Ontogenesis of thyroid function and interactions with maternal function.

Fetal and neonatal development of thyroid function involves the embryogenesis, differentiation and maturation of the thyroid gland, of the hypothalamic-pituitary-thyroid axis and of the systems controlling thyroid hormone metabolism. We focus here on aspects related to neurodevelopment. Throughout gestation, thyroxine (T4) transferred from the mother, present in embryonic fluids by 4 weeks, protects the fetal brain. Free T4 (FT4) in fetal fluids increases rapidly, approaching adult levels by midgestation, in concentrations that are determined by the maternal serum T4. T3 remains very low throughout pregnancy. In the cerebral cortex T3, generated from T4, reaches adult values by midgestation and is partly bound to specific nuclear receptor isoforms. The iodothyronine deiodinases are important for the spatial and temporal presence of T3 in different fetal brain areas. After onset of fetal thyroid secretion at midgestation, maternal transfer of T4 continues to contribute importantly to fetal serum T4, protecting neurodevelopment until birth. In rats, even a transient period of maternal hypothyroxinemia disrupts neurodevelopment irreversibly, supporting epidemiological evidence for its negative role in human neurodevelopment. The prompt treatment of maternal hypothyroidism or hypothyroxinemia should mitigate negative effects on neurodevelopment. Neurodevelopmental deficits of preterm infants might also result from an untimely interruption of the maternal transfer of T4 [Morreale de Escobar et al: J Clin Endocrinol Metab 2000;85:3975-3987; Best Pract Res Clin Endocrinol Metab 2004;18:225-248; Eur J Endocrinol 2004;151(suppl 3):U25-U37].

The role of the CAG repeat polymorphism in the androgen receptor gene and of skewed X-chromosome inactivation, in the pathogenesis of hirsutism.

The human androgen receptor (AR) gene contains a variable number of CAG repeats within exon 1. Shorter AR alleles, by increasing transactivation, may result in augmented AR-mediated sensitivity of the hair follicle. We have evaluated whether the number of CAG repeats, or if skewed inactivation of X-chromosome, favoring the presence of shorter AR alleles, influence hirsutism in women with and without hyperandrogenemia. Twenty-eight women with idiopathic hirsutism (normal serum androgens), 34 women with hyperandrogenic hirsutism (increased serum androgens), and 15 healthy control women were analyzed by evaluating the number of CAG repeats in genomic DNA, as well as the methylation pattern (after DNA digestion with HpaII), which allows identification of which allele is inactive. Despite marked differences among the groups in serum androgens, which were markedly increased in women with hyperandrogenic hirsutism as compared with women with idiopathic hirsutism and to controls, there were no significant differences among the groups in the number of CAG repeats. Moreover, skewed X-chromosome inactivation was found in 10 subjects (3 with idiopathic hirsutism, 5 with hyperandrogenic hirsutism, and 2 controls; P = 0.746) of the 67 women (14.9%) who were heterozygous for the AR gene. In several of these subjects, it was the shorter allele that was preferentially inactivated. In conclusion, neither the CAG repeat polymorphism in the AR gene, nor skewed X-chromosome inactivation, seem to play a significant role in the pathogenesis of hirsutism.

TNF-alpha and hyperandrogenism: a clinical, biochemical, and molecular genetic study.

To evaluate the role of TNF-alpha in the pathogenesis of hyperandrogenism, we have evaluated the serum TNF-alpha levels, as well as several polymorphisms in the promoter region of the TNF-alpha gene, in a group of 60 hyperandrogenic patients and 27 healthy controls matched for body mass index. Hyperandrogenic patients presented with mildly increased serum TNF-alpha levels as compared with controls (mean[median] +/- SD: 7.2[7.0] +/- 3.3 pg/ml vs. 5.6[4.4] +/- 4.0 pg/ml, P < 0.02). Although no differences in body mass index and insulin resistance indexes were observed between patients and controls, when subjects were classified by body weight, serum TNF-alpha was increased only in lean patients as compared with lean controls, but this difference was not statistically significant when comparing obese patients with obese controls. The TNF-alpha gene polymorphisms studied here (-1196C/T, -1125G/C, -1031T/C, -863C/A, -857C/T, -316G/A, -308G/A, -238G/A, and -163G/A) were equally distributed in hyperandrogenic patients and controls. However, carriers of the -308A variant presented with increased basal and leuprolide-stimulated serum androgens and 17-hydroxyprogesterone levels when considering patients and controls as a group. No differences were observed in serum TNF-alpha levels, body mass index, and insulin resistance indexes, depending on the presence or absence of these variants. In conclusion, our present results suggest that the TNF-alpha system might contribute to the pathogenesis of hyperandrogenism, independent of obesity and insulin resistance. However, elucidation of the precise mechanisms underlying the relationship between the TNF-alpha system and androgen excess is needed before considering TNF-alpha as a significant contributing factor to the development of hyperandrogenism.

Screening for mutations in the steroidogenic acute regulatory protein and steroidogenic factor-1 genes, and in CYP11A and dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome, gene-1 (DAX-1), in hyperandrogenic hirsute women.

Abstract Abnormalities in adrenal and/or ovarian steroidogenesis are found in most patients with hirsutism. The rate-limiting step in the synthesis of steroids in the ovary and the adrenal is the conversion of cholesterol into pregnenolone by cholesterol side-chain cleavage enzyme (P450scc), encoded by the gene CYP11A, after cholesterol is introduced into the mitochondria by the steroidogenic acute regulatory protein (StAR). DAX-1 is a repressor of StAR gene expression, and steroidogenic factor-1 (SF-1) is a regulator of CYP11A, DAX-1, and StAR gene. Mutations in any of these factors resulting in gain of function, or loss of repression, of StAR or P450scc might contribute to the steroidogenic abnormalities present in hirsute patients. In the present study we have screened, using heteroduplex analysis, the genes encoding StAR and SF-1 as well as DAX-1 and CYP11A for mutations in genomic DNA from 19 women presenting with hirsutism and increased serum androgen levels. When variants were found, analysis was extended to a larger group of hyperandrogenic patients and nonaffected women. Two variants were identified in the SF-1 gene. A G-->C change in exon 6, resulting in an Arg(365)Pro mutation, was found in 1 of 45 patients, but not in controls. Also, a Gly(146)Ala missense mutation, resulting from a G-->C change in exon 4, was found in 2 of 48 patients and in 2 of 50 nonaffected individuals. We identified a C-->T base pair change at position -33 of the StAR gene. Three of 48 patients and 3 of 43 controls presented this variant. No mutations were found in coding regions of the StAR gene. Analysis of CYP11A-coding regions identified a G-->A change in exon 3, resulting in a Val(179)Ile missense mutation. This mutation was found in 1 of 29 patients studied and was not present in 50 controls. Finally, analysis of DAX-1 showed no variant in any of the women studied. In conclusion, mutations in StAR, SF-1, CYP11A, and DAX-1 are seldom found in hirsute patients and do not explain the steroidogenic abnormalities found in these women.

A prospective study of the prevalence of the polycystic ovary syndrome in unselected Caucasian women from Spain.

We prospectively estimated the prevalence of the polycystic ovary syndrome (PCOS), as defined by the NIH/NICHHD 1990 endocrine criteria, in a population of 154 Caucasian women of reproductive age reporting spontaneously for blood donation. Anthropometric data; the presence of hirsutism, acne, and androgenic alopecia; and the menstrual history were recorded by a single investigator. In 145 women, blood samples were also obtained for measurement of serum androgen levels. PCOS was defined by the presence of 1) oligomenorrhea, 2) clinical and/or biochemical hyperandrogenism, and 3) exclusion of hyperprolactinemia, thyroid disorders, and nonclassic 21-hydroxylase deficiency. Hirsutism was defined by a modified Ferriman-Gallwey score of 8 or more, acne was considered as a sign of hyperandrogenism when persistent after the second decade of life, and hyperandrogenemia was defined by an increase in circulating testosterone or dehydroepiandrosterone sulfate or an increase in the free androgen index above the 95th percentile of the control values derived from the nonhirsute, nonacneic women having regular menses who were not receiving hormonal therapy. PCOS was present in 10(6.5%), hirsutism was present in 11 (7.1%), and acne was present in 19 (12.3%) of the 154 women. Our results demonstrate a 6.5% prevalence of PCOS, as defined, in a minimally biased population of Caucasian women from Spain. The polycystic ovary syndrome, hirsutism, and acne are common endocrine disorders in women.

Receiver operating characteristic analysis of the performance of basal serum hormone profiles for the diagnosis of polycystic ovary syndrome in epidemiological studies.

OBJECTIVE: We have used receiver operating characteristic (ROC) analysis to determine the diagnostic performance of several serum parameters, in order to evaluate their potential usefulness in establishing the diagnosis of polycystic ovary syndrome (PCOS) in epidemiological studies. DESIGN: Prospective study. METHODS: One hundred and fourteen women reporting spontaneously for blood donation were included in the study. Menopausal and oral contraceptive-treated women were excluded. Serum samples were obtained at the moment of donation, independently of fasting, time of day or day of menstrual cycle. Measurements included total testosterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), LH, FSH and estradiol. The free testosterone (FT) concentration and the free androgen index (FAI) were calculated from testosterone and SHBG levels. ROC curves were calculated for all these serum determinations. RESULTS: Eight patients were diagnosed with PCOS, according to the presence of oligomenorrhea, hirsutism, acne and/or hyperandrogenemia, and exclusion of non-classic congenital adrenal hyperplasia, hypothyroidism and hyperprolactinemia. Of the parameters studied SHBG, FAI, FT and DHEAS were considered adequate measures for the diagnosis of PCOS. For example, serum SHBG levels showed an area under the ROC curve of 0.875+/-(S.E.(w))0.045 (95% confidence interval 0.800-0.929). A SHBG decision threshold <37 nmol/l had a sensitivity of 87.5%, a specificity of 86.8%, a positive likelihood ratio of 6.63, and a negative likelihood ratio of 0.14, for the diagnosis of PCOS. CONCLUSIONS: Our present results strongly suggest that decreased SHBG levels, and increased FAI, free testosterone concentration and DHEAS concentrations, are highly effective as single analytical procedures in epidemiological studies for the detection of PCOS in women of reproductive age.

Thyroid hormones in human tumoral and normal nervous tissues.

We have studied T4 and T3 concentrations, DNA and protein concentrations and 5' and 5 deiodinases in samples of brain tumors obtained at surgery from 49 patients, and, in most cases, also from surrounding normal tissue. T4 concentrations in normal cortical tissue (6.19+/-0.45 ng/g) were lower than in white matter, but the difference disappeared when referred to the DNA content (2.26+/-0.27 ng/mg DNA). No other differences were found between cortical and white matter, or among cortical lobes. T4 in normal tissue was higher than previously reported, mostly from autopsy samples, whereas T3 (0.99+/-0.07 ng/g) was similar. 5'D-I activity was negligible as compared to 5'D-II (8.11+/-1.09 fmol/h/mg protein). When expressed in relation to the different DNA contents of normal vs. tumoral tissue, 5'D-II activities were the same for both. 5D activity was highly variable in the tumoral tissue, with negligible activities in meningiomas and pituitary adenomas. When referred to the DNA content, T4 and 5'D-II were the same, but T3 concentrations were lower in the tumor (0.24+/-0.03 ng/mg DNA) as compared to normal (0.35+/-0.04 ng/mg DNA) tissue samples. Whether or not this decrease of T3 affects the expression of T3-sensitive processes remains to be studied.

Role of the pentanucleotide (tttta)(n) polymorphism in the promoter of the CYP11a gene in the pathogenesis of hirsutism.

OBJECTIVE: To determine if the (tttta)(n) repeat polymorphism in the promoter region of CYP11a gene is associated with hirsutism and hyperandrogenism in women from Spain. DESIGN: Controlled clinical study. SETTING: Tertiary-care institutional hospital. PATIENT(S): Ninety-two hirsute women and 33 healthy control women. INTERVENTION(S): Basal and adrenocorticotropin-stimulated serum samples and genomic DNA extracted and purified from whole-blood samples were obtained during the follicular phase of the menstrual cycle. MAIN OUTCOME MEASURE(S): CYP11a (tttta)(n) repeat-polymorphism genotype and serum ovarian and adrenal androgen levels. RESULT(S): None of the CYP11a (tttta)(n) polymorphic alleles was associated with hirsutism. The absence of the four-repeat-units allele (4R-- genotype), which has been reported by other authors to be associated with polycystic ovary syndrome (PCOS), was found in 22.4% of the women studied here and was equally distributed among patients and controls, independently of the presence of PCOS and/or ovarian or adrenal hyperandrogenism. No differences were observed in serum hormone concentrations in 4R-- individuals as compared with subjects with at least one four-repeat-units allele. CONCLUSION(S): The (tttta)(n) repeat polymorphism in the promoter region of CYP11a does not appear to play any significant role in the pathogenesis of hirsutism and hyperandrogenism in women from Spain.

Role of the follistatin gene in women with polycystic ovary syndrome.

OBJECTIVE: To search for mutations in the coding exons of the follistatin gene of women diagnosed with polycystic ovary syndrome (PCOS). DESIGN: Controlled clinical study. SETTING: Tertiary institutional hospital. PATIENT(S): Thirty-four women diagnosed with PCOS and 15 healthy control women. INTERVENTION(S): Whole blood and serum samples were collected during the follicular phase of the menstrual cycle. MAIN OUTCOME MEASURE(S): Circulating total testosterone (T), sex hormone-binding globulin (SHBG), calculated free T (FT), androstenedione (A), dehydroepiandrosterone-sulfate (DHEAS), LH, FSH, E2, and basal and adenocorticotropic hormone (ACTH)-stimulated 17-hydroxyprogesterone (17-OHP) were determined. Insulin resistance was estimated from fasting glucose and insulin levels, using the homeostasis model assessment. The coding regions of the follistatin gene were studied by heteroduplex analysis after polymerase chain reaction amplification. RESULT(S): Women with PCOS presented with higher body-mass index, insulin resistance, T, FT, A, and ACTH-stimulated 17-OHP serum concentrations and lower SHBG serum levels, as compared with controls. No differences were observed among the groups in serum DHEAS, basal 17-OHP, E(2), LH, and FSH. No mutations were found in coding regions of the follistatin gene, with the exception of a G to A change at cDNA position 951, resulting in a silent mutation. This change was present in 2 (5.9%) of 34 patients and 1 (6.7%) of 15 controls. CONCLUSION(S): Mutations in the coding regions of the follistatin gene do not appear to be related to PCOS.

[A case of Klippel-Feil syndrome]. / Un caso de síndrome de Klippel-Feil.

We would like to present a case of Klippel-Feil Syndrome in which it is associated with multiple cervical vertebra synostosis, short neck, cervical rib, congenital sclerosis, platybasia, spina-bifida and deafness. We are revising the case history of this syndrome.

Tri-iodothyronine upregulates adiponutrin mRNA expression in rat and human adipocytes.

Adiponutrin (PNPLA3) is expressed in adipose tissue. Although its precise function is unknown, some data suggest a dual role in lipid homeostasis. We have investigated the influence of thyroid hormone (TH) on PNPLA3 mRNA, in rat and human cultured white adipocytes and in rat white adipose tissue (WAT). Pnpla3 mRNA increased during differentiation of rat adipocytes in an insulin-dependent manner. Tri-iodothyronine further increased Pnpla3 expression at any day during differentiation and its effects were time and dose-dependent. The Pnpla3 mRNA half-life was stabilized by tri-iodothyronine, but a transcriptional component was also observed. Pnpla3 mRNA decreased in WAT of hypothyroid rats and was partially restored by treatment with TH. Taqman analysis showed that tri-iodothyronine also increased human PNPLA3 expression in cultured subcutaneous adipocytes from obese patients. In conclusion, PNPLA3 mRNA expression is upregulated by tri-iodothyronine in adipocytes in vitro, in humans and rats, and in vivo in rat WAT.

Thermogenic effect of triiodothyroacetic acid at low doses in rat adipose tissue without adverse side effects in the thyroid axis.

Triiodothyroacetic acid (TRIAC) is a physiological product of triiodothyronine (T(3)) metabolism, with high affinity for T(3) nuclear receptors. Its interest stems from its potential thermogenic effects. Thus this work aimed 1) to clarify these thermogenic effects mediated by TRIAC vs. T(3) in vivo and 2) to determine whether they occurred predominantly in adipose tissues. To examine this, control rats were infused with equimolar T(3) or TRIAC doses (0.8 or 4 nmolx100 g body wt(-1) x day(-1)) or exposed for 48 h to cold. Both T(3) doses and only the highest TRIAC dose inhibited plasma and pituitary thyroid-stimulating hormone (TSH) and thyroxine (T(4)) in plasma and tissues. Interestingly, the lower TRIAC dose marginally inhibited plasma T(4). T(3) infusion increased plasma and tissue T(3) in a tissue-specific manner. The highest TRIAC dose increased TRIAC concentrations in plasma and tissues, decreasing plasma T(3). TRIAC concentrations in tissues were <10% those of T(3). Under cold exposure or high T(3) doses, TRIAC increased only in white adipose tissue (WAT). Remarkably, only the lower TRIAC dose activated thermogenesis, inducing ectopic uncoupling protein (UCP)-1 expression in WAT and maximal increases in UCP-1, UCP-2, and lipoprotein lipase (LPL) expression in brown adipose tissue (BAT), inhibiting UCP-2 in muscle and LPL in WAT. TRIAC, T(3), and cold exposure inhibited leptin secretion and mRNA in WAT. In summary, TRIAC, at low doses, induces thermogenic effects in adipose tissues without concomitant inhibition of TSH or hypothyroxinemia, suggesting a specific role regulating energy balance. This selective effect of TRIAC in adipose tissues might be considered a potential tool to increase energy metabolism.

Potent thermogenic action of triiodothyroacetic acid in brown adipocytes.

Triiodothyroacetic acid (TRIAC) is a triiodothyronine (T3) metabolite with high affinity for T3 nuclear receptors. We compared the thermogenic action of TRIAC versus T3 in brown adipocytes, by studying target genes known to mediate thermogenic action: uncoupling protein 1 (UCP-1), a marker of brown adipocytes, and type II-5'deiodinase (D2), which provides the T3 required for thermogenesis. TRIAC is 10-50 times more potent than T3 at increasing the adrenergic induction of UCP-1 mRNA and D2 activities. TRIAC action on UCP-1 is exerted at the transcriptional level. In the presence of an adrenergic stimulus, TRIAC is also more potent than T3, inducing lipoprotein lipase mRNA and 5 deiodinase (D3) activity and mRNA. Maximal effects occur at very low concentrations (0.2 nM). The greater potency of TRIAC is not due to preferential cellular or nuclear uptake. Therefore, TRIAC is a potent thermogenic agent that might increase energy expenditure and regulate T3 production in brown adipocytes.

Presence of sterile hyphae in moulds: relationship with inhibitory activity.

The relationship involving the presence of sterile hyphae in moulds and their inhibitory activity on 34 micro-organisms was investigated. From the results it was evident that antimicrobial activity varied when morphological changes were observed in the cultures.

Effect of ultraviolet light irradiation and nitrosoguanidine on viability of 46 strains of Arthrinium and their antibiotic production.

The ability of strains of the Arthrinium genus to inhibit microbial development has been previously described. In the present work different periods of mutagenic treatment using ultraviolet light, and of nitrosoguanidine treatment, on strains of Arthrinium were investigated. With nitrosoguanidine treatment the survival rate ranged from 2.17 to 8.78%. Mutant strains were only obtained with a higher antibiotic production in comparison with the wild-strain, when the mutagenic agent was UV light.

Inhibitory activity of the genus Chrysosporium in relation to the composition of the culture media used.

The effect of nutrients on the formation and accumulation of secondary metabolites in species of the genus Chrysosporium was examined. The study was performed according to the method of Campbell (1960), and among the results obtained was the fact that the only significant increase in antimicrobial capacity was observed when the strains were grown on a 2% malt extract agar and when micro-organisms were assayed using Aspergillus niger.

Virus-like particles in conidia and hypha of Arthrinium aureum.

This paper is the first report in the literature of the presence of virus-like particles in the conidia and hypha of Arthrinium aureum. These particles, detected with an electron microscope, are spherical and 52 nm in diameter.

Contribution to the study of Aspergillus fumigatus Fresenius.

50 Strains of Aspergillus fumigatus were studied for enzymatic and physiologic profile. Proteolytic and lipolytic activity were lacking. Various ribonucleases and sacchrolytic enzymes were found.