Utilisation of sperm-binding assay combined with computer-assisted sperm analysis to evaluate frozen-thawed bull semen.

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap-freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm(2) of membrane was assessed by conventional microscopy (CM) and computer-assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r(2) = 0.91 and CASA: r(2) = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm-egg-binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.

Bioactivity of prolactin in systemic sclerosis.

OBJECTIVES: To evaluate basal serum prolactin (PRL) levels in systemic sclerosis (SSc) patients with different degrees of skin involvement, and investigate its relationship with some of the clinical and serological parameters of the disease. METHODS: Basal serum PRL was measured in 44 SSc patients (38 F, 6 M) using a rat NB2 lymphoma line cell proliferation assay. Other parameters measured were: serum aminoterminal propeptide of type III procollagen (PIIINP) by RIA; soluble alpha interleukin-2 receptor (IL-2 sRalpha), serum intercellular adhesion molecule-1 (ICAM-1), von Willebrand factor (vWF) by ELISA; the erythrocyte sedimentation rate (ESR); and C-reactive protein (CRP). Skin and organ/system involvement were assessed according to Medsger et al.'s organ/system severity scale, and global disease activity index according to Valentini et al. RESULTS: The serum PRL concentration in the SSc patients was 13.8 ng/ml (95%CI from 3.2 to 49.1 ng/ml), similar than that in control subjects (12.8 ng/nl: 95%CI 3.0 to 18.4 ng/ml). Hyperprolactinemia, defined as a level > 20 ng/ml (mean 30.9 ng/ml, median 29.3) was found in a total of 6 cases (13.6%; 95%CI 5.8 to 28%) cases: in 1 out of 6 men (16.7%; 95%CI -26% to 59%) and similarly in 5/38 women (13.2%; 95%CI 1.9% to 24.4%). No correlation was found between PRL levels and SSc subgroup (lcSSc, icSSc, dcSSc), serologial parameters, or the level of disease activity. Finally, no significant correlations were found with clinical or serological variables. CONCLUSIONS: The findings confirm that mild hyperprolactinemia occurs in at subgroup of SSc patients. However, prospective studies are needed to better define the relationship between PRL and disease activity in scleroderma.

Impaired glucose oxidation and glucose-induced thermogenesis in renal transplant recipients.

BACKGROUND: Renal transplant recipients often show various metabolic abnormalities including reduced glucose tolerance, impaired insulin sensitivity and altered lipid metabolism. However, the acute effects of carbohydrate ingestion on substrate utilization and energy expenditure have not been fully elucidated. METHODS: We evaluated: (i) basal energy expenditure (EE) and substrate utilization, (ii) metabolic fate of an oral glucose load, and (iii) substrate-induced thermogenesis in: (a) 15 non-diabetic renal transplant recipients (Tx) (BMI 25+/-1) on triple immunosuppressive therapy, (b) 11 patients with primary glomerulonephritis (BMI 25+/-1) (Cort) receiving prednisone treatment, and (c) 12 healthy subjects (BMI 26+/-1) (N). Continuous indirect calorimetry was performed in the basal post-absorptive state for 60 min and continued for an additional 180 min following an oral glucose load (75 g). RESULTS: In the basal state, EE was similar in the three study groups. It averaged 14.6+/-0.7, 15.7+/-1.3, and 14.1+/-0.8 cal/kg/min in Tx, Cort, and N respectively. Glucose oxidation was higher in N (1.3+/- 0.2 mg/kg/min) than in Tx (0.7+/-0.2) and Cort (1.0+/-0.2) (P<0.05 in N vs. Tx and vs. Cort), whereas lipid oxidation was lower in N (0.6+/-0.1 mg/kg/min) than in Tx (0.9+/-0.1) and Cort (0.9+/-0.05) (P<0.03 in N vs. Tx and vs. Cort). After glucose ingestion, total carbohydrate oxidation averaged 21.2+/-2, 31.0+/-3, and 29.6+/-3 g, which represented 28+/-3, 41+/-3 and 39+/-2% of the total glucose load in Tx, Cort and N respectively (P<0.01 Tx vs Cort and N). The cumulative increase of EE (180 min) was 9.7+/-2, 13.2+/-3 and 13+/-3 kcal in Tx, Cort, and N respectively. CONCLUSIONS: The present data show that in non-diabetic renal transplant recipients basal EE is normal. However, basal lipid oxidation is higher and glucose oxidation is lower than in healthy subjects. In addition, the oxidative disposal of a glucose load and substrate-induced thermogenesis are impaired.

Twenty-four hour melatonin pattern in acromegaly: effect of acute octreotide administration.

We investigated the melatonin (MT) circadian rhythm before and after somatostatin (octreotide) acute administration in ten subjects (4 M, 6 F. 23-52 yr old) with active acromegaly due to pituitary microadenoma. Blood samples were drawn every 2 hours over a 48-h span; after 24-h basal blood collection, octreotide (Sandostatin, Sandoz) 100 micrograms sc/8 h was administered. As control, 7 healthy adult subjects (3M, 4F; 26-50 yr old) were studied in basal condition over a 24-h span. Plasma MT and GH levels were measured by RIA in each sample, IGF-1 levels were measured by immunoradiometric assay in basal and after octreotide morning samples. The comparisons were made by Mann-U-Withney and Wilcoxon test as appropriate; the existence of a MT circadian rhythm was validated by cosinor analysis; GH and MT values were correlated by Pearson's correlation coefficient. All of 7 control subjects and 2 of 10 acromegalics had significant 24-h MT rhythm. The area under curve (AUC), mesor and amplitude of the MT rhythms in acromegalics were significantly lower than in the controls (p < 0.001, 0.002 and 0.0006, respectively), with an earlier acrophase (median value: 22:14 vs 02:08 h of controls). Basal plasma IGF-1 levels and circadian GH concentrations were significantly increased in acromegalics in comparison with the control group. Octreotide administration significantly reduced GH, restoring a circadian MT rhythm in 5 of 10 acromegalics, with MT mean mesor and AUC not different from controls. Mean amplitude still remained lower than controls (p < 0.0006), with an earlier acrophase (median 00:01 h). No significant correlation was found between individual GH and MT levels. Our data indicate a reduction of MT circadian secretion in acromegaly, due especially to a blunted nocturnal increase with earlier MT peak; moreover, acute octreotide administration increase MT levels without modifying amplitude and phase of night-time secretion significantly. These findings suggest a negative interrelationship between GH and MT secretions or a facilitatory influence of somatostatin on daytime MT release only. This partial recovery of pineal secretion after octreotide in acromegalics could be a clinically significant contribution to improve their quality of life, considering that MT is involved in the regulation of several important functions.

Melatonin and the pituitary-thyroid axis status in blind adults: a possible resetting after puberty.

OBJECTIVE: Increased levels of free thyroid hormones have been previously described in prepubertal blind subjects and have been thought to be a consequence of a partial target organ refractoriness due to the early and prolonged lack of light perception. The aim of this study was to clarify whether this abnormality is permanent or transient and the interrelationships between melatonin and thyroid hormone secretion. MEASUREMENTS: Total and free thyroid hormones, TSH, thyroxine-binding globulin (TBG), reverse triiodothyronine (rT3) and melatonin were measured in plasma samples obtained at 0800 h (two hours after lights-on) in a group of 11 totally (group 1) and 16 partially (group 2) blind adult patients and in 10 age-matched healthy subjects. RESULTS: Both totally and partially blind patients showed melatonin levels higher than in controls (330 +/- 106 pmol/l, group 1 and 361 +/- 159 pmol/l, group 2, respectively; controls: 53 +/- 12 pmol/l, P < 0.001 vs both groups), but fT4, fT3, T4, T3 TSH, rT3 and TBG concentrations showed no significant differences from controls. CONCLUSIONS: A possible resetting of pituitary-thyroid axis regulation can occur in blindness after puberty; variations of melatonin secretion could play a role in this. The inhibitory effect of melatonin on thyroid gland function found in animals does not seem to occur in humans. Elevated melatonin levels, both in patients with total blindness and in those with light perception only, suggest that more complex mechanisms other than light signalling are involved in the changes of melatonin secretion in blindness.

Detection of vasopressin cell antibodies in some patients with autoimmune endocrine diseases without overt diabetes insipidus.

OBJECTIVE: Cytoplasmic autoantibodies to vasopressin cells (AVP) have been detected in patients with idiopathic central diabetes insipidus and only in one patient with endocrine autoimmune diseases without clinical diabetes insipidus. The aim of this study was to look for AVP cell antibodies (AVP-cell-Ab) in human sera of a large population of autoimmune endocrine disease patients without diabetes insipidus and to test whether an occurrence of these antibodies in some patients can be associated with partial impairment of posterior pituitary function. MEASUREMENT: Sera from 410 patients (310 females, 100 males, age range 10-46 years) with autoimmune endocrine disorders (260 with thyroid autoimmune disease, and 150 with insulin dependent diabetes mellitus) without clinical diabetes insipidus, and from 100 normal subjects, were investigated for hypothalamic autoantibodies by an indirect immunofluorescence method. Positive sera were subsequently tested with specific rabbit anti AVP serum. RESULTS: None of controls, but five out of 410 patients (1.2%) were AVP-cell-Ab positive. All positive and nine negative from the 410 screened patients were tested for posterior pituitary function. Two out of five AVP-cell-Ab positive patients showed partial diabetes insipidus. CONCLUSION: AVP cell antibodies can be shown in some patients with endocrine autoimmune disease without diabetes insipidus and can sometimes be associated with findings of partial posterior pituitary dysfunction. This suggests that clinical diabetes insipidus could be preceded by a long subclinical period characterized only by the occurrence of AVP-cell-Ab in the sera associated or followed by alterations in functional tests. Longitudinal studies are needed to confirm this hypothesis.

Normal plasma insulin-like growth factor I levels and impaired final stature in adult blind subjects.

The aim of this study was to determine if changes in IGF-1 levels and in final stature occur in blind adult subjects. Eighteen subjects (4 females and 14 males) with total blindness (Group 1) and 26 subjects (5 females and 21 males) with only light perception (Group 2), living at an Institute for blind adult subjects in Naples, Italy, were studied. Their height and weight were compared to British standards. Plasma morning IGF-1 levels, measured by IRMA method were compared to those of 18 normal controls (6 females and 12 males) matched for age and weight. A high prevalence of short stature was demonstrated especially in Group 1 (p = 0.00005 by chi-square for trend test) but was also present in Group 2 (p = 0.02). No alterations in weight distribution were observed in both groups. Basal IGF-I levels in both Group 1 and Group 2 were similar to those of controls: IGF-1 (M +/- SE), 30.9 +/- 2.9 nmol/L and 37.9 +/- 2.7 nmol/L, respectively, vs 33.3 +/- 2.9 nmol/L, NS. High prevalence of final short stature in both groups confirms a negative influence of total or partial blindness on growth. These complex mechanisms appear to involve more than alterations in GH and IGF-I secretion.

Remission of subclinical adrenocortical failure in subjects with adrenal autoantibodies.

Idiopathic Addison's disease is a chronic organ-specific autoimmune disorder with a long subclinical period characterized only by the presence of adrenal autoantibodies (AA) with or without adrenal function failure. The aim of this longitudinal study was to evaluate the behavior of AA using, an indirect fluorescence method, and adrenal function in 20 AA-positive and 50 AA-negative patients screened by an investigation of a large population of organ-specific autoimmune disease patients without clinical Addison's disease. As controls, 100 normal age-matched subjects were tested only once. In the 20 AA-positive and 50 negative patients, AA and adrenal functional tests were evaluated every 4 months for 5 yr. The AA-positive patients were grouped into 5 adrenal functional stages, specifically: stage 0, normal adrenal function; stage 1, high PRA and low (or normal) aldosterone levels alone; stage 2, along with impaired cortisol response to ACTH, stage 3, along with increased ACTH levels; and stage 4, clinically overt Addison's disease. On the basis of the behavior of AA, the 20 positive patients were grouped as follows: group A, 11 patients with AA titer of 1:8 or higher at the first observation and persistently AA positive in subsequent observations, with titers ranging from 1:8 to 1:64; group B, 6 patients with initial AA titers of 1:8 or lower and AA disappearance in subsequent observations; and group C, 3 patients with AA titer of 1:32 or higher, undergoing corticosteroid therapy after the start of the study and showing AA disappearance in subsequent observations. With respect to adrenal function in group A, 2 patients initially in stage 1 and 1 patient initially in stage 2 did not progress to the upper stages, whereas 5 patients initially in stage 0 and 3 initially in stage 1 progressed subsequently to the upper stages, in 2 cases reaching overt clinical Addison's disease (stage 4). On the other hand, all of the patients of group B showed both a spontaneous disappearance of AA and recovery of adrenal function during the study span. Also, the 3 patients of group C showed disappearance of AA after corticosteroid therapy with recovery of adrenal function. None of the 50 patients who were initially AA negative became AA positive subsequently or showed impairment of adrenal function. We reached the following conclusions. 1) AA, even if present initially in some subjects without clinical Addison's disease, can subsequently disappear. 2) Restoration of adrenal function after disappearance of AA indicates that a spontaneous remission of subclinical adrenocortical failure can occur.(ABSTRACT TRUNCATED AT 400 WORDS)

Plasma and urinary growth hormone and insulin-like growth factor I in children with chronic renal insufficiency.

We investigated the plasma and urinary growth hormone (GH) levels, and plasma insulin-like growth factor (IGF-I) in 15 (8 prepubertal and 7 pubertal) subjects with chronic renal insufficiency. Four of our 7 pubertal patients had a short stature (standard deviation score from -2.7 to -4.0). Significantly increased plasma GH and IGF-I levels were found in pubertal subjects alone (p less than 0.05). The urinary GH levels was significantly increased in prepubertal and pubertal subjects (p less than 0.001 and 0.05, respectively). A significantly inverse correlation between urinary GH excretion and creatinine clearance was found (r = -0.55; p less than 0.05). Increased plasma GH, IGF-I and urinary GH levels seem to suggest a complex alteration of GH and somatomedin metabolism in central and peripheral (receptor) cells.

Plasma thyrotropin, thyroxine, triiodothyronine, free thyroxine, free triiodothyronine, and cortisol levels in blind prepubertal boys.

Findings on thyroid function in blind subjects are lacking. The aim of this study was to investigate the thyroid hormonal pattern in prepubertal blind subjects. Six healthy and 8 blind males, aged 7-10 yr, in Tanner stage one puberty, living at Institute "Martuscelli" for blind young subjects, Napoli, Italy, were studied. Each had a TRH (200 micrograms) test at 08:00 h after nocturnal rest. Plasma TSH, T4, T3, free T4(FT4), free T3(FT3) and cortisol (F) were measured by RIA. Our blind subjects show levels of TSH (basal values and absolute peak after TRH), T4, T3 and F normal but FT4 levels significantly higher than controls (39 pg/ml +/- 4.7 vs 12 +/- 0.6, p less than 0.001; 14 pg/ml +/- 1.3 vs 4.7 +/- 0.2, p less than 0.001, respectively). Our results, similar to those found in some patients with euthyroid hyperthyroxinemia, suggest that the prolonged inability to receive light signal could influence the metabolism of thyroid hormones and/or cause a tissue resistance to their action, even if this hypothesis must be verified by future more extensive investigations.

Influence of blindness on plasma luteinizing hormone, follicle-stimulating hormone, prolactin, and testosterone levels in prepubertal boys.

The aim of this study was to determine if changes in LH, FSH, PRL, and testosterone (T) secretion occur in blind prepubertal boys. Eight blind and six normal boys, aged 7-10 yr, living at an institute for blind subjects in Naples, Italy, were studied. Each had a combined GnRH (100 micrograms) and TRH (200 micrograms) test at 0800 h after nocturnal rest. Plasma LH, FSH, PRL, and T levels were measured by RIA. The blind boys had basal plasma LH, FSH, and T levels significantly lower than those in the normal boys (P less than 0.01 for all three); plasma PRL basal levels were similar to those in the normal boys. The blind boys, moreover, had lower peak LH, FSH, and PRL (P less than 0.01 for all three peaks) levels in response to GnRH-TRH. Our results, similar to those found by others in patients with delayed puberty or with hypogonadotropic hypogonadism, suggest that light stimuli influence neuroendocrine-gonadal activity in humans, as in other mammals; and in blind prepubertal boys, impaired hormone secretion could cause a delay of pubertal development or more severe hypogonadism.

Thyroid localization in adult histiocytosis X.

Authors report on a rare case of thyroid involvement by histiocytosis X. A 27-yr-old man with diabetes insipidus and lung bullous disease of 2 yr duration was found to have thyroid gland infiltration by differentiated histiocytosis X. This was based on cytological findings consistent with the diagnosis, obtained by fine needle aspiration biopsy. Endocrine studies revealed altered hypothalamic-pituitary function accounted for by a hypothalamic lesion.

Isosexual precocious pseudopuberty secondary to a testosterone-secreting Leydig cell testicular tumour: true isosexual development early after surgery.

The paper reports on a 6-year-old boy with precocious pseudopuberty due to androgen hypersecretion by a testicular interstitial cell tumour. Steroidogenesis, characterized by high testosterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone and 17-hydroxyprogesterone plasma levels, was not modified by ACTH, dexamethasone or HCG administration. Gonadotropins were subnormal and unresponsive to LRH stimulation. TSH and prolactin levels were normal both in basal and dynamic conditions. The hormonal profile progressively returned to prepubertal value and persisted normal for 6 months after removal of the tumour. The patient entered puberty spontaneously at 7,6/12 years showing a normal pubertal basal and LRH stimulated FSH and LH and a pubertal circadian rhythm of both gonadotropins and testosterone.

Circannual variations of plasma testosterone, luteinizing hormone, follicle-stimulating hormone and prolactin in Klinefelter's syndrome.

To clarify the influence of primary testicular failure upon circannual hormone rhythmicity we have been studying, by cross-sectional design, 93 adult patients with Klinefelter's syndrome (KS) and 64 adult healthy males to look for evidence of circannual rhythms in testosterone (T), LH, FSH and PRL plasma concentrations. Plasma samples were taken from 08.00 to 09.00 h and all hormones were measured by radioimmunoassay. The data were assessed by the single cosinor method in order to obtain evidence for any significant rhythm and to estimate its parameters mesor, amplitude and acrophase. In the controls, annual rhythms were validated for the secretion of T (annual crest time in September), LH (annual crest time in February) and FSH (annual crest time in January), whereas PRL did not show a significant annual rhythm. In KS, significant circannual rhythms were validated for the secretion of T (annual crest time in April) and FSH (annual crest time in September), but not for LH and PRL secretion. Our results suggest that in KS the circannual hormone rhythmicity may be influenced by seminiferous tubule dysgenesis.

Circannual variations of plasma thyrotropin in Klinefelter's syndrome.

The aim of this study was to explore the occurrence of abnormalities in the circannual thyrotropin (TSH) rhythm in Klinefelter's syndrome (KS). For 3 years, 69 healthy adult males and 73 patients with KS, usually living in Sardinia, were studied by a cross-sectional design. Plasma samples were taken between 08.00 and 09.00 h and TSH was radioimmunoassayed. First, the mean (+/- SD) of the data, grouped for each month of the year, was estimated to seek any macroscopic annual variation. Then, a cosine function was fitted to the single serially independent data by the single cosinor method in order to test for any statistically significant rhythm and, if the no-rhythm assumption is rejected, to describe the circannual parameters. Our patients showed circannual mean TSH concentrations lower than controls (p less than 0.001) without any statistically significant circannual rhythm, which was instead apparent in normal subjects with the annual crest time in December (95% CL November-February). Our results support the hypothesis that in KS the impaired TSH secretion may be related to abnormalities in the hypothalamic-pituitary coordination of the circannual rhythmicity of this hormone.