Effects of 2-Year Physical Activity and Dietary Intervention on Adrenarchal and Pubertal Development: The PANIC Study.

CONTEXT: Childhood overweight has been linked to earlier development of adrenarche and puberty, but it remains unknown if lifestyle interventions influence sexual maturation in general populations. OBJECTIVE: To investigate if a 2-year lifestyle intervention influences circulating androgen concentrations and sexual maturation in a general population of children. METHODS: We conducted a 2-year physical activity and dietary intervention study in which 421 prepubertal and mostly normal-weight 6- to 9-year-old children were allocated either to a lifestyle intervention group (119 girls, 132 boys) or a control group (84 girls, 86 boys). The main outcome measures were serum dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), and testosterone concentrations, and clinical adrenarchal and pubertal signs. RESULTS: The intervention and control groups had no differences in body size and composition, clinical signs of androgen action, and serum androgens at baseline. The intervention attenuated the increase of DHEA (P = .032), DHEAS (P = .001), A4 (P = .003), and testosterone (P = .007) and delayed pubarche (P = .038) in boys but it only attenuated the increase of DHEA (P = .013) and DHEAS (P = .003) in girls. These effects of lifestyle intervention on androgens and the development of pubarche were independent of changes in body size and composition, but the effects of intervention on androgens were partly explained by changes in fasting serum insulin. CONCLUSION: A combined physical activity and dietary intervention attenuates the increase of serum androgen concentrations and sexual maturation in a general population of prepubertal and mostly normal-weight children, independently of changes in body size and composition.

The Mediating Role of Endocrine Factors in the Positive Relationship Between Fat Mass and Bone Mineral Content in Children Aged 9-11 Years: The Physical Activity and Nutrition in Children Study.

Introduction: We aimed to investigate whether the relationship between fat mass and bone mineral content (BMC) is mediated by insulin, leptin, adiponectin, dehydroepiandrosterone sulphate, testosterone and estradiol in children aged 9-11 years. Materials and Methods: We utilised cross-sectional data from the Physical Activity and Nutrition in Children study (n = 230 to 396; 112 to 203 girls). Fat mass and BMC were assessed with dual-energy X-ray absorptiometry. Endocrine factors were assessed from fasted blood samples. We applied the novel 4-way decomposition method to analyse associations between fat mass, endocrine factors, and BMC. Results: Fat mass was positively associated with BMC in girls (ß = 0.007 to 0.015, 95% confidence interval (CI) 0.005 to 0.020) and boys (ß = 0.009 to 0.015, 95% CI 0.005 to 0.019). The relationship between fat mass and BMC was mediated by free leptin index in girls (ß = -0.025, 95% CI -0.039 to -0.010) and boys (ß = -0.014, 95% CI -0.027 to -0.001). The relationship between fat mass and BMC was partially explained by mediated interaction between fat mass and free leptin index in boys (ß = -0.009, 95% CI -0.013 to -0.004) and by interaction between fat mass and adiponectin in girls (ß = -0.003, 95% CI -0.006 to -0.000). Conclusion: At greater levels of adiponectin and free leptin index, the fat mass and BMC relationship becomes less positive in girls and boys respectively. The positive association between fat mass with BMC was largely not explained by the endocrine factors we assessed. Clinical Trial Registration: [https://clinicaltrials.gov/ct2/show/NCT01803776], identifier NCT01803776.

A quantitative ultra-performance liquid chromatography high-resolution mass spectrometry analysis of steroids from human scalp hair.

Hair steroid analysis offers the possibility to evaluate long-term steroid metabolism. It is especially beneficial when studying the steroid milieu in newborns and children, for example it can provide new insights into steroid metabolism over months and is unaffected by diurnal fluctuations in steroid concentrations. This study describes a quantitative and highly selective high-resolution mass spectrometry method for the simultaneous analysis of multiple steroids from human scalp hair. The developed method utilizes parallel reaction monitoring in the analysis of hydroxylamine derivatized steroids from hair samples first washed with isopropanol, extracted with methanol, and further purified with solid-phase extraction and liquid-liquid extraction. The method was proven suitable for the quantitative analysis of endogenous glucocorticoids (cortisol [1.5-364 pg/mg]; cortisone [3.6-355 pg/mg]; corticosterone [0.7-347 pg/mg]; 11-deoxycortisol [0.1-343 pg/mg]), androgens (testosterone [0.1-288 pg/mg]; dehydroepiandrosterone [0.3-288 pg/mg]; androstenedione [0.1-285 pg/mg]; 11ß-hydroxyandrostenedione [0.3-304 pg/mg]), progestogens (pregnenolone [0.1-313 pg/mg]; progesterone [0.1-313 pg/mg]; 17α-hydroxypregnenolone [0.3-315 pg/mg]; 17α-hydroxyprogesterone [0.7-330 pg/mg]; 21-hydroxyprogesterone [0.6-320 pg/mg]), and estrogens (estrone [0.1-267 pg/mg]; estradiol [0.5-274 pg/mg]). In addition, 11-ketoandrostenedione [0.6-60 pg/mg]; dihydrotestosterone [0.3-290 pg/mg]; 11-ketotestosterone [0.1-12 pg/mg]; and 5α-androstanedione [0.6-281 pg/mg] could be analyzed semi-quantitatively. Aldosterone [3.5-346 pg/mg]; 11ß-hydroxytestosterone [0.3-300 pg/mg]; and 11-ketodihydrotestosterone [0.3-300 pg/mg] were also included in the method, but their concentrations were below the lower limit of quantification in all tested hair samples. The method was applied for the analysis of authentic hair samples from different age groups ranging from newborns to adults, including mothers within 48 h after delivery. The newborn hair samples displayed the widest variety and had also the highest amounts of steroids in comparison to the samples of the other groups.

Circulating Liver-enriched Antimicrobial Peptide-2 Decreases During Male Puberty.

CONTEXT: Circulating levels of liver-enriched antimicrobial peptide 2 (LEAP2), a ghrelin receptor antagonist, decrease under caloric restriction and increase in obesity. The role of LEAP2 in male puberty, a phase with accelerated energy demand, is unclear. OBJECTIVE: This work aimed to investigate whether circulating LEAP2 levels are downregulated in boys following the onset of puberty to respond to the energy need required for growth. METHODS: We determined circulating LEAP2 levels in 28 boys with constitutional delay of growth and puberty (CDGP) who participated in a randomized controlled trial (NCT01797718), and were treated with letrozole (n = 15) or intramuscular low-dose testosterone (T) (n = 13) for 6 months. Blood sampling and dual-energy x-ray absorptiometry-measured body composition were performed at 0-, 6-, and 12-month visits. RESULTS: Serum LEAP2 levels decreased statistically significantly during pubertal progression (0-6 months: mean decrease -4.3 [10.3] ng/mL, P = .036 and 0-12 months: -3.9 [9.3] ng/mL, P = .033). Between 0 and 6 months, the changes in serum LEAP2 levels correlated positively with changes in percentage of body fat (rs = 0.48, P = .011), and negatively with growth velocity and estradiol levels (rs = -0.43, P = .022, rs = -0.55, P = .003, respectively). In the T group only, the changes in serum LEAP2 correlated negatively with changes in T and estradiol levels. Between 0 and 12 months, the change in LEAP2 levels correlated negatively with the change in high-density lipoprotein levels (rs = -0.44, P = .022) and positively with the change in insulin (rs = 0.50, P = .009) and HOMA-IR (rs = 0.51, P = .007) levels. CONCLUSION: Circulating LEAP2 levels decreased after induction of puberty reciprocally with increased growth rate and energy demand, reflecting the metabolic state of the adolescent. Further, the results suggest that estradiol levels may have a permissive role in downregulating circulating LEAP2 levels.

Serum testosterone and oestradiol predict the growth response during puberty promoting treatment.

OBJECTIVE: The influence of androgens and oestrogens on growth is complex, and understanding their relative roles is important for optimising the treatment of children with various disorders of growth and puberty. DESIGN: We examined the proportional roles of androgens and oestrogens in the regulation of pubertal growth in boys with constitutional delay of growth and puberty (CDGP). The study compared 6-month low-dose intramuscular testosterone treatment (1 mg/kg/month; n = 14) with per oral letrozole treatment (2.5 mg/day; n = 14) which inhibits conversion of androgens to oestrogen. PATIENTS: Boys with CDGP were recruited to a randomized, controlled, open-label trial between 2013 and 2017 (NCT01797718). MEASUREMENTS: The patients were evaluated at 0-, 3- and 6-month visits, and morning blood samples were drawn. Linear regression models were used for data analyses. RESULTS: In the testosterone group (T-group), serum testosterone concentration correlated with serum oestradiol concentration at the beginning of the study and at 3 months, whereas in the letrozole group (Lz-group) these sex steroids correlated only at baseline. Association between serum testosterone level and growth velocity differed between the T and Lz groups, as each nmol/L increase in serum testosterone increased growth velocity 2.7 times more in the former group. Serum testosterone was the best predictor of growth velocity in both treatment groups. In the Lz-group, adding serum oestradiol to the model significantly improved the growth estimate. Only the boys with serum oestradiol above 10 pmol/L had a growth velocity above 8 cm/year. CONCLUSIONS: During puberty promoting treatment with testosterone or aromatase inhibitor letrozole, growth response is tightly correlated with serum testosterone level. A threshold level of oestrogen appears to be needed for an optimal growth rate that corresponds to normal male peak height velocity of puberty. Serum testosterone 1 week after the injection and serum testosterone and oestradiol 3 months after the onset of aromatase inhibitor treatment can be used as biomarkers for treatment response in terms of growth.

PCOS Features and Steroid Profiles Among Young Adult Women with a History of Premature Adrenarche.

CONTEXT: Premature adrenarche (PA) may increase the risk for polycystic ovary syndrome (PCOS). OBJECTIVE: To study features of PCOS in young adult women with a history of PA. METHODS: Thirty PA and 42 control females were followed from prepuberty to young adulthood (median age 18.1 years). The main outcome measures were ovarian function, the use of contraceptives, and clinical and biochemical indicators of hyperandrogenism. RESULTS: We found no differences in the use of hormonal contraceptives (50 vs 50%, PA vs controls, respectively; P > .999), indication for using contraceptives (P = .193), or in the history of oligo- (17 vs 26%, P = .392) and amenorrhea (0 vs 0%, P > .999). Among women not using hormonal contraceptives, those with a history of PA had a higher prevalence of hirsutism (27 vs 0%, P = .023) but not acne (87 vs 67%, P = .252). Steroid profiles were broadly comparable between the groups, but PA women had lower sex hormone-binding globulin (SHBG) concentrations (30.1 vs 62.4 nmol/L, P < .001) resulting in higher free androgen index (3.94 vs 2.14, P < .001). The difference in SHBG levels persisted through body mass index adjustment. SHBG correlated negatively with the homeostasis model assessment for insulin resistance (r -0.498, P = .003). Anti-Müllerian hormone concentrations were comparable between the groups (39.3 vs 32.1 pmol/L, P = .619). CONCLUSION: PA was not associated with evident ovarian dysfunction in young adult women. However, women with a history of PA had decreased SHBG levels and thus, increased bioavailability of circulating androgens.

The effect of maternal alcohol and drug abuse on first trimester screening analytes: a retrospective cohort study.

BACKGROUND: The purpose of this study was to determine whether first trimester trisomy screening (FTS) parameters are affected by alcohol and drug use. METHODS: A routine combined FTS including measurements of maternal serum levels of free ß-human chorionic gonadotropin subunit (free ß-hCG) and pregnancy-associated plasma protein A (PAPP-A) were measured at 9-11 weeks of gestation, and fetal nuchal translucency thickness (NTT) at 11-13 weeks of gestation. In total 544 women with singleton pregnancies [71 alcohol and drug abusers, 88 smokers, 168 non-smokers delivering a small for gestational age (SGA) child, and 217 unexposed control women] were assessed. RESULTS: Free ß-hCG levels were higher in alcohol and drug abusing than in unexposed pregnant women [mean 1.5 vs. 1.2 multiples of medians (MoM); P = 0.013]. However, stepwise multiple linear regression analyses suggested that smoking could explain increased free ß-hCG. Additionally, we observed lower PAPP-A levels in the smoking mothers (0.9 vs. 1.2 MoM; P = 0.045) and in those giving birth to an SGA child compared to the controls (1.1 vs.. 1.2 MoM; P < 0.001). Fetal NTT did not differ significantly between any of the groups. CONCLUSIONS: The present study shows increased free ß-hCG levels in alcohol and drug abusers, but maternal smoking may explain the result. Maternal serum PAPP-A levels were lower in smoking than non-smoking mothers, and in mothers delivering an SGA child. However, FTS parameters (PAPP-A, free ß-hCG and NTT) seem not to be applicable for the use as alcohol biomarkers because of their clear overlap between alcohol abusers and healthy controls.

Simultaneous analysis by LC-MS/MS of 22 ketosteroids with hydroxylamine derivatization and underivatized estradiol from human plasma, serum and prostate tissue.

This study describes a validated LC-MS/MS method for assaying 23 steroids within a single run from 150 µl of human plasma, serum or prostatic tissue homogenate. Isotope-labeled steroids were used as internal standards. Samples were extracted with toluene, and ketosteroids were derivatized with hydroxylamine prior to LC-MS/MS analysis. The steroids were separated on a C18 column and methanol was used as an organic solvent with the addition of 0.2 mM ammonium fluoride to improve underivatized estradiol (E2) ionization. Certified reference serums as well as plasma samples, and homogenates of prostate tissue were utilized in the method validation. The specificity of the method was inspected with a total of 27 steroids. The validation proved that the method was suitable for the quantitative analysis of a wide panel of androgens (testosterone, T (3.3 pM-13 nM); androstenedione, A4 (3.3 pM-13 nM); 5α-androstanedione, DHA4 (13 pM-13 nM); dehydroepiandrosterone, DHEA (67 pM-133 nM); dihydrotestosterone, DHT (33 pM-33 nM); 11-ketodihydrotestosterone, 11KDHT (13 pM-13nM); 11-ketotestosterone, 11KT (33 pM-6.7 nM); 11ß-hydroxyandrostenedione, 11bOHA4 (33 pM-13 nM); 11ß-hydroxytestosterone, 11OHT (13 pM-33 nM)), as well as estrogens (estrone, E1 (3.3 pM-13 nM)), progestagens (17α-hydroxypregnenolone, 17OHP5 (32 pM-127 nM); 17α-hydroxyprogesterone, 17OHP4 (67 pM-133 nM); progesterone, P4 (3.3 pM-13 nM); pregnenolone, P5 (6.6 pM-13 nM)), and glucocorticoids (cortisol, F (33 pM-134 nM); cortisone E (66 pM-131 nM); corticosterone, B (33 pM-67 nM); 11-deoxycortisol, S (33 pM-66 nM); 21-hydroxyprogesterone, 21OHP4 (32 pM-13 nM)). Furthermore, E2 (335 pM-134 nM) and 11α-hydroxyandrostenedione, 11aOHA4 (33 pM-33 nM) could be analyzed if the concentration in the sample was high enough. In addition, aldosterone, A (128 pM-64 nM) and 11-ketoandrostenedione, 11KA4 (33 pM-13 nM) could be analyzed semiquantitatively. The limits of quantification for all compounds ranged from 0.9 to 91 pg/ml, and from 0.009 to 0.9 pg/mg tissue. Compared to our previous method, this new method also permits the analysis of the more challenging steroids, like DHT, DHEA and P5, and a panel of 11-ketosteroids.

Kuopio birth cohort - design of a Finnish joint research effort for identification of environmental and lifestyle risk factors for the wellbeing of the mother and the newborn child.

BACKGROUND: A Finnish joint research effort Kuopio Birth Cohort (KuBiCo) seeks to evaluate the effects of genetics, epigenetics and different risk factors (medication, nutrition, lifestyle factors and environmental aspects) during pregnancy on the somatic and psychological health status of the mother and the child. METHODS: KuBiCo will ultimately include information on 10,000 mother-child pairs who have given their informed consent to participate in this cohort. Identification of foetal health risk factors that can potentially later manifest as disease requires a repository of relevant biological samples and a flexible open up-to-date data handling system to register, store and analyse biological, clinical and questionnaire-based data. KuBiCo includes coded questionnaire-based maternal background data gathered before, during and after the pregnancy and bio-banking of maternal and foetal samples that will be stored in deep freezers. Data from the questionnaires and biological samples will be collected into one electronic database. KuBiCo consists of several work packages which are complementary to each other: Maternal, foetal and placental metabolism and omics; Paediatrics; Mental wellbeing; Prenatal period and delivery; Analgesics and anaesthetics during peripartum period; Environmental effects; Nutrition; and Research ethics. DISCUSSION: This report describes the set-up of the KuBiCo and descriptive analysis from 3532 parturients on response frequencies and feedback to KuBiCo questionnaires gathered from June 2012 to April 2016. Additionally, we describe basic demographic data of the participants (n = 1172). Based on the comparison of demographic data between official national statistics and our descriptive analysis, KuBiCo represents a cross-section of Finnish pregnant women.

Analysis by LC-MS/MS of endogenous steroids from human serum, plasma, endometrium and endometriotic tissue.

An LC-MS/MS method was developed and validated to analyze simultaneously estrogens (estradiol, E2; estrone, E1), androgens (testosterone, T; androstenedione, A4; dehydroepiandrosterone, DHEA), progestagens (17a-hydroxypregnenolone, 17OHP5; 17a-hydroxyprogesterone, 17OHP4; progesterone, P4), glucocorticoids (cortisol, F; cortisone E; corticosterone, B; 11-deoxycortisol, S; 21-hydroxyprogesterone, 21OHP4), and mineralocorticoids (aldosterone, A) from 150 µl of human serum, plasma, or endometrium and endometriotic tissue homogenates. Samples spiked with isotope-labeled steroids as internal standards were extracted with toluene prior to LC-MS/MS analysis. The chromatographic separation of underivatized steroids was achieved on a biphenyl column with 0.2 mM NH4F as the eluent additive and a water-methanol gradient to improve E2 and E1 ionization. Method validation was performed with human plasma samples, and analysis of certified E2, T, F, and P4 reference serums (BCR-576, ERM-DA346, ERM-DA192, ERM-DA347), as well as homogenates of endometrium and endometriotic tissue. A total of 27 steroids were included in the method development to ensure the specificity of the method. After validation, the method was found suitable for quantitative analysis of 11 steroids: E2 (6.7 pM-13 nM), E1 (1.3 pM-6.6 nM), T (3.3 pM-13 nM), A4 (13 pM-33 nM), 17OHP5 (32 pM-65 nM), 17OHP4 (33 pM-13 nM), F (33 pM-133 nM), E (13 pM-130 nM), B (33 pM-134 nM), S (13 pM-129 nM), and A (32 pM-32 nM). In addition, DHEA (333 pM-32 nM), P4 (13 pM-13 nM) and 21OHP4 (13 pM-13 nM) can be analyzed semiquantitatively.

Impaired growth and intracranial calcifications in autosomal dominant hypocalcemia caused by a GNA11 mutation.

OBJECTIVE: Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia and inappropriately low PTH concentrations. ADH type 1 is caused by activating mutations in the calcium-sensing receptor (CASR), a G-protein-coupled receptor signaling through α11 (Gα11) and αq (Gαq) subunits. Heterozygous activating mutations in GNA11, the gene encoding Gα11, underlie ADH type 2. This study describes disease characteristics in a family with ADH caused by a gain-of-function mutation in GNA11. DESIGN: A three-generation family with seven members (3 adults, 4 children) presenting with ADH. METHODS: Biochemical parameters of calcium metabolism, clinical, genetic and brain imaging findings were analyzed. RESULTS: Sanger sequencing revealed a heterozygous GNA11 missense mutation (c.1018G>A, p.V340M) in all seven hypocalcemic subjects, but not in the healthy family members (n=4). The adult patients showed clinical symptoms of hypocalcemia, while the children were asymptomatic. Plasma ionized calcium ranged from 0.95 to 1.14mmol/L, yet plasma PTH was inappropriately low for the degree of hypocalcemia. Serum 25OHD was normal. Despite hypocalcemia 1,25(OH)2D and urinary calcium excretion were inappropriately in the reference range. None of the patients had nephrocalcinosis. Two adults and one child (of the two MRI scanned children) had distinct intracranial calcifications. All affected subjects had short stature (height s.d. scores ranging from -3.4 to -2.3 vs -0.5 in the unaffected children). CONCLUSIONS: The identified GNA11 mutation results in biochemical abnormalities typical for ADH. Additional features, including short stature and early intracranial calcifications, cosegregated with the mutation. These findings may indicate a wider role for Gα11 signaling besides calcium regulation.

Premature adrenarche--a common condition with variable presentation.

Adrenarche refers to a maturational increase in the secretion of adrenal androgen precursors, mainly dehydroepiandrosterone (DHEA) and its sulfate (DHEAS). In premature adrenarche (PA), clinical signs of androgen action appear before the age of 8/9 years in girls/boys, concurrently with the circulating DHEA(S) concentrations above the usually low prepubertal level. The most pronounced sign of PA is the appearance of pubic/axillary hair, but also other signs of androgen effect (adult type body odor, acne/comedones, greasy hair, accelerated statural growth) are important to recognize. PA children are often overweight and taller than their peers, and the higher prevalence of PA in girls than in boys is probably explained by higher female adiposity and peripheral DHEA(S) conversion to active androgens. PA diagnosis requires exclusion of other causes of androgen excess: congenital adrenal hyperplasia, androgen-producing tumors, precocious puberty, and exogenous source of androgens. PA has been linked with unfavorable metabolic features including hyperinsulinism, dyslipidemia, and later-appearing ovarian hyperandrogenism. Although this common condition is usually benign, PA children with additional risk factors including obesity should be followed up, with the focus on weight and lifestyle. Long-term follow-up studies are warranted to clarify if the metabolic changes detected in PA children persist until adulthood.

Premature adrenarche: etiology, clinical findings, and consequences.

Adrenarche means the morphological and functional change of the adrenal cortex leading to increasing production of adrenal androgen precursors (AAPs) in mid childhood, typically at around 5-8 years of age in humans. The AAPs dehydroepiandrosterone (DHEA) and its sulfate conjugate (DHEAS) are the best serum markers of adrenal androgen (AA) secretion and adrenarche. Normal ACTH secretion and action are needed for adrenarche, but additional inherent and exogenous factors regulate AA secretion. Inter-individual variation in the timing of adrenarche and serum concentrations of DHEA(S) in adolescence and adulthood are remarkable. Premature adrenarche (PA) is defined as the appearance of clinical signs of androgen action (pubic/axillary hair, adult type body odor, oily skin or hair, comedones, acne, accelerated statural growth) before the age of 8 years in girls or 9 years in boys associated with AAP concentrations high for the prepubertal chronological age. To accept the diagnosis of PA, central puberty, adrenocortical and gonadal sex hormone secreting tumors, congenital adrenal hyperplasia, and exogenous source of androgens need to be excluded. The individually variable peripheral conversion of circulating AAPs to biologically more active androgens (testosterone, dihydrotestosterone) and the androgen receptor activity in the target tissues are as important as the circulating AAP concentrations as determinants of androgen action. PA has gained much attention during the last decades, as it has been associated with small birth size, the metabolic and polycystic ovarian syndrome (PCOS), and thus with an increased risk for type 2 diabetes and cardiovascular diseases in later life. The aim of this review is to describe the known hormonal changes and their possible regulators in on-time and premature adrenarche, and the clinical features and possible later health problems associating with PA.

Serum androgen bioactivity is low in children with premature adrenarche.

BACKGROUND: Clinical findings in children with premature adrenarche (PA) correlate only partly with circulating levels of adrenal androgens. It is not known whether the prepubertal low circulating concentrations of testosterone (T) and dihydrotestosterone, together with those of adrenal androgens, are capable of activating the androgen receptor. METHODS: This cross-sectional study was performed at a university hospital. Circulating androgen bioactivity was measured in 67 prepubertal children with clinical signs of PA and 94 control children using a novel androgen bioassay. RESULTS: Circulating androgen bioactivity was low in the PA and control children. In the subgroup of children (n = 28) with serum T concentration over the assay sensitivity (0.35 nmol/l) and a signal in the androgen bioassay, we found a positive correlation between androgen bioactivity and serum T (r = 0.50; P < 0.01) and the free androgen index (r = 0.61; P < 0.01) and a negative correlation with serum sex hormone-binding globulin concentration (r = -0.41; P < 0.05). CONCLUSION: Peripheral metabolism of adrenal androgen precursors may be required for any androgenic effects in PA. However, the limitations in the sensitivity of the bioassay developed herein may hide some differences between the PA and control children.

Interleukins 1α and 1ß as regulators of steroidogenesis in human NCI-H295R adrenocortical cells.

Inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) regulate the activity of the hypothalamo-pituitary-adrenal (HPA) axis at several levels. Although hypothalamic CRH secretion may be the primary mechanism by which these cytokines activate the HPA axis, IL-1 expression is increased within the adrenal glands in models for systemic inflammation, and IL-1 may augment adrenal glucocorticoid production. Our aim was to investigate the direct effects of IL-1α and IL-1ß on adrenal steroidogenesis and expression of three key steroidogenic genes in human adrenocortical cells using the NCI-H295R cell line as a model. mRNAs encoding receptors for IL-1, TNF-α, and leukemia inhibitory factor (LIF) were detectable in the cell line (Affymetrix microarray analysis). Both IL-1α and IL-1ß increased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate production, and the accumulation of mRNAs for steroidogenic acute regulatory protein (STAR), 17α-hydroxylase/17,20-lyase (CYP17A1) and 3ß-hydroxysteroid dehydrogenase 2 (HSD3B2) in these cells (P<0.05 for all). Both ILs augmented TNF-α- and LIF-induced STAR and CYP17A1 mRNA accumulation, and TNF-α-induced cortisol production (P<0.05 for all). Both ILs also increased the apoptotic index of the cells (P<0.05), which was efficiently neutralized by their specific antibodies. The IL-induced changes in the STAR, HSD3B2, and CYP17A1 protein levels were not as evident as those in the respective mRNA levels. In conclusion, the combined effect of inflammatory cytokines at the adrenal level in acute or chronic inflammatory states could significantly stimulate glucocorticoid production, and thus explain the observed discrepancy between the cortisol and ACTH concentrations sometimes seen in sepsis and chronic inflammatory states.

Tracking serum lipid levels and the association of cholesterol concentrations, blood pressure and cigarette smoking with carotid artery intima-media thickness in young adults born small for gestational age.

BACKGROUND: Small birth size is associated with increased cardiovascular disease risk, but the mediating factors are poorly understood. METHODS AND RESULTS: Serum lipids, blood pressure (BP), carotid artery intima-media thickness (CA-IMT), and brachial artery flow-mediated dilatation (BA-FMD) were studied in 70 20-year-old subjects [35 sex- and age-matched pairs born small (SGA) and appropriate for gestational age (AGA)]. The SGA subjects had higher serum levels of low-density lipoprotein (LDL) cholesterol, total/high-density lipoprotein (HDL) and LDL/HDL cholesterol ratios, and lower HDL cholesterol levels than the AGA subjects (2.71 vs 2.37 mmol/L, P<0.05; 3.12 vs 2.80, P<0.01; 1.98 vs 1.61, P=0.002; 1.43 vs 1.56 mmol/L, P<0.05, respectively). In the SGA group, total and LDL cholesterol levels correlated inversely with adult height SD score (r=0.463, P=0.006 and r=0.413, P=0.015, respectively). CA-IMT or BA-FMD did not differ between the groups, but cigarette smoking, higher diastolic BP, and shorter birth length associated with higher CA-IMT in the whole study population. A clear tracking of cholesterol levels was found from 12 to 20 years. CONCLUSIONS: SGA subjects had more unfavorable lipid profiles than the controls, the shortest having the highest LDL cholesterol. Cigarette smoking, higher diastolic BP, and shorter birth length associated with higher CA-IMT in the whole study population. A clear tracking of cholesterol levels through adolescence enables early targeting of lifestyle counseling for reducing cardiovascular disease risk.

Circulating TNF-Alpha and IL-6 Concentrations and TNF-Alpha -308 G > A Polymorphism in Children with Premature Adrenarche.

Premature adrenarche (PA), the early rise in adrenal androgen production leading to prepubertal signs of androgen action, has been connected with adverse metabolic features. The metabolic syndrome is characterized by low-grade inflammation which in turn is associated with increases in circulating proinflammatory cytokines, like tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). We tested the hypothesis that serum concentrations of TNF-α and IL-6 are increased in PA by studying 73 children with PA and 98 age- and gender-matched controls. Serum TNF-α and IL-6 concentrations were measured using a multiplex bead array. The subjects were genotyped for the TNF-α gene -308 G > A polymorphism (known to affect TNF-α gene transcription), and genotype-phenotype associations were studied. The mean serum TNF-α concentration was higher in the PA than control children (20.4 vs. 18.4 pg/ml, P = 0.048), whereas there was no significant difference in the mean serum IL-6 concentrations between the study groups. The difference in TNF-α was not explained by excess body weight in the PA subjects as the difference remained significant after BMI-adjustment (P = 0.038). In the PA group, TNF-α concentration was not associated with metabolic-endocrine features, but high IL-6 was associated with lower birth weight. There was no difference in the genotype distribution of the TNF-α gene -308 G > A polymorphism between the PA and control groups. In conclusion, PA was associated with increased serum TNF-α concentrations which, unexpectedly, were not connected with BMI or insulin resistance. The TNF-α gene -308 G > A polymorphism does not seem to be associated with the development of PA.

Expression of adrenomedullin in human ovaries, ovarian sex cord-stromal tumors and cultured granulosa-luteal cells.

The aim of the present study was to characterise the expression pattern of the multifunctional vasoactive peptide adrenomedullin (ADM) in human ovarian tumors, and to find hormonal regulators of ADM expression in human ovaries. The expression of ADM messenger RNA (mRNA) was higher in granulosa cell tumors than in fibrothecomas and normal ovaries, as analysed by Northern blots. In normal ovaries, ADM immunoreactivity was localised in both granulosa and thecal cells. Eight of the 90 granulosa cell tumors (9%) showed moderate and 53 (59%) weak ADM immunoreactivity, whereas 27% (11/41) of the fibrothecomas displayed weak ADM staining. FSH, protein kinase A activator (Bu)(2)cAMP, prostaglandin E(2) (PGE(2)), activin A and the broad protein kinase regulator staurosporine decreased ADM mRNA accumulation in cultured granulosa-luteal cells time- and dose-dependently. FSH, (Bu)(2)cAMP and PGE(2) increased progesterone secretion and the accumulation of the steroidogenic acute regulatory protein mRNA in these cells. In conclusion, ADM is expressed in normal human ovaries and sex cord-stromal tumors, particularly in those of granulosa cell origin. FSH, PGE(2,) (Bu)(2)cAMP and activin A suppress ADM gene expression in granulosa-luteal cells. Expression of ADM in human ovaries and its hormonal regulation in granulosa cells suggests a paracrine role for ADM in ovarian function.

Girls with premature adrenarche have accelerated early childhood growth.

OBJECTIVE: To evaluate the effect of premature adrenarche (PA) on prepubertal growth. STUDY DESIGN: The prepubertal growth of 54 girls with PA and 52 control girls was analyzed retrospectively. Birth measures were noted, and childhood length/height and weight were measured annually until age 5 years and at the current visit (at a median age of 7.6 years). The growth variables were correlated with serum insulin-like growth factor (IGF)-1, dehydroepiandrosterone sulfate, and insulin concentrations. RESULTS: There were no significant differences in birth length or weight standard deviation scores (SDSs) between the 2 study groups. The girls with PA demonstrated a significant length SDS increment during the first 2 years of life (median +1.0 SDS; P < .001). Compared with controls, they were taller (median current height 1.2 vs 0 SDS; P < .001) and gained more weight throughout childhood. The difference in weight-for-height became significant at a later age compared with the difference in height. Median serum IGF-1 concentration adjusted for both age and body mass index SDS was higher in the PA group (24 vs 19 nmol/L; P < .031). CONCLUSIONS: PA was not associated with small birth size in our population. Girls with PA had enhanced growth already in early childhood, which was not explained by weight gain. Enhanced IGF-1 production may contribute to the prepubertal growth acceleration in PA.

Continuum of phenotypes and sympathoadrenal function in premature adrenarche.

OBJECTIVES: Premature adrenarche (PA), the early rise in adrenal androgen (AA) production, can manifest with different clinical signs of androgen effect. Premature pubarche defined as appearance of pubic hair before the age of 8/9 years in girls/boys, is the most prominent clinical sign of PA and often erroneously described as a synonym of PA. Our aim was to determine the association of circulating AA concentrations with different prepubertal signs of androgen action (SAA). Secondly, we tested whether adrenomedullary function is altered in children with SAA, as it is in congenital adrenal hyperplasia (CAH) also causing adrenal hyperandrogenism. DESIGN AND METHODS: We examined 73 Finnish prepubertal children with any hyperandrogenic sign(s) having appeared before the age of 8/9 years (girls/boys) (35 with pubic and/or axillary hair=PAH; 38 without=nonPAH), and 98 age- and sex-matched controls. Circulating adrenal steroid and catecholamine concentrations were measured and correlated with clinical parameters. RESULTS: None of the children with SAA had CAH or virilizing tumor. Serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, and androstenedione concentrations overlapped between the SAA and control children, and they were lower in the nonPAH than PAH group (P<0.01). SAA children had similar plasma epinephrine but higher norepinephrine (NE) concentrations than their controls (mean (95% confidence interval) 1.61 (1.44, 1.77) versus 1.39 (1.30, 1.49) nmol/l, P=0.03). CONCLUSIONS: PA forms a continuum with more pronounced increase in circulating androgens in children with PAH than in those without. Some children show SAA with fairly low androgen concentrations. The clinical significance of elevated NE concentrations associated with SAA needs to be confirmed in further studies.