Insomnia Scale, cognitive functions were assessed using the Mini-Mental State Examination, and symptoms of depression were measured using the Yesavage Geriatric Depression Scale. Logistic regression analysis was performed to determine if the androgens level could be related to frailty syndrome, sleep impairment, depressive symptoms, and cognitive functions. The results of the multivariate analyses show that high concentrations of androstenedione were significantly associated with frailty syndrome in both groups (p = 0.018; odds ratio = 4.66, 95% confidence interval [1.30-16.6]). There were significant relationships between frailty syndrome and the systemic concentration of androstenedione (p = 0.01), but not the concentration of testosterone (p = 0.60) or DHEA (p = 0.42). In addition, the results of the non-parametric tests show significant results between a decreased gait speed in the two groups (metastatic and localised) and the concentration of androstenedione (p = 0.015). High androstenedione levels were associated with a slow walking speed in the mCaP group (p = 0.016), while high testosterone levels were associated with a better walking speed in the localised CaP group (p = 0.03). For the concentration of androstenedione in plasma, the area under the curve was 0.72, with a 95% CI of 0.55-0.88 with acceptable values, and with a cut-off point of 4.51 pg/mL, a sensitivity of 82.9%, and specificity of 53.8%. No relationships between the concentration of androgens in plasma and sleep quality, cognitive functions, or symptoms of depression suggest that the changes were specific to frailty syndrome. Further research into the role of androstenedione should be evaluated in follow-up studies in order to recommend its use as a suitable biomarker of frailty syndrome in prostate cancer patients.

Minipuberty in Sons of Women with Low Vitamin D Status during Pregnancy.

Minipuberty is a transient phase of reproductive axis activation during the first several months of life, playing an important role in the development of reproductive organs in boys. Low 25-hydroxyvitamin D levels during pregnancy are associated with an increased risk of neonatal complications. An inadequate gestational vitamin D status is hypothesized to affect the postnatal activation of the hypothalamic-pituitary-gonadal axis. The purpose of our study was to assess whether a low vitamin D status during pregnancy determines the course of minipuberty in boys. The study included three groups of male infants born to women with different vitamin D statuses: sons of women with vitamin D deficiency (group 1), sons of women with vitamin D insufficiency (group 2), and male offspring of females with normal 25-hydroxyvitamin D levels (group 3 (the reference group)). Concentrations of testosterone, androstenedione, dehydroepiandrosterone sulfate, estradiol, progesterone, and 17-hydroxyprogesterone in saliva, as well as concentrations of gonadotropins in urine, were assayed monthly from postnatal months 1 to 6, and once every 2 months in the second half of the first year of life. Additionally, at each visit, penile length and testicular volume were assessed. Concentrations of testosterone, FSH, and LH, as well as penile length and testicular volume, were greater in group 1 than in groups 2 and 3. In turn, group 2 was characterized by higher FSH levels and a greater testicular volume than group 3. Peak concentrations of LH and testosterone were observed earlier in group 1 than in the remaining groups. The obtained results suggest that a low vitamin D status during pregnancy may have a stimulatory impact on reproductive axis activity and on the early postnatal development of male genital organs, correlating with the severity of hypovitaminosis D.

Inflammatory markers and androstenedione modify the effect of serum testosterone on obesity among men: Findings from a Chinese population.

Few studies are available on the relationship of androstenedione with inflammation and obesity and the effect of androstenedione and inflammation on the association between testosterone and obesity. This study intended to examine the mediation effect of inflammatory markers on the association of testosterone with obesity and the moderation effect of androstenedione on the association of testosterone with inflammation and obesity in Chinese rural men. This cross-sectional research enrolled 2536 male rural inhabitants from the Henan Rural Cohort study. The serum concentrations of testosterone and androstenedione were determined by liquid chromatography-tandem mass spectrometry. Linear and logistic regression were used to examine the relationships between testosterone, inflammatory markers, and obesity. Mediation and moderation analyses were carried out to evaluate the potential effects of inflammatory markers on the relationship between testosterone and obesity, as well as androstenedione on the relationships of testosterone with inflammation and obesity. After adjusting for confounding factors, the results showed that testosterone and androstenedione were negatively related to obesity, and inflammatory markers were positively associated with obesity. Besides, testosterone and androstenedione were negatively associated with inflammatory markers. Mediation analysis showed that white blood cell, neutrophil, monocyte, and high-sensitivity C-reactive protein had mediating effects on the association between testosterone and obesity. The most vital mediator was high-sensitivity C-reactive protein, and its proportion of the effect was 11.02% (defined by waist circumference), 11.15% (defined by waist-to-hip ratio), 12.92% (defined by waist-to-height ratio), and full mediating effect (defined by body mass index). Moreover, androstenedione played negative moderation effects on the associations of testosterone with inflammation and obesity. Inflammatory markers and androstenedione were first found to have modifying effects on the association of testosterone with obesity. Higher levels of testosterone and androstenedione could reduce the inflammation level and risk of obesity, indicating their potential roles in the prevention and treatment of chronic diseases.

THU574 Hsd17b1 Partially Compensates For The Lack Of Hsd17b3 In Testicular Testosterone Production In Fetal Mice, But Not In Adults

Abstract Disclosure: M. Poutanen: None. A. Junnila: None. P. Sipilä: None. I.T. Huhtaniemi: None. Hydroxysteroid (17β) dehydrogenase type 3 (Hsd17b3) is the primary enzyme converting androstenedione to testosterone in human testes. HSD17B3 deficiency is a known cause of a disorder of sex development (DSD), where XY individuals are born with a female appearance, but are virilized during puberty as testosterone starts to suddenly rise due to unknown compensatory mechanisms. We have previously established and characterized an Hsd17b3 knockout mouse line (HSD17B3KO). HSD17B3KO males were born phenotypically normal, but presented various signs of under-masculinization at later ages. However, although they have very high circulating androstenedione at adulthood, they also maintain normal-to-high concentrations of testosterone. Indeed, testosterone was only seen to be low during certain fetal and pubertal time points. We suspected that Hsd17b1, capable of catalyzing the same reaction and expressed in testes towards the end of the fetal development, could be responsible for the compensation in the testosterone production, especially in the presence of higher than normal concentrations of androstenedione. To test this, we created an Hsd17b1-Hsd17b3 double KO (DKO) mouse line. The loss of both Hsd17b1 and Hsd17b3 had a more drastic effect on the phenotype of the male animals than was observed in HSD17B3KO. The outward appearance of the DKO males was completely feminized both as newborns and at adulthood, with an anogenital distance comparable to females. The male internal genitalia were present, but compared to HSD17B3KO they were further reduced in size, as were several other androgen-sensitive tissues. However, the testes and epididymides of the DKO contained high number of mature spermatozoa. The loss of both enzymes had a clear effect on the testis steroidogenesis of the newborn animals, with a 2-fold increase in intra testis androstenedione and 6-fold decrease in testosterone concentration compared to WT and HSD17B3KO males. In contrast, at the age of 3 months, no difference in the circulating steroid concentrations was observed between the DKO and HSD17B3KO, and high concentrations of androstenedione and testosterone was present in both mice. This, along with the active spermatogenesis, imply that in a presence of high LH, a further compensatory mechanism of testosterone production is present in the adult testes. In conclusion, our results demonstrate that in the absence of Hsd17b3, Hsd17b1 is crucial for the remaining testosterone production capacity in fetal mouse testis. However, in adult animals, the compensatory role is taken over by other, so far unknown, enzymes. Compared to HSD17B3KO, the overall phenotype of the DKO male mice is more similar to that of the humans with HSD17B3 deficiency, while the phenotype of HSD17B3KO is less feminized. Presentation Date: Thursday, June 15, 2023

Crinecerfont, a CRF1 Receptor Antagonist, Lowers Adrenal Androgens in Adolescents With Congenital Adrenal Hyperplasia.

Crinecerfont, a corticotropin-releasing factor type 1 receptor antagonist, has been shown to reduce elevated adrenal androgens and precursors in adults with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD), a rare autosomal recessive disorder characterized by cortisol deficiency and androgen excess due to elevated adrenocorticotropin. To evaluate the safety, tolerability, and efficacy of crinecerfont in adolescents with 21OHD CAH. This was an open-label, phase 2 study (NCT04045145) at 4 centers in the United States. Participants were males and females, 14 to 17 years of age, with classic 21OHD CAH. Crinecerfont was administered orally (50 mg twice daily) for 14 consecutive days with morning and evening meals. The main outcomes were change from baseline to day 14 in circulating concentrations of ACTH, 17-hydroxyprogesterone (17OHP), androstenedione, and testosterone. 8 participants (3 males, 5 females) were enrolled; median age was 15 years and 88% were Caucasian/White. After 14 days of crinecerfont, median percent reductions from baseline to day 14 were as follows: ACTH, -57%; 17OHP, -69%; and androstenedione, -58%. In female participants, 60% (3/5) had ≥50% reduction from baseline in testosterone. Adolescents with classic 21OHD CAH had substantial reductions in adrenal androgens and androgen precursors after 14 days of oral crinecerfont administration. These results are consistent with a study of crinecerfont in adults with classic 21OHD CAH.

Co-culture of human cryopreserved fragmented ovarian tissue with theca progenitor cells derived from theca stem cells.

Despite the significant advances in the in vitro development of human primordial follicles, it is still a challenging approach with great potential for improvements. Therefore, the present study aimed to investigate the effect of a feeder layer of human theca progenitor cells (hTPCs) on the development of primordial follicles embedded in human ovarian tissue. Fragments of frozen-thawed ovarian tissue were activated using the vanadate-derivative dipotassium bisperoxo (5-hydroxy-pyridine-2-carboxylic) oxovanadate (V) and kit ligand for 24h. Then, the specimens were divided into the co-culture and mono-culture groups and were cultured with and without a hTPC feeder layer for 6days, respectively. Afterward, the follicles were counted and classified, and the hormone levels and expression levels of apoptosis- and folliculogenesis-related genes were assessed. Both culture groups showed significant follicle growth (P < 0.05). However, the co-culture group had a significantly higher number of growing follicles compared to the other group (P < 0.05). Moreover, the expression levels of ZP1, ZP2, ZP3, BMP-7, AMH, and GDF9 were significantly higher in the co-culture group compared to the other group (P < 0.05), while the expression levels of P53 and CASP3 were significantly lower (P < 0.05). Also, the concentrations of estradiol, progesterone, testosterone, and androstenedione were significantly higher in the co-culture group compared to the other group (P < 0.05). The present study results provided novel evidence on the direct role of hTPCs in the growth and development of human primordial follicles. However, there is a need for future studies to illustrate the underlying mechanisms. Schematic summary of the results. According to our results, the expression of ZP1, ZP2, ZP3, and GDF9 in the oocytes, AMH in the granulosa cells, and BMP4 in the theca cells of the co-culture group were significantly higher than those of the mono-culture and non-culture groups, while the expression of apoptotic genes (BAX, CASP3, and P53) was significantly lower. Moreover, the co-culture group showed significantly increased levels of estradiol, progesterone, testosterone, and androstenedione in its culture media compared to the mono-culture groups.

Liver phenotypes in PCOS: Analysis of exogenous and inherited risk factors for liver injury in two European cohorts.

Fatty liver disease (FLD) is common in women with polycystic ovary syndrome (PCOS). Here, we use non-invasive tests to quantify liver injury in women with PCOS and analyse whether FLD-associated genetic variants contribute to liver phenotypes in PCOS. Prospectively, we recruited women with PCOS and controls at two university centres in Germany and Poland. Alcohol abuse was regarded as an exclusion criterion. Genotyping of variants associated with FLD was performed using TaqMan assays. Liver stiffness measurements (LSM), controlled attenuation parameters (CAP) and non-invasive HSI, FLI, FIB-4 scores were determined to assess hepatic steatosis and fibrosis. A total of 42 German (age range 18-53 years) and 143 Polish (age range 18-40 years) women with PCOS, as well as 245 German and 289 Polish controlswere recruited. In contrast to Polish patients, Germans were older, presented with more severe metabolic profiles and had significantly higher LSM (median 5.9 kPa vs. 3.8 kPa). In the German cohort, carriers of the PNPLA3 p.I148M risk variant had an increased LSM (p=.01). In the Polish cohort, the minor MTARC1 allele was linked with significantly lower serum aminotransferases activities, whereas the HSD17B13 polymorphism was associated with lower concentrations of 17-OH progesterone, total testosterone, and androstenedione (all p < .05). FLD is common in women with PCOS. Its extent is modulated by both genetic and metabolic risk factors. Genotyping of variants associated with FLD might help to stratify the risk of liver disease progression in women suffering from PCOS.

Age-related decrease in serum dihydrotestosterone concentration is accompanied by impaired vascular status

IntroductionThe effect of androgens on the cardiovascular system in humans is ambiguous. Moreover, still little is known about the effects of the most potent androgen, dihydrotestosterone, on arterial stiffness and endothelial function. The aim of this study was to evaluate whether age-dependent alterations in serum concentration of dihydrotestosterone and its circulating metabolite are accompanied by changes in endothelial function and arterial stiffness. MethodsIn 12 young and 11 older men, basal serum concentrations of testosterone, dehydroepiandrosterone sulfate (DHAE-S), androstenedione (AE), dihydrotestosterone (DHT) and androstanediol glucuronide (ADG) were analyzed in relation to vascular status including cIMT — carotid intima media thickness, cAI — central augmentation index, crPWV — carotid radial pulse wave velocity, SI — stiffness index, endothelial and inflammatory markers. ResultsAlthough concentration of testosterone was not different between young and older group, it was demonstrated that DHT, DHEA-S, AE and ADG were significantly lower in older men in comparison to young men (p < 0.01). Interestingly the most surprising difference was found for DHT concentration, that was as much as 61 % lower in aged men that displayed significantly higher values of cIMT, AI, crPWV and SI (p < 10−4), suggestive of arterial stiffness. Furthermore, DHT was negatively correlated to all arterial wall parameters (cAI, crPWV, SI and cIMT), c-reactive protein (CRP) and hyaluronic acid (HA) concentration, as well as positively correlated to markers of endothelial function (MNA and 6-keto-PGF1α) in all studied individuals (n = 23). ConclusionsWe have shown that ageing leads to a significant decrease in DHT concentration that is accompanied by impaired arterial wall characteristics and worsened endothelial function. Therefore more attention should be paid to the DHT, DHEA-S and ADG concentrations as a biomarkers for vascular dysfunction in ageing men.

Modified-release hydrocortisone is associated with lower plasma renin activity in patients with salt-wasting congenital adrenal hyperplasia.

Poorly controlled salt-wasting (SW) congenital adrenal hyperplasia (CAH) patients often require high 9α-fluorocortisol doses as they show high levels of 17-hydroxyprogesterone (17OHP), which is a mineralocorticoid (MC)-receptor antagonist. We investigated the renin-angiotensin-aldosterone system in patients with SW-CAH receiving twice daily modified-release hydrocortisone (MR-HC, Efmody) compared with standard glucocorticoid (GC) therapy. Data were analyzed from the 6-month, phase 3 study of MR-HC (n = 42) versus standard GC therapy (n = 41). MC replacement therapy remained unchanged throughout the study. Blood pressure, serum potassium, serum sodium, plasma renin activity (PRA), and serum 17OHP and androstenedione concentrations were analyzed at baseline, 4, 12, and 24 weeks. The median serum 17OHP in the morning was significantly lower on MR-HC compared with standard GC at 24 weeks (2.5 nmol L-1 (IQR 8.3) versus 10.5 nmol L-1 (IQR 55.2), P = .001). PRA decreased significantly from baseline to 24 weeks in patients on MR-HC (0.83 ng L-1 s-1 (IQR 1.0) to 0.48 ng L-1 s-1 (IQR 0.61), P = .012) but not in patients on standard GC (0.53 ng L-1 s-1 (IQR 0.66) to 0.52 ng L-1 s-1 (IQR 0.78), P = .613). Serum sodium concentrations increased from baseline to 24 weeks in patients on MR-HC (138.8 ± 1.9 mmol L-1 to 139.3 ± 1.8 mmol L-1, P = .047), but remained unchanged on standard GC (139.8 ± 1.6 mmol L-1 to 139.3 ± 1.9 mmol L-1, P = .135). No significant changes were seen in systolic and diastolic blood pressure and serum potassium levels. 6 months of MR-HC therapy decreased PRA and increased sodium levels indicating a greater agonist action of the 9α-fluorocortisol dose, which may be due to the decreased levels of the MC-receptor antagonist 17OHP.

Does contaminant exposure disrupt maternal hormones deposition? A study on per- and polyfluoroalkyl substances in an Arctic seabird

Maternal effects are thought to be essential tools for females to modulate offspring development. The selective deposition of avian maternal hormones could therefore allow females to strategically adjust the phenotype of their offspring to the environmental situation encountered. However, at the time of egg formation, several contaminants are also transferred to the egg, including per- and polyfluoroalkyl substances (PFAS) which are ubiquitous organic contaminants with endocrine disrupting properties. It is, however, unknown if they can disrupt maternal hormone deposition. In this study we explored relationships between female PFAS burden and maternal deposition in the eggs of steroids (dihydrotestosterone, androstenedione and testosterone), glucocorticoids (corticosterone) and thyroid hormones (triiodothyronine and thyroxine) in a population of the Arctic-breeding black-legged kittiwake (Rissa tridactyla). Egg yolk hormone levels were unrelated to female hormone plasma levels. Second-laid eggs had significantly lower concentrations of androstenedione than first-laid eggs. Triiodothyronine yolk levels were decreasing with increasing egg mass but increasing with increasing females' body condition. Testosterone was the only transferred yolk hormone correlated to maternal PFAS burden: specifically, we found a positive correlation between testosterone in yolks and circulating maternal perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDcA) and perfluoroundecanoic acid (PFUnA) in first-laid eggs. This correlative study provides a first insight into the potential of some long-chain perfluoroalkyl acids to disrupt maternal hormones deposition in eggs and raises the question about the consequences of increased testosterone deposition on the developing embryo.

S-07-3: THE TESTOSTERONE-ANDROGEN RECEPTOR PATHWAY IS ASSOCIATED WITH CAROTID ATHEROSCLEROTIC PLAQUE INSTABILITY IN MEN WITH SEVERE CAROTID ATHEROSCLEROSIS

Objective: Carotid atherosclerotic plaques can be stable or unstable; the latter more likely to rupture resulting in strokes. Sex differences exist in carotid plaque morphology and composition: carotid plaques of men tend to be more unstable than of women, who tend to have more fibrous and stable plaques. Yet, women experience greater morbidity and mortality rates post-stroke. Given that sex hormones exert pleiotropic effects on the cell types involved in the atherosclerotic process, and given that estradiol is largely atheroprotective in women, we hypothesize that sex hormones and their receptors may affect plaque instability. Design and Methods: In this cross-sectional study, we measured sex hormones in the circulation and their receptors in the plaque of older men and postmenopausal women undergoing a carotid endarterectomy (n = 160). Using liquid chromatography mass spectrometry, circulating estradiol, testosterone, dehydroepiandrosterone (DHEA), and androstenedione were measured in blood samples. Plaques collected post-operatively were classified into 4 groups (n = 40/group): women with stable, women with unstable plaques, men with stable, and men with unstable plaques. Protein expression of estrogen receptor α (ER-α), estrogen receptor β (ER-β), G protein-coupled estrogen receptor (GPER), and androgen receptor (AR) was quantified using immunohistochemistry and western blot analysis. Plaque sex hormone receptor mRNA expression was assessed using qRT-PCR. Results: Men with unstable plaques demonstrated greater circulating concentrations of testosterone and androstenedione compared to men with stable plaques (P = 0.02 and P = 0.047, respectively). Unstable plaques in men demonstrated significantly less AR and ER-α mRNA expression compared to stable plaques from men (P = 0.0003 and P = 0.042, respectively) and compared to unstable plaques from women (P = 0.0088 and P = 0.002). Yet, AR, ER-α, ER-β, and GPER protein expression was significantly greater in unstable plaques from men compared to stable plaques from men (P = 0.039, P = 0.006, P &lt; 0.0001, and P = 0.0002, respectively) and compared to unstable plaques from women (P = 0.005, P = 0.041, P = 0.028, and P = 0.002, respectively). Expression was observed in macrophages, foam cells, endothelial cells, and smooth muscle cells. Moreover, unstable plaques from men demonstrated greater features of plaque instability compared to unstable plaques from women, including hemorrhage, large lipid core, abundance of inflammatory cells, and fibrous cap infiltration by inflammatory cells (P &lt; 0.05 for all). Conclusion: Our preliminary findings indicate a possible association between androgens and the androgen receptor pathway and plaque instability. With further investigations, our ongoing work will unravel the underlying mechanisms, and may ultimately lead to hormone-specific therapies aimed at stabilizing plaques and reducing the incidence of stroke for men and women.

Steroid profiling using liquid chromatography mass spectrometry during adrenal vein sampling in patients with primary bilateral macronodular adrenocortical hyperplasia.

Adrenal vein sampling (AVS) is not a routine procedure in patients with primary bilateral macronodular adrenocortical hyperplasia (PBMAH), but has been used to determine lateralization of cortisol secretion in order to guide decision of unilateral adrenalectomy. Our aim was to characterize the steroid fingerprints in AVS samples of patients with PBMAH and hypercortisolism and to identify a reference hormone for AVS interpretation. Retrospectively, we included 17 patients with PBMAH from the German Cushing's registry who underwent AVS. 15 steroids were quantified in AVS and peripheral blood samples using LC-MS/MS. We calculated lateralization indices and conversion ratios indicative of steroidogenic enzyme activity to elucidate differences between individual adrenal steroidomes and in steroidogenic pathways. Adrenal volume was negatively correlated with peripheral cortisone (r=0.62, p<0.05). 24-hour urinary free cortisol correlated positively with peripheral androgens (rDHEA=0.57, rDHEAS=0.82, rA=0.73, rT=0.54, p<0.05). DHEA was found to be a powerful reference hormone with high selectivity index, which did not correlate with serume cortisol and has a short half-life. All investigated steroids showed lateralization in single patients indicating the heterogenous steroid secretion pattern in patients with PBMAH. The ratios of corticosterone/aldosterone (catalyzed by CYP11B2), androstenedione/dehydroepiandrosterone (catalyzed by HSD3B2) and cortisone/cortisol (catalyzed by HSD11B2) in adrenal vein samples were higher in smaller adrenals (p<0.05). ARMC5 mutation carriers (n=6) showed lower androstenedione/17-hydroxyprogesterone and higher testosterone/androstenedione (p<0.05) ratios in peripheral blood, in line with lower peripheral androstenedione concentrations (p<0.05). Steroid profiling by LC-MS/MS led us to select DHEA as a candidate reference hormone for cortisol secretion. Lateralization and different steroid ratios showed that each steroid and all three steroidogenic pathways may be affected in PBMAH patients. In patients with germline ARMC5 mutations, the androgen pathway was particularly dysregulated.

The impact of diabetes mellitus type 2 on the steroidogenesis of male Zucker Diabetic Fatty rats.

The aim of this study was to evaluate the impact of diabetes mellitus type 2 (DM2) on the male endocrine system of Zucker Diabetic Fatty (ZDF) rats. Sexually mature ZDF rats were divided to a lean (control) and obese group, and had diabetes confirmed by blood tests. For the in vivo experiment, fasting blood was collected to obtain blood plasma. In case of the in vitro experiments, testicular fragments were cultured for 24 h, and the culture medium was collected. The concentrations of testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEA-S), estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were quantified in the blood plasma and the medium by the ELISA method, while cholesterol (CHOL) was assessed spectrophotometrically. A significant decline of T (36.31 %), A4 (25.11 %) and FSH (26.99 %) as well as a significant increase of CHOL and E2 (36.17 %) was observed in the blood plasma of obese ZDF rats in comparison to the control. Under in vitro conditions, a significant decrease of FSH (23.35 %) accompanied by an increase of E2 was observed in the obese group compared to the control. In the case of CHOL, LH, T, DHEA and A4 no significant differences were observed. Our results suggest that except for FSH and E2 all steroid biomolecules were synthetized normally by the testicular tissue, however a dramatic endocrine disturbance was observed at the system level. We may conclude that DM2 has negative effects on systemic hormone secretion and these alterations are more pronounced in combination with obesity.

Testosterone Deficiency and Nutritional Parameters as Predictors of All-Cause Mortality among Male Dialysis Patients.

Chronic kidney disease (CKD) is associated with an accelerated risk of cardiovascular mortality. Hormonal and metabolic disorders in CKD may constitute novel risk factors. Our objective was to characterize and evaluate prognostic implications of circulating sex steroids and selected nutritional parameters in patients at different stages of CKD. Studied groups were composed of 78 men: 31 on hemodialysis (HD), 17 on peritoneal dialysis (PD), 30 with CKD stage G3-G4. Total testosterone (TT), dehydroepiandrosterone sulphate (DHEA-S), androstenedione, luteinizing hormone (LH), prolactin (PRL), and biochemical parameters were measured; Free testosterone (FT) was calculated. The lowest TT and FT were observed in HD, the highest- in CKD (p = 0.006 for TT, p = 0.005 for FT). TT positively correlated with total cholesterol in HD (p = 0.012), FT negatively correlated with BMI in CKD (p = 0.023). During the 12 months, 9 patients died (5 in the HD, 4 in the PD group). The deceased group had significantly lower concentrations of albumin (p = 0.006) and prealbumin (p = 0.001), and a significantly higher concentration of androstenedione (p = 0.019) than the surviving group. In the group of men on dialysis, a serum TT concentration &lt;2.55 ng/mL (Q1-first quartile) was associated with a 3.7-fold higher risk of death, although statistical significance was not achieved (p = 0.198). After analysis of the ROC curves, the FT level was the best prognostic marker in HD (AUC = 0.788; 95% CI: 0.581-0.996; p = 0.006) Conclusions: Total and free testosterone levels were lower in the HD group than in the CKD group. The nutritional status undoubtedly affects the survival of dialysis patients but also the concentrations of testosterone significantly contributes to further worsening the prognosis.

Attainment of Sexual Maturity and Gonadotropin Priming in Gilts Determine Follicular Development, Endocrine Milieu and Response to Ovulatory Triggers.

The routine procedure of estrous cycle synchronization in pigs allows for the use of gonadotropins to stimulate ovarian activity. The applied protocols of eCG and hFSH priming similarly affected development of ovarian follicles in two classes 3–6 mm and >6 mm of diameter, however, the number of small follicles (<3 mm) was 2-fold higher in hFSH- than in eCG-primed prepubertal gilts. The attainment of sexual maturity increased concentration of estradiol, testosterone and androstenedione in the follicular fluid of hFSH/eCG-primed gilts, however, prostaglandin E2 and F2α metabolite increased in mature hFSH- and eCG-primed gilts, respectively. The maturity increased mRNA and/or protein expression of key steroidogenic enzymes, prostaglandin synthases or luteinizing hormone receptors in follicular walls. Both hormonal primers played a moderate role in affecting expression of steroidogenic enzymes in follicular walls. In vitro studies showed higher estradiol production in r-hLH (p = 0.04)- and r-hCG (p = 0.049)-stimulated follicular walls of mature gilts than in prepubertal hFSH-primed gilts. Both ovulatory triggers decreased the abundance of LHCG/FSH mRNA receptors in follicular walls, which mimic downregulation of these receptors by a preovulatory LH surge, confirmed in vivo. These data revealed the importance of sexual maturity in the protection of the estrogenic environment, and the selective, moderate role of eCG and FSH in the activation of steroidogenic enzymes in preovulatory follicles.

Simultaneous Determination of Steroids and Glucocorticoids in Human Urine by Ultra-High-Performance Liquid Chromatography – Tandem Mass Spectrometry (UPLC–MS/MS)

Prostate cancer is a hormone-dependent disease and therefore androgens play a critical role in its development. For both research on clinical conditions as well as diagnostic and monitoring purposes, accurate measurement of steroid hormones is essential. This study aimed to develop a sensitive and selective method for simultaneously detecting nine sex steroids and two glucocorticoids in human urine and to evaluate the alterations of urinary endogenous steroid hormones induced by prostate cancer. A total of 97 Chinese subjects were randomly recruited for this study, including 66 patients with prostate cancer and 31 healthy controls. The concentrations of steroid hormones, including dehydroepiandrosterone, dehydroepiandrosterone sulfate (DHEAS), androstenedione, testosterone, dihydrotestosterone (DHT), estrone, estradiol, estriol (E1), progesterone, cortisol (F), and cortisone (E), were simultaneously determined by ultra-high-performance liquid chromatography – tandem mass spectrometry with atmospheric pressure chemical ionization in the positive mode (UPLC–APCI+–MS/MS). The population analysis indicated that prostate cancer patients showed significantly higher DHEAS, DHT, E1, and E than healthy controls. Furthermore, the levels of DHEAS and E1 were significantly positively correlated with serum levels of prostate-specific antigen (PSA), which is considered to be the best conventional prostate cancer marker in the current study. In summary, we validated the use of urine samples for multiple steroid profiling by UPLC–MS/MS. Two steroids were significantly altered and correlated with PSA in the prostate cancer population. These findings open further investigation of the potential role of sex steroids in prostate cancer diagnosis. Studying large samples and determining these steroid hormones in the urine are still necessary.