Impaired estrogen-induced negative feedback on gonadotropin secretion in patients with gonadotropin-secreting and nonfunctioning pituitary adenomas.

BACKGROUND: Several in vitro studies suggest that gonadotropin-secreting pituitary adenomas (Gn-omas) and non functioning pituitary adenomas (NFPA) originate from gonadotroph cells. Patients with Gn-oma and NFPA frequently show abnormal gonadotropin response to TRH. The aim of the study was to investigate whether the estrogen-induced negative feedback is operating in either patients with Gn-oma or NFPA. MATERIALS AND METHODS: Serum gonadotropin levels were evaluated at 24 h after ethinylestradiol administration (1 mg per os; EE2 test) in seven patients with a diagnosis of Gn-oma, based on the presence of high follicle-stimulating hormone (FSH) and/or lutenising hormone (LH) levels with normal or high levels of sex steroids, in 22 patients with NFPA with normal or low levels of gonadotropin and sex steroids, and 30 sex- and age-matched healthy subjects. A normal response to EE2 test was arbitrarily defined as a serum LH and FSH decrease of at least 40 and 30% below basal levels. RESULTS: Among patients with Gn-oma, only one had a normal FSH inhibition and another, a normal LH inhibition. Among the 22 patients with NFPA, the EE2 test caused a normal FSH or LH reduction in 10 and 15, respectively, while a normal reduction of both FSH and LH was observed in nine. CONCLUSIONS: The study demonstrates that estrogen-induced negative feedback of gonadotropin secretion is disrupted in almost all patients with Gn-oma and in half of those with NFPA. This defective feedback is reminiscent of the resistance to thyroid hormones and glucocorticoids observed in patients with thyroid-stimulating hormone- (TSH-) and adrenocorticotropic hormone- (ACTH-)secreting adenomas, respectively.

Multigenerational familial medullary thyroid cancer (FMTC): evidence for FMTC phenocopies and association with papillary thyroid cancer.

BACKGROUND: Occurrence in a familial setting is well established for medullary thyroid carcinoma (MTC) and has been more recently reported for papillary thyroid cancer (PTC). Germline mutations or rearrangements of the RET proto-oncogene are the genetic background of the majority of hereditary MTCs and of about 25-40% of PTCs. PATIENTS: A large multigenerational familial medullary thyroid cancer (FMTC) family, comprised of four generations and a total of 60 subjects, has been fully evaluated. Studies on germline RET mutations and polymorphisms, on somatic RET activation and on haplotyping with RET-linked markers, were performed. RESULTS: RET mutational analysis revealed a rare missense point mutation in exon 15 of RET (A891S), associated with FMTC. Haplotype analysis showed a co-segregation between the allelic variant 5 of D10S578 marker (which is tightly linked to the RET locus) and the RET mutation. Two patients, from different branches of the family, did not harbour the point mutation A891S despite histological confirmation of MTC. In these cases, haplotype analysis excluded the involvement of the RET gene itself in the pathogenesis of the MTC. In three patients, the coexistence, in different foci, of medullary and papillary thyroid cancer was documented. The genetic studies did not show ret/PTC rearrangements. The microsatellite analysis excluded co-segregation of RET locus with the MTC/PTC phenotype. CONCLUSIONS: We report a full clinical and molecular analysis of a large FMTC kindred with an uncommon RET mutation. In two family members, phenotype and genotype were not concordant, representing the first evidence of FMTC phenocopies. Furthermore, the association of familial forms of medullary and papillary thyroid cancers has been found in 30% of patients undergoing thyroidectomy for MTC. In these situations, genetic analyses excluded the possible germline involvement of RET. Though FMTC phenocopies are likely to represent an exceptional finding, such a possibility should be taken into account in the genetic counselling for MEN 2 syndromes.

Absence of thyroid transcription factor-1 expression in human parathyroid and pituitary glands.

Thyroid transcription factor-1 (TTF-1), a tissue-specific nuclear transcription factor involved in the embryogenesis and differentiation of human thyroid, lung and brain, has been recently identified in other rat tissues, including parafollicular C cells and parathyroid chief cells. Based on this distribution, a possible role for this factor in calcium homeostasis has been suggested. This study investigated the presence of TTF-1 transcripts and protein in human tissues expressing the calcium sensing receptor (CaSR). Using a RT-PCR technique, complemented by Southern blot analysis, TTF-1 expression was detected in human C cells (two medullary thyroid carcinomas), but not in normal and adenomatous (four adenomas and three hyperplasia) parathyroid, and normal and adenomatous (six adenomas) pituitary tissues. CaSR was expressed in all samples. The absence of expression was confirmed by Western blot. In contrast to previous studies in the rat, this study demonstrates the absence of TTF-1 transcripts in the human adult parathyroid and pituitary glands, although a role for this factor during the ontogeny of these organs cannot be excluded.

Mutations of LH and FSH receptors.

Gonadotropins control male and female gonadal function by acting through specific receptors. The recent description of several mutations in LH and FSH receptors has significantly improved our understanding of the pathophysiology of several sexual disorder. Both gain- and loss-of-function germline mutations leading to constitutive receptor activation or to hormone resistance have been described. The clinical impact of these mutant receptors can be markedly different, depending upon the sex of the affected patient and the degree of functional alteration. Numerous mutations were described in LH receptor gene. Constitutive activation of this receptor leads to male-limited precocious pseudopuberty, whereas hypergonadotropic hypogonadism is the clinical phenotype of LH resistance. On the other hand, few mutations of FSH receptor were described so far. Inactivating mutations of FSH receptor are involved in some cases of hypergonadotropic hypogonadism with a more severe impairment of fertility in female patients. Only one gain-of-function mutation of FSH receptor was reported to maintain fertility in one hypophysectomized man. This review is focused on the known genetic alterations of gonadotropic receptors in humans and their impact on male sexual differentiation and fertility.

Mutational analysis of GNAS1 in patients with pseudohypoparathyroidism: identification of two novel mutations.

Pseudohypoparathyroidism (PHP) refers to two major variants that generally coexist in the same family, PHP type Ia (PHP Ia), in which both PTH resistance and a constellation of physical features, termed Albright's hereditary osteodystrophy (AHO), are present, and pseudopseudohypoparathyroidism (PPHP), in which AHO occurs without PTH resistance. Most patients with PHP Ia show a partial deficiency (50%) of Gs activity, due to loss of function mutations in Gsalpha gene (GNAS1). The present study reports clinical, biochemical, and molecular data of 8 unrelated families with PHP Ia and PPHP. The 13 exons of GNAS1 were screened for mutations by PCR and direct sequencing of the amplified products. We detected heterozygous mutations in the affected members of the 4 families in which PHP Ia was present. In 2 families 2 previously reported deletions in exons 5 and 7 were found, whereas in the other 2 families, 2 novel frameshift deletions were identified in exons 1 and 11, causing a premature stop codon in the mutant allele. No mutation was detected in the families in which PPHP was the only clinical manifestation. In conclusion, we report the first mutational analysis of Italian patients with PHP Ia and PPHP, and we describe two novel deletions in GNAS1. Furthermore, we confirm that these mutations cannot be detected in families with isolated PPHP, suggesting that these forms of AHO are genetically distinct from PHP Ia.

Induction of specific phosphodiesterase isoforms by constitutive activation of the cAMP pathway in autonomous thyroid adenomas.

Thyrocytes largely depend on cAMP signaling for replication and differentiation. This pathway may be constitutively activated by mutations of the TSH receptor (TSHR) and Gsalpha in autonomous thyroid adenomas (ATAs). Because steady state cAMP results from production by adenylyl cyclase and degradation by phosphodiesterases (PDEs), we evaluated PDE activity and expression in ATAs with wild-type and mutant TSHR and Gsalpha. Activating mutations of TSHR and Gsalpha were identified in 7 and 1 of 18 ATAs, respectively. No difference was observed in the cAMP content in ATAs with or without activating mutants. In the surrounding normal thyroid tissue (NTs), PDE activity was 80% isobutylmethylxanthine sensitive, with the major contribution by PDE1 and a minor contribution by PDE4. No differences were observed in PDE activities between NTs and ATAs with wild-type TSHR and Gsalpha. In contrast, in the presence of mutant TSHRs or Gsalpha, total PDE activity was higher. This increase was primarily due to PDE4 induction (917 +/- 116% over NTs), associated with a minor PDE1 increase only in ATAs with mutant TSHR. By RT-PCR, increments of PDE4D and 4C messenger ribonucleic acids were found in the ATAs with mutant TSHR or Gsalpha, whereas messenger ribonucleic acids encoding other cAMP-specific PDEs were not significantly increased. This study provides a characterization of the PDEs expressed in human thyroid and demonstrates a dramatic PDE4 induction in the ATAs bearing mutant TSHR or Gsalpha genes. The increase in cAMP-degrading activity may represent a marker of constitutive adenylyl cyclase activation and constitutes an intracellular feedback mechanism with significant impact on the phenotypic expression of the activating mutations.

Effect of aging on serum gonadotropin levels in healthy subjects and patients with nonfunctioning pituitary adenomas.

OBJECTIVE: Nonfunctioning pituitary adenomas (NFPA), which represent about one-quarter of human pituitary tumors, occur in middle or old age. Determination of gonadotropin levels, which are not expected to be high during the early postmenopause in normal women and which are low in women with NFPA, is important to distinguishing hypogonadal status due to the normal decline of gonadal function from that due to hypothlalamic-pituitary dysfunction. The aim of the study was to verify whether this difference still persists in old subjects, despite the physiological decline of gonadotropins in the last decades of life. DESIGN AND METHODS: The study included 154 healthy subjects (aged 50-104 years) and 47 patients with NFPA (aged 50-80 years). Blood samples were collected after an overnight fast and hormone levels were measured by two immunofluorimetric assays. RESULTS: In healthy women the highest serum levels of gonadotropins were present in the 50-60 year age group, with a slight but progressive age-associated decrease in serum FSH and LH being observed thereafter. In healthy men serum gonadotropin levels were stable up to 70 years, increased up to 75-85 years and thereafter gradually decreasing. Female patients with NFPA showed levels of gonadotropins which were far lower than controls. Only three patients had levels of both FSH and LH above the 2.5 centile for normal subjects. A high sensitivity and specificity of gonadotropin measurements (about 90%) for the diagnosis of NFPA was observed in female patients aged 50-80 years. In male subjects, a large overlap of gonadotropin values in NFPA and controls, namely over the 50-70 years age range, was observed. CONCLUSIONS: Our study demonstrates that despite the gradual decline of gonadotropin levels in healthy postmenopsausal women, the reduction of both FSH and LH persists in old patients with NFPA, suggesting that measurement of gonadotropin levels could prove useful in the evaluation of pituitary lesions even in old women. More subtle differences seem to occur in male subjects.

Blood pressure measurement in haemodialysis patients.

Several studies suggest that the 24 hour ambulatory blood pressure monitoring (ABPM) predicts left ventricular hypertrophy more accurately than conventional blood pressure measurement (CBPM) with mercury sphygmomanometer. We estimated the left ventricular mass by M-mode echocardiography in 58 patients on regular haemodialysis treatment during the midweek haemodialysis (HD) interval. ABPM was recorded during the 24 hours preceding the dialysis session and the average of values were compared with the average of the 13 pre HD CBPM recorded by nurses during the month preceding the echocardiography study. The two types of BP measurements correlated significantly with each other, (systolic BP r = 0.62; p < 0.001 and diastolic BP r = 0.74; p < 0.001). The correlation of left ventricular mass with pre-HD systolic BP was stronger (r = 0.54; p < 0.001) than with 24h-systolic BP (r = 0.33; p < 0.01). The overall accuracy of prediction was also similar (68% for pre HD-CBPM; 67% for 24h-ABPM). Measurements of diastolic BP did not correlate significantly with LVM. Our data suggest that 24h-ABPM does not offer any advantage over pre HD-CBPM in predicting left ventricular hypertrophy in HD patients.

Expression of calcium-sensing receptor and characterization of intracellular signaling in human pituitary adenomas.

Extracellular Ca(2+)-sensing receptor (CaSR) has been recently identified in rat and mouse pituitary and in AtT-20 cells. The aim of the study was to investigate the presence of CaSR in the human pituitary and its signaling pathway. Normal parathyroid biopsies, autoptic normal pituitaries, and seven nonfunctioning and six GH-secreting adenomas were studied. Southern blot analysis of the RT-PCR products from pituitary adenomas indicated that the PCR fragments obtained were products of specific amplification of CaSR messenger ribonucleic acid. Sequence analysis showed nucleotide identity of these products with the available human parathyroid CaSR. By immunoblotting analysis CaSR, was detected in normal and adenomatous pituitary tissues. In all tumors studied, extracellular Ca2+ (2.5 mmol/L) induced a significant increase in intracellular Ca2+, mainly due to Ca2+ mobilization (from 82.7+/-11 to 148+/-36 nmol/L; P < 0.001). Similar results were obtained with the CaSR activators gadolinium and neomycin. Moreover, CaSR activators significantly increased cAMP levels; this effect was not mimicked by other agents able to increase intracellular Ca2+, such as TRH. CaSR agonists did not increase resting GH secretion in any GH-secreting adenomas, but amplified the GH response to GHRH. In this study we first demonstrate CaSR expression in the human pituitary and provides evidence for an additional mechanism by which calcium might regulate pituitary cell function.

Prenatal diagnosis of thyroid hormone resistance.

A 29-yr-old woman with pituitary resistance to thyroid hormones (PRTH) was found to harbor a novel point mutation (T337A) on exon 9 of the thyroid hormone receptor beta (TRbeta) gene. She presented with symptoms and signs of hyperthyroidism and was successfully treated with 3,5,3'-triiodothyroacetic acid (TRIAC) until the onset of pregnancy. This therapy was then discontinued in order to prevent TRIAC, a compound that crosses the placental barrier, from exerting adverse effects on normal fetal development. However, as the patient showed a recurrence of thyrotoxic features after TRIAC withdrawal, we sought to verify, by means of genetic analysis and hormone measurements, whether the fetus was also affected by RTH, in order to rapidly reinstitute TRIAC therapy, which could potentially be beneficial to both the mother and fetus. At 17 weeks gestation, fetal DNA was extracted from chorionic villi and was used as a template for PCR and restriction analysis together with direct sequencing of the TRbeta gene. The results indicated that the fetus was also heterozygous for the T337A mutation. Accordingly, TRIAC treatment at a dose of 2.1 mg/day was restarted at 20 weeks gestation. The mother rapidly became euthyroid, and the fetus grew normally up to 24 weeks gestation. At 29 weeks gestation mild growth retardation and fetal goiter were observed, prompting cordocentesis. Circulating fetal TSH was very high (287 mU/L) with a markedly reduced TSH bioactivity (B/I: 1.1 +/- 0.4 vs 12.7 +/- 1.2), while fetal FT4 concentrations were normal (8.7 pmol/L; normal values in age-matched fetuses: 5-22 pmol/L). Fetal FT3 levels were raised (7.1 pmol/L; normal values in age-matched fetuses: <4 pmol/L), as a consequence of 100% cross-reactivity of TRIAC in the FT3 assay method. To reduce the extremely high circulating TSH levels and fetal goiter, the dose of TRIAC was increased to 3.5 mg/day. To monitor the possible intrauterine hypothyroidism, another cordocentesis was performed at 33 weeks gestation, showing that TSH levels were reduced by 50% (from 287 to 144 mU/L). Furthermore, a simultaneous ultrasound examination revealed a clear reduction in fetal goiter. After this latter cordocentesis, acute complications occured, prompting delivery by cesarean section. The female neonate was critically ill, with multiple-organ failure and respiratory distress syndrome. In addition, a small goiter and biochemical features ofhypothyroidism were noted transiently and probably related to the prematurity of the infant. At present, the baby is clinically euthyroid, without goiter, and only exhibits biochemical features of RTH. In summary, although further fetal studies in cases of RTH are necessary to determine whether elevated TSH levels with a markedly reduced bioactivity are a common finding, our data suggest transient biochemical hypothyroidism in RTH during fetal development. Furthermore, we advocate prenatal diagnosis of RTH and adequate treatment of the disease in case of maternal hyperthyroidism, to avoid fetal thyrotrope hyperplasia, reduce fetal goiter, and maintain maternal euthyroidism during pregnancy.

[Activating mutations of the gonadotrophin receptors]. / Mutations activantes des récepteurs des gonadotrophines.

The different types of activating mutations of LH and FSH receptors genes are described. They result in a constitutive permanent activation of the LH or FSH function responsible for functional disorders which is also observed in some ovarian tumours. Two types of functional disorders have been reported: male precocious puberty through activating mutation of the LH receptor, male fertility in the absence of FSH through activating mutation of the FSH receptor. Activating mutations of the FSH receptor observed in certain ovarian tumours result in hypersecretion of oestrogens.

Serum FSH bioactivity and inhibin levels in patients with gonadotropin secreting and nonfunctioning pituitary adenomas.

It has been reported that serum FSH bioactivity and inhibin levels can be used as markers of the presence of true gonadotropin-secreting pituitary adenoma (Gn-oma). To verify this hypothesis, we have investigated the bioactivity of FSH and serum inhibin alpha-alpha and alpha-beta A levels in a series of patients with either Gn-oma or nonfunctioning pituitary adenoma (NFPA). Nine patients with Gn-oma (6 men and 3 women), 21 with NFPA (9 men and 12 women) and 30 normal subjects were included in the study. We studied FSH biological activity (FSH-B) by using Sertoli cell aromatase bioassay (SAB) and alpha-alpha and alpha-beta A inhibin levels by two noncompetitive immunometric assays (IEMA). In male patients with Gn-oma, serum immunoreactive FSH (FSH-I) and FSH-B levels ranged from 5.1 to 35.5 U/L and from 8.3 to 48 U/L, respectively, FSH B/I ratio being elevated in 2 (2.5 and 4.1; normal male range: 0.3-1.5), while female patients with Gn-oma had serum FSH-I and FSH-B levels ranging from 43.2 to 162 U/L and from 41.2 to 112.8 U/l, respectively, with a normal FSH B/I ratio. In male patients with NFPA, FSH-I and FSH-B levels ranged from 2.7 to 10.7 U/l and from 2.4 to 11.4 U/l while in females they ranged from 3.4 to 67.9 and from 4.6 to 60.8 U/l, respectively. FSH B/I ratio was elevated in 1 male (3.3) and normal in the remaining patients with NFPA. Serum alpha-alpha inhibin levels were normal or low in patients with Gn-oma and NFPA, while alpha-beta A inhibin concentrations were slightly elevated in 1 of 6 postmenopausal women (0.9; normal range < 0.7 U/ml). The present study confirms and extends previous reports indicating that male patients with Gn-oma may secrete FSH molecules with increased bioactivity. However, this abnormality was also observed in one male patient with NFPA. Moreover, the measurement of inhibin levels does not appear to be a reliable in vivo marker of pituitary tumors of gonadotroph origin, as it was normal or low in almost all patients with either Gn-oma or NFPA.

Changes in the degree of sialylation of carbohydrate chains modify the biological properties of circulating thyrotropin isoforms in various physiological and pathological states.

Variation in asparagine-linked carbohydrate chains have a major impact on TSH biological properties. In particular, highly sialylated TSH is characterized by impaired intrinsic bioactivity and prolonged half-life. The aim of the present study was to investigate the changes in the degree of sialylation of circulating TSH isoforms that may occur in several physiological and clinical situations. Bioactivity and terminal sugar residues of immunopurified TSH were studied in 26 normal adults (day- and nighttime serum pools), 2 cord serum pools from normal fetuses during the third trimester, 1 fetus with primary hypothyroidism (PH; 27th week), 1 fetus with resistance to thyroid hormone (RTH; 28th and 33rd weeks), 24 patients with PH (before and during L-T4 treatment), and 5 patients with RTH before and during triiodothyrocetic acid (TRIAC) treatment. Nighttime TSH isoforms have an increased degree of sialylation compared to daytime TSH (35.8 +/- 9.7% vs. 23.8 +/- 5.8%; P < 0.03), thus accounting for the lower bioactivity [biological/immunological TSH ratio (TSH B/I), 1.3 +/- 0.4 vs. 2.0 +/- 0.2; P < 0.0007]. In adult PH, TSH isoforms are highly sialylated (45.4 +/- 7.6%; P < 0.007), showing an impaired bioactivity (0.7 +/- 0.3; P < 0.001). L-T4 therapy was accompanied by a trend toward normalization of TSH biological properties; TSH B/I was higher (1.0 +/- 0.3; P < 0.01), and the degree of sialylation was lower (36.8 +/- 7.0%; P < 0.02). A significant inverse correlation between TSH B/I values and the degree of sialylation was observed (P < 0.001). In normal fetuses, extremely bioactive asialo-TSH isoforms are circulating during the 3rd trimester. The impaired thyroid hormone action, such as that occurring in hypothyroid or RTH fetuses, induces an early expression of alpha-2,6-sialyltransferase activity within thyrotropes and results in the secretion of high amounts of sialylated TSH isoforms (34.6% and 26.3%). A hybrid TSH with peculiar terminal sugar residues and enhanced bioactivity is circulating in patients with RTH (TSH B/I, > or = 2.2). Treatment with low doses of TRIAC can initially reduce thyroid hormone secretion in RTH, mainly through the secretion of TSH isoforms with changed terminal sugar residues and reduced bioactivity (TSH B/I, 0.9-1.7). In conclusion, changes in the terminal sialic acid residues modulate the biological properties of circulating TSH, play a relevant physiopathological role in various situations, and contribute to adjust thyroid-stimulating activity to temporary needs.

Thyrotropin-secreting pituitary tumors in hyper- and hypothyroidism.

Thyrotropin (TSH)-secreting pituitary tumors may be found in two opposite clinical situations: the hyperthyroidism secondary to thyrotroph adenomas, also called central hyperthyroidism, and the long-standing primary hypothyroidism which can be accompanied by a compensatory pituitary enlargement. TSH-secreting pituitary adenomas belong to the syndromes of "inappropriate secretion of TSH" (IST). The adjective "inappropriate" indicates the lack of the expected suppression of TSH secretion when free thyroid hormone levels are actually elevated, as in the other forms of thyrotoxicosis. Moreover, TSH-omas have to be differentiated from the non-neoplastic form of IST which is due to resistance to thyroid hormone. Differently, pituitary hyperplasia, which is reversible on thyroid hormone replacement, is the more frequent cause of a pituitary mass occurring in the context of untreated primary hypothyroidism. Failure or delay in the recognition of the above clinical situations may cause dramatic consequences, such as unnecessary pituitary surgery in hypothyroid patients or improper thyroid ablation in those with central hyperthyroidism. In contrast, early diagnosis and proper treatment of TSH-secreting pituitary tumors prevents the appearance of signs and symptoms of mechanical compression of the adjacent structures by the expanding tumor mass (visual field defects, headache and hypopituitarism).