OBJECTIVE

The aim was to monitor carefully follicular growth arrest in polycystic ovaries by assay of hormones in individual follicles.

METHODS

Fluid from follicles less than or equal to 10 mm was obtained from ovaries of 16 regularly cycling women between days 1 and 12 of the follicular phase (controls, n = 120 follicles), polycystic ovaries of five women with polycystic ovary syndrome (n = 43), and polycystic ovaries from 14 long-term testosterone treated female to male transsexuals (n = 120).

METHODS

Fluid was assayed for oestradiol, androstenedione, and immunoactive inhibin. Luteinizing hormone, follicle-stimulating hormone, and testosterone levels were estimated in serum.

RESULTS

Median serum LH was lower in transsexuals than in controls (P less than 0.05), and in polycystic ovary syndrome (P less than 0.01). Median serum testosterone was not significantly different between polycystic ovary syndrome and transsexuals, and was elevated in both groups as compared to controls (P less than 0.01). Oestradiol was present in all follicles obtained from polycystic ovaries of polycystic and transsexual patients, in which no follicle greater than 10 mm could be detected. In the three groups, between-patient differences in mean oestradiol, androstenedione, inhibin, and androstenedione/oestradiol ratio were significantly larger than expected in view of the variation between follicles within individuals. Taking into account this between-patient difference, no significant differences could be established between the three groups for all endocrine parameters. The percentage of presumed healthy follicles (androstenedione/oestradiol ratio less than or equal to 4) was 12% in controls, 17% in polycystics, and 14% in transsexuals, and was not significantly different between groups.

CONCLUSIONS

The results may indicate that (1) abnormally high circulating androgen concentrations with or without elevated LH levels disturb the process of selection, and could therefore play a role in the pathogenesis of polycystic ovaries; (2) in polycystic ovaries from polycystic ovary syndrome and transsexual patients, aromatase activity is present in vivo in small antral follicles, and the proportion of presumed healthy follicles is not different from that encountered in normal ovaries; (3) oestradiol levels are not different between non-dominant follicles of normal and polycystic ovaries, suggesting that only enhancement of aromatase activity by FSH may be disrupted in polycystic ovaries, (4) because androstenedione levels are not different comparing follicles of normal and polycystic ovaries, hyperandrogenaemia in the syndrome seems to originate from the abnormally high number of cystic atretic follicles generally observed in polycystic ovaries; (5) marked variation in the endocrine follicular microenvironment within and between-women precludes pooling fluid from several follicles.

The hypothalamic-pituitary-gonadal and -adrenal axes are regarded as the main sites of the actions of alcohol on steroids. In the present study the effect of alcohol (0.<em>4</em>-0.5 g/kg, orally) on venous plasma and urinary androgens was investigated in 21 premenopausal women using oral contraceptives as well as in 10 premenopausal nonusers. After intake of alcohol, an acute elevation in plasma testosterone, a decline in <em>androstenedione</em> levels, and an elevation in the ratio of testosterone to <em>androstenedione</em> were observed in both groups. The effects lasted throughout the period of ethanol elimination and were abolished during pretreatment with <em>4</em>-methylpyrazole (10-15 mg/kg, orally). The acute effects were higher in the group using oral contraceptives than in the nonusers. The testosterone effect in plasma was reflected in the free testosterone fraction. A decline in urinary androsterone and etiocholanolone levels, the principal catabolic products of androgens, was observed during alcohol intoxication. In conclusion, the present acute effects on plasma and urinary steroid hormones seem to be explained by an inhibited catabolism mediated by the alcohol-induced change in the redox state in the liver. Our results suggests that the liver should be included as a major site in the acute endocrinological effects of alcohol on steroid hormones in women.

Studies on projection of the sensory neurons onto the olfactory bulb in fish have revealed a clear subdivision into spatially different areas that each responded specifically to different classes of odorants. Amino acids induce activity in the lateral part, bile salts induce activity in the medial part, and alarm substances induce activity in the posterior part of the medial olfactory bulb. In the present study, we demonstrate a new feature of the bulbar chemotopy showing that neurons specifically sensitive to sex pheromones are located in a central part of the ventral olfactory bulb in crucian carp. Extensive single-unit recordings were made from these neurons, stimulating with four sex pheromones, 17,20beta-dihydroxy-<em>4</em>-pregnen-3-one, 17,20beta-dihydroxy-<em>4</em>-pregnen-3-one-20-sulfate, <em>androstenedione</em>, and prostaglandin F(2alpha), known to induce specific reproductive behaviors in males of carp fish. All substances were applied separately to the sensory epithelium at a concentration of 10(-9) M. Of the 297 neurons recorded in males, the majority (236 or 79.5%) responded exclusively to one of the four sex pheromones and thus showed a high specificity. Of the 96 neurons recorded from the olfactory bulb in females, only 1 unit showed such a specific activation. These findings reflect remarkable differences between males and females in the discriminatory power of the olfactory neurons toward these sex pheromones. The gender differences are discussed in relation to behavior studies, expression of olfactory receptors, and the convergence of sensory neurons onto the secondary neurons in the olfactory bulb.

A sensitive in vitro 3H2O microassay for aromatase activity was used to evaluate the potency and selectivity of three aromatase inhibitors in mammalian (gerbil) and avian (ring dove) hypothalamus. The steroidal inhibitors, 1,<em>4</em>,6-androstatrien-3,17-dione (ATD) and <em>4</em>-hydroxy-<em>androstenedione</em> (<em>4</em>-OH-A) were compared with a new non-steroidal imidazole inhibitor, CGS 169<em>4</em>9A [<em>4</em>-(5,6,7,8-tetrahydroimidazo-[1,5-a]-pyridin-5-yl)benzonitrile HCl]. Adult male dove hypothalamic aromatase is highly active [Vmax = 5.3 pmol testosterone (T) converted/h/mg protein], has high substrate binding affinity (Km = <em>4</em>.0 nM), and direct involvement in control of sexual behaviour. With [1 beta-3H]T or [1 beta-3H]A as substrate, male dove preoptic aromatase activity was inhibited more effectively and selectively by CGS 169<em>4</em>9A. Thus, Kis and IC50s for aromatization were approximately 50 times lower for the non-steroidal inhibitor, and inhibition of the other major androgen-metabolizing enzymes (5 alpha/beta-reductase) occurred at concentrations at least one order of magnitude greater than for ATD and <em>4</em>-OH-A. Neonatal male gerbil hypothalamic aromatase activity (Vmax = 1.3 pmol T converted/h/mg protein) was lower than in the dove. Aromatase inhibition by CGS 169<em>4</em>9A is more potent in the neonatal gerbil than in the dove (Kis of 0.03 and 0.60 nM, respectively, with A as substrate). We conclude that the imidazole is an effective aromatase inhibitor in both the adult and developing brain.

Plasma testosterone (T), dihydrotestosterone (DHT), 17 beta-estradiol (E2), 17-hydroxyprogesterone (17-OHP), <em>androstenedione</em> (delta), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), 5-androstene-3 beta-17 beta-diol (A-diol) and cortisol (F) have been measured in a group of normal males and in a group of patients with Klinefelter's syndrome (KS) before and after hCG stimulation. Significantly lower baseline levels of T and DHT and significantly higher baseline levels of E2 were found in patients with KS. No significant differences were found between baseline levels of 17-OHP, delta, DHEA, DHEAS, A-diol, and F. After hCG stimulation between T, DHT, E2 and 17-OHP levels showed a significant increase in the two groups of subjects. The percentage variation of T and DHT, however, was much less important in Klinefeiter patients, while E2 and 17-OHP did not show a significantly different pattern from that of normal controls, hCG administration did not produce any significant variation of delta, DHEA, DHEAS, and F in the two groups of subjects, while A-diol levels increased significantly in normal subjects, but not in Klinefelter patients. Our data may be consistent with the hypothesis that testicular steroidogenesis in Klinefelter patients is impaired below the 21-C-steriod level not only at delta <em>4</em> but also at the delta 5 pathway.

To determine the role of newly synthesized estrogens in LH receptor expression, granulosa cells from diethylstilbestrol-implanted immature rats were cultured with FSH plus aromatase inhibitors. When present throughout the <em>4</em>8-h culture period, <em>4</em>-hydroxy-<em>4</em>-androstene-3,17-dione (<em>4</em>-OHA; greater than or equal to 100 microM) and 1,<em>4</em>,6-androstatriene-3,17-dione (greater than or equal to 5 microM) inhibited FSH-induced LH receptor formation by <em>4</em>0% and 90%, respectively. Both aromatase inhibitors caused relatively greater inhibition of LH receptor formation when added from 20-<em>4</em>8 h of culture, the period during which FSH-stimulated estrogen synthesis occurs (85% maximal inhibition with <em>4</em>-OHA and 95% with 1,<em>4</em>,6-androstatriene-3,17-dione). Addition of estradiol, but not <em>androstenedione</em>, reversed the reduction of LH receptor formation by <em>4</em>-OHA, indicating that the effects of the aromatase inhibitors were specifically related to their blockade of estradiol synthesis. The stimulation of estrogen production by FSH alone (8-fold) or with <em>androstenedione</em> (80-fold) during the <em>4</em>8-h culture period was prevented by <em>4</em>-OHA. FSH-stimulated cAMP production was initially enhanced by <em>4</em>-OHA from 0-20 h of culture, but was reduced from 20-<em>4</em>8 h. Lower concentrations of <em>4</em>-OHA (less than or equal to 50 microM) amplified FSH-stimulated cAMP production and LH receptor formation. However, these responses were blocked by the antiestrogen keoxifene or the antiandrogen flutamide, indicating that <em>4</em>-OHA or a metabolite may have partial estrogenic or androgenic properties. The inhibitory effects of higher concentrations of <em>4</em>-OHA on LH receptor expression were potentiated by keoxifene or flutamide. These results indicate that estrogen production and action are necessary for LH receptor expression in the granulosa cell.

OBJECTIVE

Androgens such as dehydroepiandrosterone sulfate (DHEAS) and testosterone are markedly lower in postmenopausal women with rheumatoid arthritis (RA) than in controls. In contrast, compared to controls, serum levels of estrogens are normal or elevated in women with RA. Since tumor necrosis factor (TNF) alters production of these hormones, we investigated changes of these hormones during anti-TNF antibody (anti-TNF) therapy with adalimumab in longstanding RA.

METHODS

In this longitudinal anti-TNF therapy study in 13 patients with long-standing RA without prior prednisolone (7 infusions of anti-TNF: Week 0, 2, <em>4</em>, 6, 8, 10, and 12), we measured serum concentrations of interleukin 6 (IL-6), <em>androstenedione</em>, DHEA, DHEAS, free testosterone, estrone, and 17ss-estradiol. Levels of these hormones in patients were compared to serum levels of 31 age and sex matched healthy controls.

RESULTS

Upon treatment with anti-TNF, there was an impressive decrease of clinical markers of inflammation, erythrocyte sedimentation rate, and serum levels of IL-6. Serum levels of DHEAS and free testosterone were markedly lower at baseline in patients compared to controls, but this did not change during anti-TNF therapy. Serum levels of DHEA and 17ss-estradiol were significantly elevated in patients compared to controls, but similarly, anti-TNF therapy did not change initially increased levels. Molar ratios of hormones, which reflect hormone shifts via converting enzymes, showed typical alterations at baseline, but did not change markedly during anti-TNF therapy.

CONCLUSIONS

Longterm therapy with anti-TNF did not change altered serum levels of typical sex hormones in patients with RA, although baseline values were largely different. In patients with RA, this indicates that alterations of sex hormones and altered activity of respective converting enzymes are imprinted for a long-lasting period over at least 12 weeks.

OBJECTIVE

Adult-type hypolactasia (ATH) is related to lower calcium and milk intake, which might be associated with obesity and metabolic disturbances. Women with polycystic ovary syndrome (PCOS) frequently suffer from metabolic disturbances including central obesity. We aimed to examine the association of ATH and calcium intake with anthropometric, metabolic and endocrine parameters in a cohort of PCOS and control women.

METHODS

Metabolic, endocrine and anthropometric measurements and oral glucose tolerance tests were performed in 50<em>4</em> PCOS and 366 control women. Genotyping of ATH, defined by the -13910 variant of the MCM6 gene, was performed. Calcium intake was assessed by questionnaires.

RESULTS

Adult-type hypolactasia was more prevalent in PCOS women (29·8%) than in controls (23·5%) (P = 0·0<em>4</em>0). PCOS women with ATH had higher waist-to-hip ratio (WHR) (0·80 [0·75-0·88] vs 0·78 [0·73-0·85], P = 0·0<em>4</em>6), glucose 2 h (5·28 [<em>4</em>·57-6·33] mmol/l vs 5·67 [<em>4</em>·68-6·78] mmol/l, P = 0·037), HbA1c (5·2 [5·0-5·<em>4</em>]%vs 5·1 [5·0-5·3]%, P = 0·009), parathyroid hormone (3·72(2·91-<em>4</em>·86] pmol/l vs 3·61 [2·9<em>4</em>-<em>4</em>·63] pmol/l, P = 0·030) and Ferriman-Gallwey-Scores (FG Scores) (7 [3-12] vs <em>4</em> [1-9], P = 0·002) and lower 25(OH)D levels (5<em>4</em>·<em>4</em> [35·2-80·6] nmol/l vs 68·<em>4</em> [<em>4</em>9·7-89·<em>4</em>] nmol/l, P < 0·001) than PCOS women without ATH. The association of 25(OH)D and FG-Scores with ATH remained significant in age-, BMI- and WHR-adjusted analyses. PCOS women within the highest quartile of calcium intake had significantly lower testosterone (P = 0·023) and <em>androstenedione</em> (P = 0·032) and significantly higher high-density lipoprotein (HDL) levels (P = 0·035) than PCOS women with lower calcium intake.

CONCLUSIONS

Our results indicate an association of ATH with PCOS susceptibility. Moreover, ATH might influence WHR, HbA1c and FG-Scores as well as 25(OH)D levels. Higher calcium intake was associated with lower androgens and higher HDL levels.

Recent evidence suggests the presence of a functional endothelin-angiotensin-atrial natriuretic peptide system at the ovarian level. This study aimed to investigate 1) the local interrelationships among angiotensin II (Ang II), endothelin-1 (ET-1), and atrial natriuretic peptide (ANP); 2) the possible effect of each vasoactive peptide on the secretion of steroid hormones and prostaglandins (PGs) in isolated bovine mature follicles; and 3) the expression of mRNAs for Ang II, ET-1, and ANP receptors in the theca layer of follicles at different developmental stages. Each preovulatory follicle obtained before the LH surge (based on the concentrations of steroids and PGs) received implants of <em>4</em> capillary dialysis membranes into the theca layer. The follicles were then incubated in organ culture chambers and perfused with Ringer's solution for 12 h. Stimulation by infusion of the different substances into the microdialysis system was carried out between <em>4</em> and 8 h. The infusion of ET-1 (10(-7) M) stimulated the release of ANP and estradiol but inhibited the release of <em>androstenedione</em> and progesterone. The infusion of ANP (10(-7) M) stimulated the release of Ang II, progesterone, and <em>androstenedione</em>. Moreover, the infusion of Ang II (10(-5) M) inhibited the release of ANP but stimulated the release of ET-1, progesterone, and estradiol. All three peptides examined increased PGE(2) and PGF(2) release. In the reverse transcription-polymerase chain reaction analysis, expression of the mRNAs for ET type A and type B, and Ang II type 1 receptors did not change with the follicular size and the intrafollicular estradiol concentrations. Expression of the mRNA for the Ang II type 2 receptor dropped in follicles when the estradiol concentration ranged from 20 to 180 ng/ml and increased again when the estradiol concentration was>> 180 ng/ml. The levels of expression of ANP type C receptor mRNA were slightly greater in follicles with estradiol concentrations>> 20 ng/ml than in follicles with estradiol concentrations < 20 ng/ml. These results demonstrate a complex interaction among Ang II, ET-1, and ANP that may contribute to increasing the follicular production of PGs and modulate steroidogenesis in the bovine mature follicle, thus providing evidence for a local functional endothelin-angiotensin-ANP system.

Genome-wide association studies (GWAS) have identified many single nucleotide polymorphisms (SNPs) associated with prostate cancer risk. There is limited information on the mechanistic basis of these associations, particularly about whether they interact with circulating concentrations of growth factors and sex hormones, which may be important in prostate cancer etiology. Using conditional logistic regression, the authors compared per-allele odds ratios for prostate cancer for 39 GWAS-identified SNPs across thirds (tertile groups) of circulating concentrations of insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein 3 (IGFBP-3), testosterone, <em>androstenedione</em>, androstanediol glucuronide, estradiol, and sex hormone-binding globulin (SHBG) for 3,0<em>4</em>3 cases and 3,<em>4</em>78 controls in the Breast and Prostate Cancer Cohort Consortium. After allowing for multiple testing, none of the SNPs examined were significantly associated with growth factor or hormone concentrations, and the SNP-prostate cancer associations did not differ by these concentrations, although <em>4</em> interactions were marginally significant (MSMB-rs1099399<em>4</em> with <em>androstenedione</em> (uncorrected P = 0.008); CTBP2-rs<em>4</em>962<em>4</em>16 with IGFBP-3 (uncorrected P = 0.003); 11q13.2-rs12<em>4</em>18<em>4</em>51 with IGF-1 (uncorrected P = 0.006); and 11q13.2-rs10896<em>4</em><em>4</em>9 with SHBG (uncorrected P = 0.005)). The authors found no strong evidence that associations between GWAS-identified SNPs and prostate cancer are modified by circulating concentrations of IGF-1, sex hormones, or their major binding proteins.

The effect of flutamide on basal and ACTH-stimulated plasma levels of adrenal androgens was investigated in 6 patients with untreated advanced prostate cancer, aged 52-75 yr. Flutamide was administered (250 mg three times daily) for 10 days; before and after treatment, a synthetic ACTH1-2<em>4</em> stimulation test (250 micrograms im, with blood sampling immediately before and 60 min after the stimulus) was performed. Basal plasma 17OH-pregnenolone (delta 5-17OHP), 170H-progesterone (delta <em>4</em>-17OHP), <em>androstenedione</em> (A), dehydroepiandrosterone (DHEA) and its sulphate (DHEAS) were unchanged by flutamide treatment. In contrast, basal plasma testosterone (T) concentrations significantly increased (p less than 0.05). The response of cortisol delta <em>4</em>-17OHP, delta 5-17OHP, A and DHEA to ACTH, as well as the ACTH-stimulated delta 5-17OHP/delta <em>4</em>-17OHP, delta 5-17OHP/DHEA, delta <em>4</em>-17OHP/A and DHEA/A ratios, were unchanged by flutamide treatment. These findings indicate that: a) Short-term flutamide administration enhances testicular steroidogenesis, via augmented LH pulse frequency; b) Adrenal steroidogenesis seems to be not affected by the drug, since ACTH-stimulated plasma levels of adrenal androgens and precursors/products ratios were unchanged.

Cultured thecal preparations from human ovarian follicles obtained from patients during the follicular phase of the menstrual cycle produced large amounts of delta <em>4</em>-<em>androstenedione</em> and smaller amounts of testosterone and other androgens when stimulated by hCG, (Bu)2cAMP, and prostaglandin E2, but not when stimulated by FSH. Prostaglandin E2 also stimulated thecal cAMP production under the same conditions. By contrast, androgen production by granulosa cells was negligible with or without gonadotropins in the culture medium. Granulosa cells also did not produce 17 beta-estradiol, even when exposed to FSH and hCG, unless supplied with exogenous delta <em>4</em>-<em>androstenedione</em>. On the other had, thecal, preparations did not produce significant amounts of 17 beta-estradiol even in the presence of FSH, hCG, and relatively large amounts of exogenous delta <em>4</em>-<em>androstenedione</em>. These observations suggest that delta <em>4</em>-<em>androstenedione</em> produced by LH or hCG-stimulated thecal cells is the main substrate for the synthesis of 17 beta-estradiol by FSH-stimulated granulosa cells in vivo.

A method is described for the determination of anabolic steroids including testosterone, 19-nor-<em>4</em>-androstene-3,17-dione, <em>4</em>-androstene-3,17-dione and nandrolone in food supplements. Initial clean-up is done by HPLC followed by determination with GC/MS. A 'contaminated' food supplement was analysed and appeared to contain 19-nor-<em>4</em>-androstene-3,17-dione and <em>4</em>-androstene-3,17-dione. One capsule of this nutritional supplement was ingested by five male volunteers. Urine samples were collected and analysed by GC/MS and GC/MS-MS. Neither the ratio testosterone/epitestosterone, nor the ratio <em>androstenedione</em>/epitestosterone increased significantly. Concentrations above 2 ng/ml for norandrosterone, the major metabolite of nandrolone, were detected until <em>4</em>8-1<em>4</em><em>4</em> h after ingestion of the food supplement.

BACKGROUND

Increases of adrenal hormone concentrations other than cortisol have been reported in dogs with hyperadrenocorticism (HAC).

OBJECTIVE

Measuring noncortisol adrenal hormone concentrations will help identify HAC in dogs. The objective was to determine plasma cortisol, androstenedione, estradiol, progesterone, testosterone, and 17-hydroxyprogesterone concentrations during ACTH stimulation testing of dogs with clinical signs of HAC to ascertain their utility in diagnosis of the disease.

METHODS

Ninety dogs with clinical findings consistent with HAC had ACTH stimulation tests performed. Results from 29 dogs were excluded from analysis because diagnoses were inconclusive for a variety of reasons. Results from 32 dogs with HAC and 29 dogs with disease other than HAC were analyzed.

METHODS

Prospective observational study. Concentrations of adrenocortical hormones were determined before and 1 hour after injecting 5 μg/kg ACTH IM. Diagnoses were determined by response to therapy, histopathology or both.

RESULTS

Post-ACTH cortisol (P < .001), progesterone (P = .001), and 17-hydroxyprogesterone (P < .001) concentrations were associated with a diagnosis of HAC. Sensitivity and specificity, respectively, for diagnosing HAC for post-ACTH cortisol were 84 and 59%, progesterone 88 and 55%, and 17-hydroxyprogesterone 91 and 59%, and for post-ACTH cortisol, progesterone and 17-hydroxyprogesterone combined were 88 and 55%. Of 5 dogs with HAC and normal post-ACTH cortisol concentrations, 5 had increased progesterone and 4 had increased 17-hydroxyprogesterone.

CONCLUSIONS

Serum progesterone and 17-hydroxyprogesterone concentrations were useful to diagnose HAC in this study, but were not more sensitive or specific than cortisol concentration.

Ovarian function was studied in 17 women with active ankylosing spondylitis (AS). Levels of FSH, LH, prolactin and <em>androstenedione</em> were normal in menstruating patients and FSH and LH were elevated in menopausal patients. In menstruating patients with active AS the estradiol levels were lower than in patients with inactive AS and significantly (p less than 0.05) lower than controls. Progesterone levels in menstruating patients were lower (P = NS) than controls. In menopausal patients estrogen levels were lower than their controls (P = NS). There was a significant (p less than 0.05) inverse correlation between the sedimentation rate and the estrogen level. Seven patients accepted oral estrogen therapy (average duration <em>4</em> months) and peripheral arthritis subsided within one month, all variables of clinical activity of AS improved and at the end of the study all patients were in functional class I.

This study compared immediate and carryover effects of mastitis induced by Gram-negative endotoxin (E. coli LPS) and Gram-positive exosecretions (Staph. aureus ex.) on preovulatory follicle function. Synchronized, uninfected cyclic lactating Holstein cows were treated with PGF(2α) on day 6 of the cycle and 36 h later, a dose of either E. coli LPS (n = 8), S. aureus ex. (n = 10), or saline (n = 9) was administered into the mammary gland. Follicular fluids and granulosa cells were aspirated 6 h later from the preovulatory follicles and cows were treated with GnRH. This (cycle 1; immediate effect) was repeated three times (excluding the mammary injections) to induce three 7 d cycles (cycles 2, 3, and <em>4</em>; carryover effect). E. coli LPS increased body temperature, plasma cortisol concentration, and somatic cell count (SCC), whereas S. aureus ex. induced a minor, subclinical elevation of SCC and slight rise (NS) in body temperature and cortisol concentration. Follicular estradiol, <em>androstenedione</em>, and progesterone concentrations in the E. coli LPS group decreased (P < 0.05) in cycle 1 to about <em>4</em>0%, 13%, and 35%, respectively, of control levels, whereas in the S. aureus ex. group, only estradiol decreased (P < 0.05), to 56% of control concentrations. In cycles 3 and <em>4</em>, follicular steroids in the E. coli LPS group returned to control concentrations, whereas in the S. aureus ex. group, follicular concentrations of estradiol and <em>androstenedione</em> were lower (P < 0.10) than in controls. In the control group, the concentrations of all follicular and circulating steroids remained stable (P>> 0.05) throughout the study. Follicle size was similar in all groups, but the S. aureus ex. treatment caused a decrease (P < 0.02) in the number of follicles developed in cycles 3 and <em>4</em>. The mRNA expression of steroidogenic genes and LHCGR in the granulosa cells was not affected (P>> 0.05) by either treatment during the study, except for a tendency toward lower (P < 0.1) expression in cycle 1 and lower (P < 0.05) expression in cycle <em>4</em> of the latter in the S. aureus ex. group. Strain levels, such as SCC and body temperature, following toxin injection correlated well with the magnitude of the immediate decline in follicular steroids. As is typical for Gram-negative clinical events, E. coli LPS-induced acute mastitis caused immediate, short-term, but not long-term impairment of follicular responses, whereas the Gram-positive S. aureus ex.-induced subclinical mastitis exhibited both immediate and carryover disruptive effects on preovulatory follicle function.

Steroid hormones are crucial substances that mediate a wide range of vital physiological functions of the body. Determination of the levels of steroid hormones plays an important role in understanding the mechanism of the steroid hormone-related diseases. In this study, we present a novel targeted metabolic profiling method based on the introduction of an easily protonated stable isotope tag to a hydroxyl-containing steroid hormone with a synthesized derivatization reagent, deuterium <em>4</em>-(dimethylamino)-benzoic acid (d(<em>4</em>)-DMBA), and liquid chromatography-mass spectrometry (LC-MS). Different from other reported derivatization reagents that have been used to enhance the sensitivities for estrogens or androgens, our method is comprehensive with the capability of covering hydroxyl-containing androgens, estrogens, corticoids, and progestogens. Furthermore, the nonderivatized steroid hormones (e.g., 17α-hydroxyprogesterone, progesterone, and <em>androstenedione</em>) were not destroyed during the derivatization process, and their levels could still be obtained in one LC-MS run. We were able to detect 2<em>4</em> steroid hormones at subng/mL levels (the lower limit of detection could reach 5 pg/mL for estrone and 16α-hydroxy estrone, which is equivalent to 0.1 pg on column) with maximum sensitivity enhancement factors of more than 10(3)- to 10(<em>4</em>)-fold after derivatization. The method was successfully applied to the measurement of free (unconjugated) steroid hormones in urine samples of males, females, and pregnant women. Because the significant role the steroid hormone pathway plays in humans, a comprehensive, sensitive, specific, and accurate method for profiling the steroid hormone metabolome shall offer new insights into hormone-related diseases.

OBJECTIVE

Aim of this study is to report on basal clinical phenotype and follow up after diagnosis, of patients with 17beta-hydroxysteroid-dehydrogenase type 3 (17beta-HSD3) deficiency in Italy.

METHODS

Pediatric Endocrine Departments, University Hospitals.

METHODS

The cases of 5 Italian subjects affected by 17beta-HSD3 deficiency are presented in this study.

METHODS

Laboratory and genetic assessment. Gonadectomy and female sex assignment (<em>4</em> patients) or GnRH analog therapy to regress puberty and gender identity disorder (1 patient).

RESULTS

Presentation lasted from pregnancy (pre-natal diagnosis of a <em>4</em>6,XY fetus with female external genitalia) to infancy (inguinal hernia containing testes/clitoromegaly) and adolescence (virilisation). All subjects but one (subject 1, Central-Northern Italy) were from small areas of Southern Italy. Endocrine data (baseline and/or stimulated testosterone/ Delta<em>4</em>-androstenedione ratio) were informative. Two girls were homozygous for 17beta-HSD3 gene mutations (G289S/G289S; R80W/R80W), while the others were compound heterozygous (IVS325+<em>4</em> A>T/A203V; L212Q/M235V; R80W/A235E). Four patients were confirmed as females and were well-adjusted with assigned sex; gender identity disorder improved during treatment with GnRH analog in the last subject.

CONCLUSIONS

17betaHSD3 deficiency may present from pregnancy to puberty for different clinical issues. Albeit testosterone/Delta<em>4</em>-androstenedione ratio represents the most accurate endocrine marker to diagnose the disorder, hCGstimulation is mandatory in pre-puberty. Molecular analysis of 17beta-HSD3 gene should be performed to confirm the diagnosis. Temporary GnRH analog treatment may regress gender identity disorder and provide time to confirm or change the birth sex assignment. Female individuals seems to be compliant with their sex, providing that virilisation does not occur. In Italy, the disorder seems to be more prevalent in the Southern regions and shows genetic heterogeneity.

The patient was born with ambiguous genitalia (stade III of Prader). The karyotype revealed a normal female genotype. A defect in 21-hydroxylase, at first suspected, was denied by the hormonal studies. Indeed, extremely high levels of pregnenolone, pregnenolone sulfate, progesterone were found in association with low plasma levels of delta <em>4</em>-<em>androstenedione</em>, testosterone, dehydroepiandrosterone and its sulfate, while cortisol 17OH-progesterone and ACTH levels and plasma renin activity were normal. The hormonal pattern was thus consistent with 17,20-desmolase deficiency. The dynamic studies further supported this contention: all the progestagens rose further after ACTH stimulation and were suppressed by dexamethasone. Meanwhile, all androgens failed to rise after ACTH: the responses of cortisol were normal. The in utero virilization of the female fetus was not understood until an history of virilization was allegedly found in the mother (luteoma of pregnancy). This is the first case of 17-20 desmolase defect recognized in a female newborn. This child, born with ambiguous genitalia had presented the concurrence of two very rare conditions. The in utero virilization of maternal origin enabled us to make the diagnosis of the 17-20 desmolase defect, which otherwise would have been ignored in a XX subject in the neonatal period because it would obviously be unsymptomatic at this age.

OBJECTIVE

Newborn screening for congenital adrenal hyperplasia (CAH) is generally performed using 17- hydroxyprogesterone dissociation-enhanced, lanthanide fluorescence immunoassay (DELFIA®). The primary screening results must be confirmed due to high false-positive rates; however, the need to obtain a separate specimen can hamper early recognition, differential diagnosis and treatment. We aimed to develop a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that allows both the confirmation and differential diagnosis of CAH using the same dried blood spot (DBS) as in primary screening.

METHODS

An LC-MS/MS assay for cortisol, 21-deoxycortisol, 11-deoxycortisol, <em>4</em>-<em>androstenedione</em> and 17-hydroxyprogesterone was developed, validated and applied to a total of 163 DBS samples tested positive in primary newborn screening in a cross-border cooperation.

RESULTS

Excellent baseline resolution and reliable determination of all analytes were achieved in DBS samples following simple sample preparation without derivatization. Of a total of 163 DBS samples tested positive in primary screening, the 21-hydroxylase-deficient form of CAH was confirmed in 1 sample.

CONCLUSIONS

The present LC-MS/MS assay was successfully applied as a second-tier test in a cross-border cooperation for newborn screening. The assay allows concurrent confirmation and differential diagnosis of CAH and can be performed on the same DBS samples as in primary screening, enabling early diagnosis and treatment.

Insight into the mechanisms by which steroid hormones are released from the testes was sought by examining the concentrations of progesterone, 17alpha-hydroxyprogesterone, and <em>androstenedione</em> as well as testosterone in spermatic vein blood every 15 min for <em>4</em> h in men with varicocele-associated infertility. Coincident discrete secretory episodes of all four steroids were found, and spermatic vein concentrations of testosterone were highly positively correlated to the concentrations of progesterone (r = 0.79), 17alpha-hydroxyprogesterone (r = 0.81), and <em>androstenedione</em> (r = 0.82), respectively. The sum of the four measured steroids per mL plasma was calculated, and testosterone was found to account for 70%, 17alpha-hydroxyprogesterone for 2<em>4</em>%, <em>androstenedione</em> for 5%, and progesterone for 1% of the total. In a previous study of the intratesticular steroids in a separate population of men with varicocele-associated infertility, the sum of these four steroids per g tissue was similarly calculated. Testosterone accounted for 70% of the four measured steroids, 17alpha-hydroxyprogesterone for 22%, <em>androstenedione</em> for <em>4</em>%, and progesterone for 3% of the total. Thus, the relative concentrations of these four steroids are nearly identical in testicular tissue and spermatic vein plasma. From these data we hypothesize that steroids in the testicular interstitium are cosecreted into peripheral plasma in response to stimulation by LH and propose that the mechanism initiating this pulsatile mode of secretion oftestosterone and its precursor steroids may not be coupled to testosterone biosynthesis.

OBJECTIVE

To determine the effect of oral melatonin (MT) administration on serum concentrations of sex hormones, prolactin, and thyroxine in dogs.

METHODS

Prospective study.

METHODS

8 male and 8 female adult sexually intact dogs.

METHODS

5 male and 5 female dogs were treated with MT (1.0 to 1.3 mg/kg [0.<em>4</em>5 to 0.59 mg/lb] of body weight), PO, every 12 hours for 28 days; the other 6 dogs were used as controls. Blood samples were collected on days 0, 1<em>4</em>, and 28, and serum concentrations of estradiol-17 beta, progesterone, testosterone, <em>androstenedione</em>, 17-hydroxyprogesterone (17-HP), dihydroepi<em>androstenedione</em> sulfate (DHEAS), prolactin, and thyroxine were determined. On day 5, serum MT concentrations were measured before and periodically for up to 8 hours after MT administration in <em>4</em> treated dogs.

RESULTS

Female dogs treated with MT had significant decreases in serum estradiol, testosterone, and DHEAS concentrations between days 0 and 28. Male dogs treated with MT had significant decreases in serum estradiol and 17-HP concentrations between days 0 and 28. Serum MT concentrations increased significantly after MT administration and remained high for at least 8 hours. Prolactin and thyroxine concentrations were unaffected by treatment.

CONCLUSIONS

Melatonin is well absorbed following oral administration and may alter serum sex hormone concentrations.

An analytical procedure for the simultaneous determination of twelve endogenous steroids (testosterone, <em>androstenedione</em>, 17beta-estradiol, estrone, pregnenolone, progesterone, dihydro<em>androstenedione</em>, dihydrotestosterone, 11alpha-ketotestosterone, 17alpha-hydroxyprogesterone, 17alpha-hydroxypregnenolone, 17alpha,20beta-dihydroxy-<em>4</em>-pregnen-3-one) in plasma and bile samples by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC-MS) has been developed. After enzymatic hydrolysis for bile samples only, samples were concentrated and purified using two successive SPE (C(18) and NH(2)) cartridges. Analytes were derivatized with a mixture of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) / mercaptoethanol / ammonium iodide (NH(<em>4</em>)I) and determined by GC-MS in selective ion monitoring mode. For most of the steroids monitored, recoveries were in the range 90-120% in plasma and in the range 60-70% in bile, and the reproducibility was below 10% for the complete procedure. Limits of detection obtained ranged from 0.1 to 0.<em>4</em> ng/g in fish plasma and from 1.6 to 1<em>4</em> ng/g in fish bile. The developed method was successfully applied to the determination of plasma steroids in flounders (Platichthys flesus) collected from two French estuaries.

An experiment was conducted in order to determine the pattern of, and the relationships between, the secretion of inhibin, estradiol, and <em>androstenedione</em> by the ovary and the concentration of LH, FSH, and PRL during the estrous cycle of sheep. The estrous cycles of 6 Finn-Merino ewes in which the left ovary had been autotransplanted to the neck were synchronized by two injections of cloprostenol (100 micrograms im) a potent analog of prostaglandin F2 alpha (PG) given 1<em>4</em> days apart. The ewes had ovarian and jugular venous blood samples taken at four hourly intervals from <em>4</em>2 h before the second PG injection until day 6 of the following cycle. All animals responded to PG with the preovulatory LH surge occurring within 58 +/- 2 h (mean +/- SEM). The concentration of FSH in jugular venous plasma fell (P less than 0.001) after the induction of luteolysis and then exhibited 3 peaks, the first coincident with the LH surge, the second on day 1, and the third on day 6. After injection of PG the secretion rates of inhibin, estradiol, and <em>androstenedione</em> increased (P less than 0.05) within <em>4</em>-8 h. After this increase in the early follicular phase the secretion rate of estradiol continued to rise until the time of the LH surge (P less than 0.001). Although the secretion of <em>androstenedione</em> and inhibin increased in the 36 h before the LH surge the magnitude of this rise was less marked than for estradiol and was not statistically significant. Within <em>4</em>-8 h of the start of the LH surge the secretion of estradiol and <em>androstenedione</em> declined rapidly reaching barely detectable levels within 16 h (P less than 0.001). In contrast the secretion of inhibin increased after the LH surge reaching a broad peak (P less than 0.05) of approximately 16-h duration, coincident with the second peak of FSH. From days 2-6 mean secretion of inhibin remained relatively stable at 2-6 ng/min although considerable variation was observed in individual profiles. The rate of estradiol secretion increased steadily from its nadir on day 1 to a broad peak centered around day 3 (3-6 ng/min, P less than 0.001) followed by a decline until by day 6 the estradiol secretion rate was less than 1 ng/min (P less than 0.01). The secretory profile for PRL showed a close relationship with estradiol secretion.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)