The cyto-protective effects of LH on ovarian reserve and female fertility during exposure to gonadotoxic alkylating agents in an adult mouse model.

STUDY QUESTION: Does LH protect mouse oocytes and female fertility from alkylating chemotherapy? SUMMARY ANSWER: LH treatment before and during chemotherapy prevents detrimental effects on follicles and reproductive lifespan. WHAT IS KNOWN ALREADY: Chemotherapies can damage the ovary, resulting in premature ovarian failure and reduced fertility in cancer survivors. LH was recently suggested to protect prepubertal mouse follicles from chemotoxic effects of cisplatin treatment. STUDY DESIGN, SIZE, DURATION: This experimental study investigated LH effects on primordial follicles exposed to chemotherapy. Seven-week-old CD-1 female mice were randomly allocated to four experimental groups: Control (n = 13), chemotherapy (ChT, n = 15), ChT+LH-1x (n = 15), and ChT+LH-5x (n = 8). To induce primary ovarian insufficiency (POI), animals in the ChT and ChT+LH groups were intraperitoneally injected with 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan, while control mice received vehicle. For LH treatment, the ChT+LH-1x and ChT+LH-5x animals received a 1 or 5 IU LH dose, respectively, before chemotherapy, then a second LH injection administered with chemotherapy 24 h later. Then, two animals/group were euthanized at 12 and 24 h to investigate the early ovarian response to LH, while remaining mice were housed for 30 days to evaluate short- and long-term reproductive outcomes. The effects of LH and chemotherapy on growing-stage follicles were analyzed in a parallel experiment. Seven-week-old NOD-SCID female mice were allocated to control (n = 5), ChT (n = 5), and ChT+LH-1x (n = 6) groups. Animals were treated as described above, but maintained for 7 days before reproductive assessment. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the first experiment, follicular damage (phosphorylated H2AX histone (γH2AX) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay), apoptotic biomarkers (western blot), and DNA repair pathways (western blot and RT-qPCR) were assessed in ovaries collected at 12 and 24 h to determine early ovarian responses to LH. Thirty days after treatments, remaining mice were stimulated (10 IU of pregnant mare serum gonadotropin (PMSG) and 10 IU of hCG) and mated to collect ovaries, oocytes, and embryos. Histological analysis was performed on ovarian samples to investigate follicular populations and stromal status, and meiotic spindle and chromosome alignment was measured in oocytes by confocal microscopy. Long-term effects were monitored by assessing pregnancy rate and litter size during six consecutive breeding attempts. In the second experiment, mice were stimulated and mated 7 days after treatments and ovaries, oocytes, and embryos were collected. Follicular numbers, follicular protection (DNA damage and apoptosis by H2AX staining and TUNEL assay, respectively), and ovarian stroma were assessed. Oocyte quality was determined by confocal analysis. MAIN RESULTS AND THE ROLE OF CHANCE: LH treatment was sufficient to preserve ovarian reserve and follicular development, avoid atresia, and restore ovulation and meiotic spindle configuration in mature oocytes exposed at the primordial stage. LH improved the cumulative pregnancy rate and litter size in six consecutive breeding rounds, confirming the potential of LH treatment to preserve fertility. This protective effect appeared to be mediated by an enhanced early DNA repair response, via homologous recombination, and generation of anti-apoptotic signals in the ovary a few hours after injury with chemotherapy. This response ameliorated the chemotherapy-induced increase in DNA-damaged oocytes and apoptotic granulosa cells. LH treatment also protected growing follicles from chemotherapy. LH reversed the chemotherapy-induced depletion of primordial and primary follicular subpopulations, reduced oocyte DNA damage and granulosa cell apoptosis, restored mature oocyte cohort size, and improved meiotic spindle properties. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was a preliminary study performed with mouse ovarian samples. Therefore, preclinical research with human samples is required for validation. WIDER IMPLICATIONS OF THE FINDINGS: The current study tested if LH could protect the adult mouse ovarian reserve and reproductive lifespan from alkylating chemotherapy. These findings highlight the therapeutic potential of LH as a complementary non-surgical strategy for preserving fertility in female cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Regional Valencian Ministry of Education (PROMETEO/2018/137), the Spanish Ministry of Science and Innovation (CP19/00141), and the Spanish Ministry of Education, Culture and Sports (FPU16/05264). The authors declare no conflict of interest.

High progesterone levels during the luteal phase related to the use of an aromatase inhibitor in breast cancer patients.

OBJECTIVE: To evaluate the hormonal profile in three breast cancer patients who underwent controlled ovarian stimulation in the presence of the aromatase inhibitor letrozole. PATIENTS AND METHODS: In IVF University referral center, a case series of three breast cancer patients who underwent controlled ovarian stimulation (COS) with recombinant FSH and letrozole were investigated. Ovulation was induced with hCG (case No. 1) or with GnRH agonist (case No. 2-3). The primary outcome of our study was the detection of progesterone levels in the luteal phase. RESULTS: Very high progesterone values (mean 186.6 ± 43.6 ng/mL) during the luteal phase were recorded in all three cases. CONCLUSIONS: High progesterone levels can be related to the use of letrozole independently of the most commonly used trigger regimen. Although progesterone has long been considered a protective factor against breast cancer, several studies have demonstrated that progesterone could expand a transformation-sensitive stem cell population in the mammary glands. The estrogen negative feedback effect on the hypothalamus-pituitary axis and the disruption of steroid biosynthesis and could represent an intriguing reason behind this phenomenon. Our results highlight the need to evaluate further the increase in progesterone levels in the luteal phase in women with breast cancer undergoing COS with letrozole.

Weekly docetaxel versus CMF as adjuvant chemotherapy for older women with early breast cancer: final results of the randomized phase III ELDA trial.

BACKGROUND: Evidence on adjuvant chemotherapy in older women with breast cancer is poor. We tested whether weekly docetaxel is more effective than standard chemotherapy. PATIENTS AND METHODS: We carried out a multicenter, randomized phase III study. Women aged 65-79, operated for breast cancer, with average to high risk of recurrence, were allocated 1 : 1 to CMF (cyclophosphamide 600 mg/m², methotrexate 40 mg/m², fluorouracil 600 mg/m², days 1, 8) or docetaxel (35 mg/m(2) days 1, 8, 15) every 4 weeks, for four or six cycles according to hormone receptor status. Primary end point was disease-free survival (DFS). A geriatric assessment was carried out. Quality of life (QoL) was assessed with EORTC C-30 and BR-23 questionnaires. RESULTS: From July 2003 to April 2011, 302 patients were randomized and 299 (152 allocated CMF and 147 docetaxel) were eligible. After 70-month median follow-up, 109 DFS events were observed. Unadjusted hazard ratio (HR) of DFS for docetaxel versus CMF was 1.21 [95% confidence interval (CI) 0.83-1.76, P = 0.32]; DFS estimate at 5 years was 0.69 with CMF and 0.65 with docetaxel. HR of death was 1.34 (95% CI 0.80-2.22, P = 0.26). There was no interaction between treatment arms and geriatric scales measuring patients' ability or comorbidities. Hematological toxicity, mucositis and nausea were worse with CMF; allergy, fatigue, hair loss, onychopathy, dysgeusia, diarrhea, abdominal pain, neuropathy, cardiac and skin toxicity were worse with docetaxel. One death was attributed to CMF and two to docetaxel. Increasing age, impairment in instrumental daily living activities, number of comorbidities and docetaxel treatment were independently associated with severe nonhematological toxicity. QoL was worse with docetaxel for nausea-vomiting, appetite loss, diarrhea, body image, future perspective, treatment side-effects and hair loss items. CONCLUSIONS: Weekly docetaxel is not more effective than standard CMF as adjuvant treatment of older women with breast cancer and worsens QoL and toxicity. CLINICALTRIALSGOV: NCT00331097.

Bone effect of adjuvant tamoxifen, letrozole or letrozole plus zoledronic acid in early-stage breast cancer: the randomized phase 3 HOBOE study.

BACKGROUND: To measure bone mineral density (BMD) reduction produced by letrozole as compared with tamoxifen and the benefit of the addition of zoledronic acid. PATIENTS AND METHODS: A phase 3 trial comparing tamoxifen, letrozole or letrozole+zoledronic acid in patients with hormone receptor-positive early breast cancer was conducted; triptorelin was given to premenopausal patients. Two comparisons were planned: letrozole versus tamoxifen and letrozole+zoledronic acid versus letrozole. Primary end point was the difference in 1-year change of T-score at lumbar spine (LTS) measured by dual energy X-ray absorptiometry scan. RESULTS: Out of 483 patients enrolled, 459 were available for primary analyses. Median age was 50 (range 28-80). The estimated mean difference (95% confidence interval [CI]) in 1-year change of LTS was equal to -0.30 (95% CI -0.44 to -0.17) in the letrozole versus tamoxifen comparison (P<0.0001) and to +0.60 (95% CI +0.46 to +0.77) in the letrozole+zoledronic acid versus letrozole comparison (P<0.0001). Bone damage by letrozole decreased with increasing baseline body mass index in premenopausal, but not postmenopausal, patients (interaction test P=0.004 and 0.47, respectively). CONCLUSIONS: In the HOBOE (HOrmonal BOne Effects) trial, the positive effect of zoledronic acid on BMD largely counteracts damage produced by letrozole as compared with tamoxifen. Letrozole effect is lower among overweight/obese premenopausal patients.

Adenoviral gene transfer of Akt enhances myocardial contractility and intracellular calcium handling.

The serine-threonine kinase Akt/PKB mediates stimuli from different classes of cardiomyocyte receptors, including the growth hormone/insulin like growth factor and the beta-adrenergic receptors. Whereas the growth-promoting and antiapoptotic properties of Akt activation are well established, little is known about the effects of Akt on myocardial contractility, intracellular calcium (Ca(2+)) handling, oxygen consumption, and beta-adrenergic pathway. To this aim, Sprague-Dawley rats were subjected to a wild-type Akt in vivo adenoviral gene transfer using a catheter-based technique combined with aortopulmonary crossclamping. Left ventricular (LV) contractility and intracellular Ca(2+) handling were evaluated in an isolated isovolumic buffer-perfused, aequorin-loaded whole heart preparations 10 days after the surgery. The Ca(2+)-force relationship was obtained under steady-state conditions in tetanized muscles. No significant hypertrophy was detected in adenovirus with wild-type Akt (Ad.Akt) versus controls rats (LV-to-body weight ratio 2.6+/-0.2 versus 2.7+/-0.1 mg/g, controls versus Ad.Akt, P, NS). LV contractility, measured as developed pressure, increased by 41% in Ad.Akt. This was accounted for by both more systolic Ca(2+) available to the contractile machinery (+19% versus controls) and by enhanced myofilament Ca(2+) responsiveness, documented by an increased maximal Ca(2+)-activated pressure (+19% versus controls) and a shift to the left of the Ca(2+)-force relationship. Such increased contractility was paralleled by a slight increase of myocardial oxygen consumption (14%), while titrated dose of dobutamine providing similar inotropic effect augmented oxygen consumption by 39% (P<0.01). Phospholamban, calsequestrin, and ryanodine receptor LV mRNA and protein content were not different among the study groups, while sarcoplasmic reticulum Ca(2+) ATPase protein levels were significantly increased in Ad.Akt rats. beta-Adrenergic receptor density, affinity, kinase-1 levels, and adenylyl cyclase activity were similar in the three animal groups. In conclusion, our results support an important role for Akt/PKB in the regulation of myocardial contractility and mechanoenergetics.

[Myocardial metabolism of calcium in heart failure: from physiology to new therapeutic perspectives]. / Metabolismo miocardico del calcio nell'insufficienza cardiaca: dalla fisiologia ai nuovi orizzonti terapeutici.

Development of heart failure is associated with an impairment of intracellular calcium handling. The precise mechanisms involved are still obscure. When membrane depolarization occurs, a small amount of extracellular calcium enters the intracellular milieu through the L-type channels. Such "trigger" calcium acts on specific receptors of the sarcoplasmic reticulum, that, in turn, according to the so-called calcium entry-calcium release mechanism, allows the release of a larger amount of calcium from the sarcoplasmic reticulum. Removal of calcium from the cytosol is the key event of the diastolic phase. Calcium removal from cytosol occurs through specific membrane pumps. Recent therapeutic approaches involving gene targeting of calcium pumps have yielded promising results. Specifically, increased levels of SERCA 2 in the myocardium have shown to enhance cardiac contractility under normal circumstances and in experimental heart failure. Future research is needed to confirm these findings in human heart failure.

Effects of two years of growth hormone (GH) replacement therapy on bone metabolism and mineral density in childhood and adulthood onset GH deficient patients.

The aim of the current study was to evaluate bone metabolism and mass before and after 2 years of GH replacement therapy in adults with childhood or adulthood onset GH deficiency. Thirty-six adults with GH deficiency, 18 with childhood onset, 18 with adulthood onset GH deficiency and 28 sex-, age-, height- and weight-matched healthy subjects entered the study. Biochemical indexes of bone turnover such as serum osteocalcin, serum carboxyterminal telopeptide of type-I procollagen, urinary hydroxyproline/creatinine and deoxypyridinoline/creatinine, of soft tissue formation such as aminoterminal propeptide of type-III and bone mineral density were evaluated. Childhood onset GH deficient patients had significantly decreased bone (osteocalcin: 2.5+/-1.3 vs 6.6+/-4.8 mcg/l, p<0.001) and soft tissue formation (aminoterminal propeptide of type III: 273+/-49 vs 454+/-23 U/I, p<0.001) indexes and normal bone resorption indexes (serum carboxyterminal telopeptide of type-I procollagen: 105+/-48 vs 128+/-28 mcg/l p=NS; urinary hydroxyproline/creatinine: 0.19+/-0.16 vs 0.28+/-0.16 mmol/mol, p=NS; urinary deoxypyridinoline/creatinine: 21 +/-10 vs 25+/-8 mcmol/mol, p=NS) compared to healthy subjects. On the contrary, no significant difference in bone turnover indexes between adulthood onset GH deficient patients and healthy subjects was found. Moreover, significantly decreased bone mineral density at any skeletal site and at whole skeleton was found in GH deficient patients compared to healthy subjects (e.g. femoral neck: 0.74+/-0.13 vs 0.97+/-0.11 g/cm2, p<0.001). In addition, a significant reduction of bone mineral density was found in childhood compared to adulthood onset GH deficient patients at any skeletal site, except at femoral neck. After 3-6 months of treatment, both groups of patients had a significant increase in bone turnover and in soft tissue formation. In particular, in childhood onset GH deficient patients after 3 months osteocalcin increased from 2.5+/-1.3 to 7.9+/-2.1 mcg/l, p<0.001 aminoterminal propeptide of type-III from 273+/-49 to 359+/-15 U/I p<0.001; serum carboxyterminal telopeptide of type-I procollagen from 105+/-48 to 201+/-45 mcg/l, p<0.001; urinary hydroxyproline/creatinine from 0.19+/-0.16 to 0.81+/-0.17 mmol/mol, p<0.001; urinary deoxypyridinoline/creatinine from 21 +/-10 to 54+/-20 mcmol/mol, p<0.001; while in adulthood onset GH deficient patients after 6 months osteocalcin increased from 4.2+/-3.6 to 6.5+/-1.9 mcg/l, p<0.05; aminoterminal propeptide of type- III from 440+/-41 to 484+/-37 U/I, p<0.05; serum carboxyterminal telopeptide of type-I procollagen from 125+/-40 to 152+/-22 mcg/l, p<0.05; urinary hydroxyproline/creatinine from 0.24+/-0.12 to 0.54+/-0.06 mmol/mol, p<0.001; urinary deoxypyridinoline/creatinine from 23+/-8 to 42+/-5 mcmol/mol, p<0.001. No significant difference in bone turnover between pre- and post-treatment period was found after 18-24 months of GH therapy. Conversely, bone mineral density was slightly reduced after 3-6 months of GH therapy, while it was significantly increased after 18-24 months. In fact, femoral neck bone mineral density values significantly rose from 0.74+/-0.13 g/cm2 to 0.87+/-0.11 g/cm2 (pre-treatment vs 2 years of GH treatment values). In conclusion, patients with childhood or adulthood onset GH deficiency have osteopenia that can be improved by long-term treatment with GH.

Bone mineral density and circulating cytokines in patients with acromegaly.

Acromegalic patients present an increase of osteoblastic and osteoclastic activity, showing a different effect on the axial and appendicular skeletal structures. At this regard controversial data about bone mineral density (BMD) have been published in literature. In fact an increase of BMD levels in femoral neck and Ward's triangle without any difference in lumbar spine has been described. On the other hand normal BMD levels at forearm and reduced BMD levels at lumbar spine were found. These patients seem to have a reduction of trabecular BMD similar to postmenopausal osteoporotic patients despite normal or slightly elevated cortical BMD. Recently, it has been described that cytokines, in particular tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), are implicated in the pathogenetic mechanism of postmenopausal osteoporosis. Taking into account that growth hormone (GH) can increase TNF-alpha and IL-1 secretion by mononuclear blood cells, the evaluation of possible relationship between the reduced BMD at lumbar spine and circulating cytokines levels was carried out in acromegalic patients. In addition we evaluated the effect of acute octreotide administration on serum TNF-alpha and IL-I concentrations. Eleven patients with active acromegaly and eleven healthy age-, sex-, weight- and heightmatched subjects were enrolled in this study. BMD was significantly reduced at lumbar spine (0.80 +/- 0.29 g/cm2 vs 1.02 +/- 0.11 g/cm2; p < 0.01), but not at femoral neck level or at Ward's triangle level (0.92 + 0.15 g/cm2 vs 0.97 + 0.11 g/cm2, p = NS; and 0.74 +/- 0.16 g/cm2 vs 0.85 +/- 0.1 g/cm2, p = NS) when compared to controls. Baseline serum levels of TNF-alpha and IL-1 were in the normal range both in patients and controls. After acute octreotide administration, no differences in circulating TNF-alpha and IL-1 levels were found. In conclusion, acromegalic patients present a reduced BMD at lumbar spine but not at femoral neck level and Ward's triangle. Circulating cytokines such as TNF-alpha and IL-1 are in the normal range. These data suggest that cytokines are not involved in the pathogenesis of GH-excess induced osteoporosis. The possibility that the GH excess might affect bone turnover inducing an increase of cytokines acting by a paracrine/autocrine mechanism cannot be ruled out.

Left ventricular function in young adults with childhood and adulthood onset growth hormone deficiency.

OBJECTIVE: The impairment of heart structure and function in adults with childhood onset GH deficiency has been recently described. However, previous echocardiographic studies have reported no differences in cardiac mass and function between adulthood onset GH deficient patients and healthy subjects. DESIGN: The aim of this study was to evaluate cardiac performance in adult patients with childhood and adulthood onset GH deficiency, using equilibrium radionuclide angiography, a method more accurate than echocardiography. PATIENTS: Eleven patients with childhood onset GH deficiency, 9 patients with adulthood onset GH deficiency and 12 age-, gender-, height- and weight-matched healthy subjects entered the study. MEASUREMENTS: All the study population underwent equilibrium radionuclide angiography at rest and during physical exercise. RESULTS: Both childhood and adulthood onset GH deficient patients had an impaired left ventricular systolic performance both at rest (ejection fraction was 55 +/- 6%, 55 +/- 10% and 66 +/- 6% in childhood and adulthood onset GH deficient patients and control group, respectively; P < 0.0001) and during physical exercise (ejection fraction was 54 +/- 9% in childhood onset GH deficient patients, 53 +/- 9% in adulthood onset GH deficient patients and 76 +/- 7% in normal subjects; P < 0.0001). Peak ejection rate was 3.2 +/- 0.8 end-diastolic volume/second, 3.0 +/- 0.6 end-diastolic volume/second and 3.9 +/- 0.8 end-diastolic volume/ second in childhood and adulthood onset GH deficient patients and control group, respectively (P < 0.01). Exercise-induced changes in end-systolic volume were increased in both groups of patients compared with healthy subjects. In contrast, exercise-induced end-diastolic volume changes were not different between GH deficient patients and controls. Resting peak filling rate was 2.6 +/- 0.7 end-diastolic volume/second, 2.5 +/- 0.7 end-diastolic volume/ second and 3.1 +/- 0.3 end-diastolic volume/second in the 2 groups of patients and healthy subjects, respectively (P < 0.05). Reduced exercise tolerance in all patients, as shown by the significantly lower values of peak workload (P < 0.0001), peak rate-pressure product (P < 0.01) and exercise duration (P < 0.0001) was observed. CONCLUSION: Patients affected by GH deficiency have left ventricular systolic dysfunction at rest and during physical exercise, suggesting that GH plays a physiological role in maintaining normal cardiac performance in humans. Furthermore, no difference between childhood and adulthood onset GH deficient patients was found indicating that both group of patients have an impairment of cardiac function.

Increased arterial intima-media thickness in childhood-onset growth hormone deficiency.

Very little is known about the atherosclerotic risk in patients with childhood-onset growth hormone deficiency (GHD). Such data may be relevant to reconstructing the natural course of the cardiovascular abnormalities associated with GHD. To this end, the intima-media thickness (IMT) of the carotid arteries and the vascular risk factors were evaluated in 14 childhood-onset GHD patients (age 25 +/- 1 yr, BMI 22 +/- 0.6 Kg/m2) and in 14 age-, sex-, and BMI-matched control subjects. IMT was greater in GHD patients (0.83 +/- 0.06 and 0.81 +/- 0.06 mmol/L for the right and left carotid artery) than in controls (0.64 +/- 0.03 and 0.64 +/- 0.04 mmol/L, P < 0.01 and P < 0.02, respectively). Serum total and lipoprotein cholesterol, and serum total triglycerides did not differ between the two groups. However, a significant increase in low density lipid triglycerides was present in GHD patients (0.27 +/- 0.02 mmol/L) compared with controls (0.19 +/- 0.01; P = 0.007). No difference was found in plasma fibrinogen and serum Lp(a) levels. Plasma glucose and insulin concentrations were similar in GHD and control subjects both in the fasted state and after an oral glucose load. In conclusion, young patients with childhood-onset GHD show an increased IMT in the absence of clear-cut abnormalities of the classic vascular risk factors. This suggests a role for GH deficiency per se in increasing the atherosclerotic risk.

Lung volumes and respiratory muscle strength in adult patients with childhood- or adult-onset growth hormone deficiency: effect of 12 months' growth hormone replacement therapy.

We have described impairment of the respiratory function in adult patients with childhood-onset growth hormone (GH) deficiency. The aim of the present study was to evaluate lung volumes and respiratory muscle strength in patients diagnosed as GH deficient before and after 6 and 12 months of recombinant GH treatment. Ten adults diagnosed as GH deficient in childhood, ten adults diagnosed as GH deficient in adulthood and ten healthy subjects entered the study. For each subject, evaluation of respiratory function followed the same standard approach, consisting of respiratory muscle strength assessment, record of flow-volume curves, measurement of static lung volumes and lung diffusing capacity. Childhood-onset GH-deficient patients had a significant reduction of maximal inspiratory (p < 0.01) and maximal expiratory (p < 0.05) mouth pressures. Total lung capacity, vital capacity and functional residual capacity were significantly reduced compared to healthy subjects (p < 0.05). Conversely, residual volume and diffusing lung capacity did not show any significant change. No significant change of the ratio between the percentage forced expiratory volume in 1 s and the forced vital capacity was observed. The decrease of respiratory mouth pressures was not correlated to the decrease of lung volumes. Adult-onset GH-deficient patients had only a significant reduction of maximal expiratory pressure compared to healthy subjects (p < 0.05). After 6 months of treatment no significant differences in any of the evaluated parameters were found. After 12 months of treatment patients with childhood-onset GH deficiency show a significant improvement of lung volumes (p < 0.01) and maximal respiratory mouth pressures (p < 0.005), whereas adult-onset GH-deficient patients show a significant improvement of maximal expiratory pressure (p < 0.05). In conclusion, the results of this study showed that adult patients affected with childhood-onset GH deficiency suffer from an impairment of the ventilatory function due to a reduction of lung volumes and a decrease of respiratory pressures probably due to a reduction of respiratory muscle strength. This impairment was reversed after 12 months of treatment with recombinant GH. Conversely, adult-onset GH-deficient patients had only an impairment of the maximal expiratory pressure, probably due to respiratory muscle weakness re-established after 12 months of GH therapy.

Tumor necrosis factor-alpha increases after corticotropin-releasing hormone administration in Cushing's disease. In vivo and in vitro studies.

The aim of this study was to evaluate the effect of acute human corticotropin (ACTH)-releasing hormone (CRH) administration (100 micrograms, as i.v. bolus) on tumor necrosis factor-alpha (TNF alpha) levels in the inferior petrosal sinuses and in the peripheral blood of 7 patients with Cushing's disease subjected to diagnostic inferior petrosal sinus sampling. Blood samples for ACTH, beta-endorphin (beta-EPH) and TNF alpha were collected from inferior petrosal sinuses and periphery simultaneously. In addition, TNF alpha concentrations were measured after CRH administration (10 nmol/l, 100 nmol/l and 1 mumol/l) in culture medium from primary cultures obtained in 3 of 7 patients. At baseline, plasma ACTH and beta-EPH levels were significantly higher in the inferior petrosal sinus ipsilateral to the ACTH-secreting adenoma than in the contralateral one and in the periphery (p < 0.001) whereas no significant difference was found as far as serum TNF alpha levels were concerned. CRH administration caused a significant increase of ACTH (p < 0.001), beta-EPH (p < 0.01) and TNF alpha (p < 0.01) levels greater in the ipsilateral inferior petrosal sinus than in the contralateral one and in the periphery. In addition, CRH increased ACTH, beta-EPH and TNF alpha levels in the culture medium of three ACTH-secreting tumors at the doses of 100 nmol/l and 1 mumol/l (greater than 300, 200 and 110% of baseline pretreatment incubation levels, respectively). These data suggest that CRH may increase TNF alpha concentrations in the inferior petrosal sinus ipsilateral to the ACTH-secreting adenoma and in the peripheral blood as well. In addition, it stimulated TNF alpha release both in vivo and in vitro. These findings suggest the possibility that an imbalanced intrapituitary TNF alpha production can be detected in ACTH-secreting adenomas.

Reevaluation of growth hormone (GH) secretion in 69 adults diagnosed as GH-deficient patients during childhood.

At present, the most appropriate method for diagnosing GH deficiency (GHD) in adults remains unclear. Recently, it has been demonstrated that GHD in adults can be identified by insulin tolerance test (ITT). Moreover, it has been described that the GHRH plus pyridostigmine (GHRH+PD) test is more accurate than an arginine, glucagon, levodopa, or GHRH test to diagnose GHD in adults. In the current study, firstly we reevaluated GH secretion by the GHRH+PD test in adults previously diagnosed as GH deficient in childhood. The study included 69 patients and 38 healthy subjects. After the GHRH+PD test, the patients and the healthy subjects had peak GH levels of 10.6 +/- 11.2 and 56.7 +/- 28.1 micrograms/L, respectively (P < 0.001). The patients were divided into two groups, responder patients and nonresponder patients, considering an arbitrary cut-off of 10 micrograms/L as the GH peak after the GHRH+PD test. Thirty-nine patients had GH peak below 10 micrograms/L (1.9 +/- 1.7 micrograms/L), whereas the remaining 30 patients above 10 micrograms/L (21.6 +/- 8.] micrograms/L; P < 0.001). Secondly, we compared the GHRH+PD test and the ITT in diagnosing GHD. Twenty-one of the 39 patients with a GH peak below 10 micrograms/L and 29 of the 30 patients with a GH peak above 10 micrograms/L after the GHRH+PD test underwent an ITT. The GH peak after insulin administration was 2.1 +/- 1.7 micrograms/L in nonresponder patients and 21.1 +/- 9.3 micrograms/L in responder patients after the GHRH+PD test (P < 0.001). Three of the responder patients to the GHRH+PD test were identified as GH deficient by the ITT. The relative diagnostic accuracies of the two tests to discriminate GH-deficient patients from healthy subjects were similar (ITT vs. GHRH test: sensitivity, 100%, specificity, 90%; GHRH+PD vs. ITT; sensitivity, 88%; specificity, 100%). In conclusion, in adults previously diagnosed as GH deficient, it is mandatory to reevaluate GH secretion. GHRH+PD and/or ITT are able to diagnose GHD in adults with similar accuracies. Taking into account the potential side-effects of the ITT, however, the GHRH+PD test is the most reliable and safe test to accurately diagnose GHD status in adults.