Avaliação clínica-epidemiológica e laboratorial do eixo hipotálamo-hipósise-goodal de pacientes com obesidade classe III

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

Avaliação dos níveis séricos de cortisol e de hidroepiandrosterona em pacientes com malária por Plasmodium falciparum não-complicada

O objetivo desta pesquisa foi avaliar o comportamento dos níveis séricos de cortisol e dehidroepiandrosterona (DHEA) em pacientes com malária por Plasmodium falciparum. Como o cortisol apresenta um efeito imunossupressor e o DHEA um efeito imunoestimulador, estudou- se a correlação entre os níveis destes esteróides e a condição clínica do paciente de malária. A amostra constou de 24 pacientes com malária por P. falciparum não-complicada, sendo 18 do sexo masculino e 6 do sexo feminino, com idade variando de 15 a 47 anos, 12 primoinfectados e 12 multi-infectados, provenientes de área endêmica de malária da Amazônia. Coletaram-se amostras diárias de sangue de 20 em 20 minutos no pré-tratamento (D0), 24 horas após o início da medicação (D1) e no 8º dia de acompanhamento (D7), quando o paciente já se encontrava assintomático. Todos os pacientes apresentavam parasitemia negativa em D7. Dosaram-se: os níveis séricos de cortisol em D0, D1 e D7; DHEA em D0 e D7; os níveis de anticorpos totais IgG anti-P. falciparum, anti-P. vivax, e anticorpos IgM anti-P. falciparum em D0. Comparam-se os níveis séricos de cortisol dos três dias, concluindo-se que os níveis de cortisol eram significativamente mais elevados em D0 do que nos outros dias. Foram correlacionados os níveis de cortisol com a parasitemia, obtendo-se como significativas as correlações entre cortisol D0 e parasitemia D1, assim como cortisol D1 com parasitemia D1, levando-se a deduzir que o cortisol pode interferir na resposta inicial à terapêutica de pacientes com malária por P. falciparum. O cortisol foi correlacionado com a temperatura, tempo de evolução da doença, níveis de anticorpos IgG anti-P. falciparum, não se obtendo resultados estatisticamente significativos, levando a inferir que a temperatura não interfere nos níveis de cortisol e o mesmo não interfere nos níveis de anticorpos, e não apresenta variações importantes com o tempo de evolução da doença. Os níveis de DHEA em D0, foram significativamente mais elevados do que em D7, apesar dos pacientes estarem sintomáticos há mais de um dia, já que um estímulo mantido do eixo hipotálamo-hipófise-adrenal (HPA) leva a uma diminuição deste esteróide. O DHEA foi correlacionado com a parasitemia obtendo-se um resultado significativo na correlação DHEA D0 com parasitemia D1. A correlação entre cortisol e DHEA em D0 não foi significativa (p = 0,057), porém este resultado leva a crer que o DHEA acompanha o aumento dos níveis de cortisol. Obteve-se uma correlação negativa entre DHEA e tempo de evolução de doença, apesar destes níveis estarem aumentados no pré-tratamento. Calculou-se a correlação parcial entre cortisol, DHEA e temperatura, concluindo-se que a temperatura interfere positivamente na correlação cortisol e DHEA. Uma vez que a febre reflete o momento em que ocorre a lise das hemácias secundária a esquizogonia, provavelmente esta lise com conseqüente liberação de citocinas serve como um fator agudizador da estimulação do eixo HPA, sugerindo que a liberação dos dois hormônios apresenta mecanismo comum. A correlação entre DHEA e anticorpos não foi significativa, portanto o DHEA não deve interferir na produção de anticorpos de pacientes com malária por P. falciparum.

Alterações endócrinas e metabólicas em pacientes HIV positivos com lipodistrofia

A lipodistrofia do HIV e evento comum em pacientes em uso de terapia antiretroviral fortemente ativa (HAART). O presente estudo avalia as alterações metabolicas e endocrinas em pacientes que apresentam lipodistrofia. Foram avaliados 40 pacientes com lipodistrofia e classificados em lipoatrofia, lipohipertrofia (lipoacumulacao) e lipodistrofia mista e analisadas as alterações endocrinas e metabolicas. As variáveis estudadas foram: resistência insulínica pelo HOMA-IR (Homeostasis Model Assesment Insulin Resistance), teste oral de tolerancia a glicose, TSH, T4 livre, cortisol, DHEA, prolactina, testosterona em homens, FSH, LH e estradiol nas mulheres, colesterol total e fracoes (HDL e LDL), acido úrico, ureia, creatinina, AST e ALT, medidas antropométricas (peso, estatura, índice de massa corpórea, circunferência abdominal, quadril, relação cintura/quadril), densitometria óssea e avaliação de esteatose hepática por ultrassonografia abdominal. A lipo-hipertrofia isolada ocorreu em sua totalidade no sexo feminino (p <0,05). A média de idade foi semelhante nos três grupos, sem variação na distribuição por faixa etária. Dislipidemia ocorreu em todos os grupos após o inicio da terapia anti-retroviral (lipodistrofia mista, 100%, lipo-hipertrofia, 80% e lipoatrofia 70%). Variáveis antropométricas mostram elevação da cintura abdominal em mulheres com lipo-hipertrofia. (98,2 + 13,4) e menores em mulheres com lipoatrofia (76,8 + 6,1). Pacientes com lipoatrofia apresentam-se eutroficos (82,4%), e pacientes com lipo-hipertrofia apresentam sobrepeso ou obesidade (60%). Foi encontrada associação entre osteoporose e sexo masculino (p < 0,05) alta prevalência de dislipidemia (85%) e síndrome metabólica (37,5%) em todos os grupos estudados. A síndrome metabólica esteve associada com a lipo-hipertrofia. Não foram encontradas alterações significativas na tireoide, cortisol, relação cortisol/DHEA, testosterona, FSH, LH e prolactina. Conclusão: O sexo feminino apresenta forte associação com a lipo-hipertrofia isolada. A lipo-hipertrofia parece estar associado com a síndrome metabólica e resistência insulínica. Os homens tem maiores risco de desenvolvimento de osteopenia e osteoporose. Esteatose hepática, apesar de bastante comum não esta associado a um tipo particular de lipodistrofia. Alterações hormonais não são eventos comuns em pacientes com lipodistrofia do HIV.

Contribuição do hiperandrogenismo para o desenvolvimento de síndrome metabólica em mulheres obesas com síndrome dos ovários policísticos

OBJETIVO: Avaliar a contribuição do hiperandrogenismo para o desenvolvimento da síndrome metabólica (SM) em mulheres obesas com ou sem Síndrome dos Ovários Policísticos (SOP). MÉTODOS: Estudo transversal retrospectivo no qual foram incluídas 60 mulheres obesas com fenótipo clássico da SOP - Consenso de Rotterdam - e 70 obesas sem SOP. A SM foi diagnosticada pelos critérios do NCEP-ATP III. A obesidade foi definida pelo índice de massa corpórea e o hirsutismo, pelo Índice de Ferriman-Gallwey (IFG). As dosagens realizadas foram: testosterona total, sulfato de dehidroepiandrosterona (SDHEA), insulina e glicose, colesterol total, HDL e triglicerídios. A resistência insulínica (RI) foi avaliada pelo HOMA-IR e pelo índice de sensibilidade à insulina de Matsuda e De Fronzo. A analise estatística foi realizada com o teste t de Student, teste do χ² e análise de regressão logística multivariada (p<0,05). RESULTADOS: As obesas com SOP apresentaram significativamente maiores valores de IFG (15,4±6,1), circunferência da cintura (105,6±11,4 cm), testosterona (135,8±71,4 ng/dL), SDHEA (200,8±109,2 µg/dL), HOMA-IR (8,4±8,5) e menores valores de ISI (2,0±1,8) quando comparadas às obesas não SOP (3,2±2,1; 101,4±9,2 cm; 50,0±18,2 ng/dL; 155,0±92,7 µg/dL; 5,1±4,7; 3,3±2,7, respectivamente) (p<0,05). A frequência de SM foi significativamente maior nas obesas com SOP (75%) do que nas obesas não SOP (52,8%) (p=0,01). A análise multivariada não demonstrou contribuição das variávies IFG, testoterona total e SDHEA para o desenvolvimento da SM (p>0,05). CONCLUSÃO: Mulheres obesas com SOP apresentam maior frequência de SM quando comparadas às obesas não SOP. O hiperandrogenismo não mostrou influência nesse grupo de mulheres estudadas.

Clinical Assessment of Tribulus terrestris Extract in the Treatment of Female Sexual Dysfunction

This is a qualitative-quantitative study based on hospital records of female patients of reproductive age, presenting sexual dysfunction, and treated with 250 mg Tribulus terrestris extract (1 tablet thrice daily for 90 days). Safety monitoring included vital signs, physical examination, laboratory tests, and occurrence of adverse events. Efficacy analysis included results of the Female Sexual Function Index (FSFI), dehydroepiandrosterone (DHEA) levels together with total and free testosterone, and the patient and physician assessments. There was a statistically significant improvement in total FSFI scores (P < 0.0001) post-treatment, with improvement among 106 (88.33%) subjects. There was a statistically significant (P < 0.0001) increase in the level of DHEA, while the levels of both serum testosterone (P = 0.284) and free testosterone decreased (P < 0.0001). Most adverse events recorded were related to the gastrointestinal tract. Physical examination showed no significant changes post-treatment. Based on the results, it is concluded that the T. terrestris extract is safe and effective in the treatment of female sexual dysfunction.

Avaliação adrenocortical, tireoideana e do estresse oxidativo de cães naturalmente infectados por Ehrlichia canis

Pós-graduação em Medicina Veterinária - FCAV

A novel DAX1/NR0B1 mutation in a patient with adrenal hypoplasia congenita and hypogonadotropic hypogonadism

We report a case of adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) due to a novel DAX1 mutation. A 19-month-old boy with hyperpigmentation and failure to thrive came to our service for investigation. Three brothers of the patient had died due to adrenal failure, and a maternal cousin had adrenal insufficiency. Adrenoleukodystrophy was excluded. MRI showed normal pituitary and hypothalamus. Plasma hormone evaluation revealed high ACTH (up to 2,790 pg/mL), and low levels of androstenedione, DHEA-S, 11-deoxycortisol, and cortisol. At 14 years of age the patient was still prepubescent, his weight was 43.6 kg (SDS: -0.87) and his height was 161 cm (SDS: -0.36), with normal body proportions. In the GnRH test, basal and maximum values of LH and FSH were respectively 0.6/2.1 and < 1.0/< 1.0 U/L. Molecular investigation identified a novel mutation that consists of a deletion of codon 372 (AAC; asparagine) in exon 1 of DAX1. This mutation was not found in a study of 200 alleles from normal individuals. Prediction site analysis indicated that this alteration, located in the DAX1 ligand-binding domain, may damage DAX1 protein. We hypothesize that the novel (p.Asp372del) DAX1 mutation might be able to cause a disruption of DAX1 function, and is probably involved in the development of AHC and HH in this patient. Arq Bras Endocrinol Metab. 2012;56(8):496-500

Der Wirkungsmechanismus von Neurosteroiden auf das Cytoskelett neuronaler Zellen

Neurosteroide können langsame genomische und schnelle nicht-genomische Effekte zeigen. Die Synthese und der Metabolismus von Neurosteroiden werden entwicklungsbedingt reguliert. In den letzten Jahren sind immer mehr schnelle Steroideffekte bekannt geworden, die sowohl über klassische als auch über nicht-klassische Rezeptoren laufen. Zum heutigen Stand der Forschung sind die morphologischen Effekte von Neurosteroiden auf das neuronale Cytoskelett und die involvierten Signalkaskaden noch weitgehend unerforscht. In diesem Zusammenhang stellen sich auch die Fragen nach den verantwortlichen Rezeptoren und dem Transportmechanismus sowie der subzellulären Lokalisation der Steroide. Die im Rahmen meiner Promotion erhaltenen Ergebnisse zeigen, dass die Steroide DHEA und Testosteron eine Reorganisation des Aktincytoskeletts in neuronalen Zellen induzieren und dass diese Effekte diesen Steroiden und nicht ihren Folgemetaboliten zuzuordnen sind. DHEA bewirkt die Kontraktion der Zellen, eine erhöhte Ausbildung von Stressfasern und fokalen Adhäsionskomplexen sowie die Bildung von Filopodien. Der diesen Effekten zu Grunde liegende Signalweg konnte eindeutig identifiziert werden. DHEA induziert in neuronalen Zellen die Aktivierung des Rho-Signalwegs. Diese Aktivierung führt zu einem erhöhten Phosphorylierungsstatus der regulatorischen leichten Kette von Myosin II (MRLC) an Serin 19 und der damit verbundenen erhöhten Myosin-Aktin-Interaktion. Die Ausbildung von Filopodien wird vermutlich über eine Aktivierung der GTPase Cdc42 vermittelt. Testosteron induziert das Auswachsen langer Neuriten sowie eine Verminderung von Stressfasern in neuronalen Zellen. Diese Effekte sind abhängig von der Aktivität der PI3-Kinase. Die im Rahmen dieser Arbeit gewonnenen Erkenntnisse deuten darauf hin, dass Testosteron über die PI3-Kinase und FAK den Rac-Signalweg induziert, da es zu einer Inhibierung des Rho-Signalwegs kommt. Zahlreiche Erkenntnisse weisen darauf hin, dass DHEA und Testosteron die Aktivierung der beteiligten Signalwege über einen G-Protein gekoppelten Rezeptor induzieren. DHEA und Testosteron beeinflussen auch die Expression und die Lokalisation der regulatorischen leichten Ketten von Myosin II. Im Gegensatz zu DHEA (Lokalisation der MRLC in der kortikalen Region der Zelle), induziert Testosteron eine Umlokalisation der MRLC in den Zellkern. Daher ist es denkbar, dass die MRLCs, wie auch Aktin, als Transkriptionsfaktoren wirken können. Die Synthese eines funktionalen, fluoreszierenden DHEA-Derivats (DHEA-Bodipy) ermöglichte erstmals, den Transport und die subzelluläre Lokalisation von DHEA in neuronalen Zellen zu beobachten. DHEA-Bodipy wird in neuronalen Zellen in den Mitochondrien lokalisiert. Diese Lokalisation ergibt völlig neue Ansätze im Verständnis zellulärer Wirkungsorte von Steroiden und beteiligter Rezeptoren. Das in meiner Arbeit vorgestellte Verfahren zur Fluoreszenzmarkierung von Steroiden bietet vielfältige Möglichkeiten im Einsatz zellbiologischer Methoden. Nach diesem Verfahren hergestellte, fluoreszierende Steroide eignen sich aufgrund ihrer Stabilität sehr gut für die Untersuchung des Transports und der subzellulären Lokalisation von Steroiden an fixierten und lebenden Zellen sowie für Colokalisationsexperimente. Diese Methode grenzt somit auch die Anzahl möglicher molekularer Interaktionspartner ein. Für Testosteron konnte ebenfalls ein fluoreszierendes Testosteron-Derivat (Testosteron-Bodipy) synthetisiert werden. Die Aufklärung der Effekte von Steroiden auf das neuronale Cytoskelett und der beteiligten Signalkaskaden sowie die Identifizierung der zellulären Wirkungsorte ermöglichen therapeutische Ansätze zur Behandlung neurodegenerativer Erkrankungen, deren Ursachen in Abnormitäten des Cytoskeletts oder fehlregulierter Neurosteroidogenese zu begründen sind.

Dehydroepiandrosterone sulfate in the assessment of the hypothalamic-pituitary-adrenal axis

The role of dehydroepiandrosterone-sulfate (DHEA-S) in assessing the integrity of the hypothalamic-pituitary-adrenal (HPA) axis in patients with suspected insufficiency is uncertain.

Hexose-6-phosphate dehydrogenase modulates 11beta-hydroxysteroid dehydrogenase type 1-dependent metabolism of 7-keto- and 7beta-hydroxy-neurosteroids

BACKGROUND: The role of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in the regulation of energy metabolism and immune system by locally reactivating glucocorticoids has been extensively studied. Experiments determining initial rates of enzyme activity revealed that 11beta-HSD1 can catalyze both the reductase and the dehydrogenase reaction in cell lysates, whereas it predominantly catalyzes the reduction of cortisone to cortisol in intact cells that also express hexose-6-phosphate dehydrogenase (H6PDH), which provides cofactor NADPH. Besides its role in glucocorticoid metabolism, there is evidence that 11beta-HSD1 is involved in the metabolism of 7-keto- and 7-hydroxy-steroids; however the impact of H6PDH on this alternative function of 11beta-HSD1 has not been assessed. METHODOLOGY: We investigated the 11beta-HSD1-dependent metabolism of the neurosteroids 7-keto-, 7alpha-hydroxy- and 7beta-hydroxy-dehydroepiandrosterone (DHEA) and 7-keto- and 7beta-hydroxy-pregnenolone, respectively, in the absence or presence of H6PDH in intact cells. 3D-structural modeling was applied to study the binding of ligands in 11beta-HSD1. PRINCIPAL FINDINGS: We demonstrated that 11beta-HSD1 functions in a reversible way and efficiently catalyzed the interconversion of these 7-keto- and 7-hydroxy-neurosteroids in intact cells. In the presence of H6PDH, 11beta-HSD1 predominantly converted 7-keto-DHEA and 7-ketopregnenolone into their corresponding 7beta-hydroxy metabolites, indicating a role for H6PDH and 11beta-HSD1 in the local generation of 7beta-hydroxy-neurosteroids. 3D-structural modeling offered an explanation for the preferred formation of 7beta-hydroxy-neurosteroids. CONCLUSIONS: Our results from experiments determining the steady state concentrations of glucocorticoids or 7-oxygenated neurosteroids suggested that the equilibrium between cortisone and cortisol and between 7-keto- and 7-hydroxy-neurosteroids is regulated by 11beta-HSD1 and greatly depends on the coexpression with H6PDH. Thus, the impact of H6PDH on 11beta-HSD1 activity has to be considered for understanding both glucocorticoid and neurosteroid action in different tissues.

Androgen and gonadotropin patterns differ in HIV-1-infected men who develop lipoatrophy during antiretroviral therapy: a case-control study

OBJECTIVES: We compared androgen and gonadotropin values in HIV-infected men who did and did not develop lipoatrophy on combination antiretroviral therapy (cART). METHODS: From a population of 136 treatment-naïve male Caucasians under successful zidovudine/lamivudine-based cART, the 10 patients developing lipoatrophy (cases) were compared with 87 randomly chosen controls. Plasma levels of free testosterone (fT), dehydroepiandrosterone (DHEA), follicle-stimulating hormone and luteinizing hormone (LH) were measured at baseline and after 2 years of cART. RESULTS: At baseline, 60% of the cases and 71% of the controls showed abnormally low fT values. LH levels were normal or low in 67 and 94% of the patients, respectively, indicating a disturbance of the hypothalamic-pituitary-gonadal axis. fT levels did not significantly change after 2 years of cART. Cases showed a significant increase in LH levels, while controls showed a significant increase in DHEA levels. In a multivariate logistic regression model, lipoatrophy was associated with higher baseline DHEA levels (P=0.04), an increase in LH levels during cART (P=0.001), a lower body mass index and greater age. CONCLUSIONS: Hypogonadism is present in the majority of HIV-infected patients. The development of cART-related lipoatrophy is associated with an increase in LH and a lack of increase in DHEA levels.

STAR splicing mutations cause the severe phenotype of lipoid congenital adrenal hyperplasia: insights from a novel splice mutation and review of reported cases

OBJECTIVE The steroidogenic acute regulatory protein (StAR) transports cholesterol to the mitochondria for steroidogenesis. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH) which is characterized by impaired synthesis of adrenal and gonadal steroids causing adrenal insufficiency, 46,XY disorder of sex development (DSD) and failure of pubertal development. Partial loss of StAR activity may cause adrenal insufficiency only. PATIENT A newborn girl was admitted for mild dehydration, hyponatremia, hyperkalemia and hypoglycaemia and had normal external female genitalia without hyperpigmentation. Plasma cortisol, 17OH-progesterone, DHEA-S, androstendione and aldosterone were low, while ACTH and plasma renin activity were elevated, consistent with the diagnosis of primary adrenal insufficiency. Imaging showed normal adrenals, and cytogenetics revealed a 46,XX karyotype. She was treated with fluids, hydrocortisone and fludrocortisone. DESIGN, METHODS AND RESULTS Genetic studies revealed a novel homozygous STAR mutation in the 3' acceptor splice site of intron 4, c.466-1G>A (IVS4-1G>A). To test whether this mutation would affect splicing, we performed a minigene experiment with a plasmid construct containing wild-type or mutant StAR gDNA of exons-introns 4-6 in COS-1 cells. The splicing was assessed on total RNA using RT-PCR for STAR cDNAs. The mutant STAR minigene skipped exon 5 completely and changed the reading frame. Thus, it is predicted to produce an aberrant and shorter protein (p.V156GfsX19). Computational analysis revealed that this mutant protein lacks wild-type exons 5-7 which are essential for StAR-cholesterol interaction. CONCLUSIONS STAR c.466-1A skips exon 5 and causes a dramatic change in the C-terminal sequence of the protein, which is essential for StAR-cholesterol interaction. This splicing mutation is a loss-of-function mutation explaining the severe phenotype of our patient. Thus far, all reported splicing mutations of STAR cause a severe impairment of protein function and phenotype.

Gonadotrophin stimulation for in vitro fertilization significantly alters the hormone milieu in follicular fluid: a comparative study between natural cycle IVF and conventional IVF.

STUDY QUESTION Is the steroid hormone profile in follicular fluid (FF) at the time of oocyte retrieval different in naturally matured follicles, as in natural cycle IVF (NC-IVF), compared with follicles stimulated with conventional gonadotrophin stimulated IVF (cIVF)? SUMMARY ANSWER Anti-Mullerian hormone (AMH), testosterone (T) and estradiol (E2) concentrations are ∼3-fold higher, androstenedione (A2) is ∼1.5-fold higher and luteinizing hormone (LH) is ∼14-fold higher in NC-IVF than in cIVF follicles, suggesting an alteration of the follicular metabolism in conventional gonadotrophin stimulated IVF. WHAT IS KNOWN ALREADY In conventional IVF, the implantation rate of unselected embryos appears to be lower than in NC-IVF, which is possibly due to negative effects of the stimulation regimen on follicular metabolism. In NC-IVF, the intrafollicular concentration of AMH has been shown to be positively correlated with the oocyte fertilization and implantation rates. Furthermore, androgen treatment seems to improve the ovarian response in low responders. STUDY DESIGN, SIZE, DURATION This cross-sectional study involving 36 NC-IVF and 40 cIVF cycles was performed from 2011 to 2013. Within this population, 13 women each underwent 1 NC-IVF and 1 cIVF cycle. cIVF was performed by controlled ovarian stimulation with HMG and GnRH antagonists. PARTICIPANTS/MATERIALS, SETTING, METHODS Follicular fluid was collected from the leading follicles. AMH, T, A2, dehydroepiandrosterone (DHEA), E2, FSH, LH and progesterone (P) were determined by immunoassays in 76 women. Aromatase activity in follicular fluid cells was analysed by a tritiated water release assay in 33 different women. For statistical analysis, the non-parametric Mann-Whitney U or Wilcoxon tests were used. MAIN RESULTS AND ROLE OF CHANCE In follicular fluid from NC-IVF and from cIVF, median levels were 32.8 and 10.7 pmol/l for AMH (P < 0.0001), 47.2 and 18.8 µmol/l for T (P < 0.0001), 290 and 206 nmol/l for A2 (P = 0.0035), 6.7 and 5.6 pg/ml for DHEA (n.s.), 3292 and 1225 nmol/l for E2 (P < 0.0001), 4.9 and 7.2 mU/ml for FSH (P < 0.05), 14.4 and 0.9 mU/ml for LH (P < 0.0001) and 62 940 and 54 710 nmol/l for P (n.s.), respectively. Significant differences in follicular fluid concentrations for AMH, E2 and LH were also found in the 13 patients who underwent both NC-IVF and cIVF when they were analysed separately in pairs. Hormone analysis in serum excluded any relevant impact of AMH, T, A2, and E2 serum concentration on the follicular fluid hormone concentrations. Median serum concentrations were 29.4 and 0.9 mU/ml for LH (P < 0.0001) and 2.7 and 23.5 nmol/l for P (P < 0.0001) after NC-IVF and c-IVF, respectively. Positive correlations were seen for FF-AMH with FF-T (r = 0.35, P = 0.0002), FF-T with FF-LH (r = 0.48, P < 0.0001) and FF-E2 with FF-T (r = 0.75, P < 0.0001). The analysis of aromatase activity was not different in NC-IVF and cIVF follicular cells. LIMITATION, REASONS FOR CAUTION Any association between the hormone concentrations and the implantation potential of the oocytes could not be investigated as the oocytes in cIVF were not treated individually in the IVF laboratory. Since both c-IVF and NC-IVF follicles were stimulated by hCG before retrieval, the endocrine milieu in the natural cycle does not represent the pure physiological situation. WIDER IMPLICATIONS OF THE FINDINGS The endocrine follicular milieu and the concentration of putative markers of oocyte quality, such as AMH, are significantly different in gonadotrophin-stimulated conventional IVF compared with natural cycle IVF. This could be a cause for the suggested lower oocyte quality in cIVF compared with naturally matured oocytes. The reasons for the reduced AMH concentration might be low serum and follicular fluid LH concentrations due to LH suppression, leading initially to low follicular androgen concentrations and then to low follicular AMH production. STUDY FUNDING/COMPETING INTERESTS Funding for this study was obtained from public universities (for salaries) and private industry (for consumables). Additionally, the study was supported by an unrestricted grant from MSD Merck Sharp & Dohme GmbH and IBSA Institut Biochimique SA. The authors are clinically involved in low-dose monofollicular stimulation and IVF therapies, using gonadotrophins from all gonadotrophin distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. Otherwise, the authors have no competing interests. TRIAL REGISTRATION NUMBER Not applicable.

A novel form of CYP3A from rat brain: cDNA cloning, protein characterization and gene regulation of cytochrome P450 3A9

One full length cDNA clone, designated 3aH15, was isolated from a rat brain cDNA library using a fragment of CYP3A2 cDNA as a probe. 3aH15 encoded a protein composed of 503 amino acid residues. The deduced amino acid sequence of 3aH15 was 92% identical to mouse Cyp3a-13 and had a 68.4% to 76.5% homology with the other reported rat CYP3A sequences. Clone 3aH15 was thus named CYP3A9 by Cytochrome P450 Nomenclature Committee. CYP3A9 seems to the major CYP3A isozyme expressed in rat brain. Sexual dimorphism of the expression of CYP3A9 was shown for the first time in rat brain as well as in rat liver. CYP3A9 appears to be female specific in rat liver based on the standards proposed by Kato and Yamazoe who defined sex specific expression of P450s as being a 10-fold or higher expression level in one sex compared with the other. CYP3A9 gene expression was inducible by estrogen treatment both in male and in female rats. Male rats treated with estrogen had a similar expression level of CYP3A9 mRNA both in the liver and brain. Ovariectomy of adult female rats drastically reduced the mRNA level of CYP3A9 which could be fully restored by estrogen replacement. On the other hand, only a two-fold induction of CYP3A9 expression by dexamethasone was observed in male liver and no significant induction of CYP3A9 mRNA was observed in female liver or in the brains. These results suggest that estrogen may play an important role in the female specific expression of the CYP3A9 gene and that CYP3A9 gene expression is regulated differently from other CYP3A isozymes. ^ P450 3A9 recombinant protein was expressed in E. coli using the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity (10.1 nmol P450/mg protein) from solubilized fractions using two chromatographic steps. The purified P450 3A9 protein was active towards the metabolism of many clinically important drugs such as imipramine, erythromycin, benzphetamine, ethylmorphine, chlorzoxazone, cyclosporine, rapamycin, etc. in a reconstituted system containing lipid and rat NADPH-P450 reductase. Although P450 3A9 was active towards the catabolism of testosterone, androstenedione, dehydroepiandrosterone (DHEA) and 17β-estradiol, P450 3A9 preferentially catalyzes the metabolism of progesterone to form four different hydroxylated products. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture and GSH. The possible mechanisms of the stimulatory effects of GSH on P450 3A9 activities are discussed. Sexually dimorphic expression of P450 3A9 in the brain and its involvement in many neuroactive drugs as well as neurosteroids suggest the possible role of P450 3A9 in some mental disorders and brain functions. ^

Steroidogenesis of the testis - new genes and pathways

Defects of androgen biosynthesis cause 46,XY disorder of sexual development (DSD). All steroids are produced from cholesterol and the early steps of steroidogenesis are common to mineralocorticoid, glucocorticoid and sex steroid production. Genetic mutations in enzymes and proteins supporting the early biosynthesis pathways cause adrenal insufficiency (AI), DSD and gonadal insufficiency. The classic androgen biosynthesis defects with AI are lipoid CAH, CYP11A1 and HSD3B2 deficiencies. Deficiency of CYP17A1 rarely causes AI, and HSD17B3 or SRD5A2 deficiencies only cause 46,XY DSD and gonadal insufficiency. All androgen biosynthesis depends on 17,20 lyase activity of CYP17A1 which is supported by P450 oxidoreductase (POR) and cytochrome b5 (CYB5). Therefore 46,XY DSD with apparent 17,20 lyase deficiency may be due to mutations in CYP17A1, POR or CYB5. Illustrated by patients harboring mutations in SRD5A2, normal development of the male external genitalia depends largely on dihydrotestosterone (DHT) which is converted from circulating testicular testosterone (T) through SRD5A2 in the genital skin. In the classic androgen biosynthetic pathway, T is produced from DHEA and androstenedione/-diol in the testis. However, recently found mutations in AKR1C2/4 genes in undervirilized 46,XY individuals have established a role for a novel, alternative, backdoor pathway for fetal testicular DHT synthesis. In this pathway, which has been first elucidated for the tammar wallaby pouch young, 17-hydroxyprogesterone is converted directly to DHT by 5α-3α reductive steps without going through the androgens of the classic pathway. Enzymes AKR1C2/4 catalyse the critical 3αHSD reductive reaction which feeds 17OH-DHP into the backdoor pathway. In conclusion, androgen production in the fetal testis seems to utilize two pathways but their exact interplay remains to be elucidated.