The seminiferous tubule caliber pattern as evaluated at high magnification during microdissection testicular sperm extraction predicts sperm retrieval in patients with non-obstructive azoospermia.

BACKGROUND: microTESE proved to be the gold standard surgical approach for patients with non-obstructive azoospermia (NOA), but sperm retrieval rates (SRRs) vary considerably among centers. Some authors compared their SRRs with the pattern of seminiferous tubule caliber found at high magnification, but none provided diagnostic accuracy measures. OBJECTIVE: The present retrospective study sought to verify the diagnostic accuracy of the pattern of seminiferous tubule caliber in predicting the sperm retrieval in NOA patients. MATERIALS AND METHODS: Data from 143 infertile NOA men undergoing unilateral (64) or bilateral (79) microTESE (222 testes) were retrospectively evaluated. During microTESE, if present, dilated tubules (DTs) were retrieved, otherwise tubules with slightly larger caliber (SDT) (×24) than that of the surroundings were removed. When no DT or SDT were found, not dilated tubules (NDTs) were excised. RESULTS: Spermatozoa were retrieved in 95 of 222 testes (42.8%); sperm retrieval was successful in 90% of testes with DTs, in 47% of those with SDTs, and only in 7% of those with NDTs (p < 0.0001). Stepwise binary logistic regression revealed that the combination of seminiferous tubule pattern and testis histology was significantly predictive of SSR, being able to classify 86.8% of testes, with an excellent diagnostic accuracy (AUC 0.93). The median number of spermatozoa retrieved was significantly higher in DTs compared with SDTs and NDTs. DISCUSSION: The results of the present study provide reliable accuracy measures in support of the relationship between seminiferous tubule caliber pattern and SSR in patients with non-obstructive azoospermia. We are proposing for the first time that spermatozoa may be retrieved even from slightly dilated tubules in about half of cases. The pattern of tubules retrieved, together with histology, may represent an additional outcome measure of microTESE. CONCLUSION: The pattern of seminiferous tubules together with testis histology predicts sperm retrieval with an excellent diagnostic accuracy.

Temperature dependence of electric transport in few-layer graphene under large charge doping induced by electrochemical gating.

The temperature dependence of electric transport properties of single-layer and few-layer graphene at large charge doping is of great interest both for the study of the scattering processes dominating the conductivity at different temperatures and in view of the theoretically predicted possibility to reach the superconducting state in such extreme conditions. Here we present the results obtained in 3-, 4- and 5-layer graphene devices down to 3.5 K, where a large surface charge density up to about 6.8·10(14) cm(-2) has been reached by employing a novel polymer electrolyte solution for the electrochemical gating. In contrast with recent results obtained in single-layer graphene, the temperature dependence of the sheet resistance between 20 K and 280 K shows a low-temperature dominance of a T(2) component - that can be associated with electron-electron scattering - and, at about 100 K, a crossover to the classic electron-phonon regime. Unexpectedly, this crossover does not show any dependence on the induced charge density, i.e. on the large tuning of the Fermi energy.

Sperm retrieval for intra-cytoplasmic sperm injection in non-obstructive azoospermia.

Surgical testicular sperm retrieval for intra-cytoplasmic sperm injection (ICSI) purposes is the only possibility of biological fathering in case of non-obstructive azoospermia (NOA). Successful retrieval only correlates with histology, not with FSH values or testicular volume. Concurrent AZFa and AZFb microdeletion predict unsuccessful recovery. Testicular sperm extraction (TESE) (mean of successful retrievals in literature: 52.7%) is the technique of choice: we had successful retrievals in 100% of cases of hypospermatogenesis with > 5 spermatids/tubule (spd/tub), 81.8% of cases of hypospermatogenesis with < 4 spd/tub, 50% of cases of maturation arrest, and 25% of cases of histologically pure Sertoli cell-only syndrome. Microsurgical TESE (mTESE) has been reported to increase successful retrievals: from 16.7-45% for standard TESE to 42.9-63.6% for mTESE, depending on the distribution of testicular histology in the various case studies; from 9 to 14 cases out of 22, respectively, in the only study in which TESE and mTESE were performed simultaneously on the same testis. Improvements in biological procedures for TESE retrievals can increase positive findings. TeFNA does not appear to be indicated in NOA, both because of its low success rates--which, in practice, are only positive in hypospermatogenesis--and because it is unable to detect any carcinomas in situ. Previous surgery of left varicocele in NOA could increase the chances of subsequent recovery. ICSI from TESE has lower birth rates in NOA than in obstructive azoospermia (OA) (19% vs 28%). Abortion rates are significantly higher following ICSI from NOA (11.5%) than from OA (2.5%) (P=0.001). Therefore, the prognostic fertility of a couple with an NOA male is quite lower than for a couple with an OA male.

myo-[3H]-inositol loaded erythrocytes and white ghosts: two models to investigate the phosphatidylinositol synthesis in human red cells.

Human erythrocytes were loaded with myo-[(3)H]-inositol in the presence or absence of cytidine trisphosphate to investigate the synthesis of membrane phosphoinositides in the intact red cell. The addition of cytidylic nucleotides to the loading mixture yielded a four-fold increase in the [(3)H]-labeling of the membranes. The [(3)H]-labeling of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was distinguished by two chromatographic techniques. Experiments performed on white ghosts demonstrated the presence of CDP-diacylglycerol synthase and phosphatidylinositol synthase. These results and those already reported allow to discuss a possible turnover of the inositol polar head.

Effects of UV-C irradiation on phosphoinositide turnover in plant cells: similarities with those occurring via the formation of reactive oxygen intermediates in animal cells.

With the aim of examining the response of plant cells to UV-C irradiation, we investigated the behaviour of the phosphatidylinositol 4,5 bisphosphate (PtdIns 4,5-P2) molecule (the precursor of the phosphoinositide signal transduction cascade) by exposing callus cells from Peucedanum verticillare to UV-C (130 J m-2) and by examining the level and the fatty acid composition of PtdIns 4,5-P2 at different times after irradiation. We show that a pathway for the UV-C response includes transient PtdIns 4,5-P2 breakdown. The effect of ultraviolet rays is mimicked by H2O2 suggesting that in this plant it may be brought about by reactive oxygen intermediates (ROI), as already underlined in experimental animal models.

Stimulation of in vitro rat hepatocyte proliferation by conditioned medium obtained from an immortalized macrophage cell line.

The hepatomitogenic effect of conditioned medium (CDM), obtained from the N-11 mouse macrophage cell line was analysed in rat hepatocyte primary cultures. CDM concentrations from 0.01% to 100% were used and the stimulating action in terms of mitotic index (MI) was evaluated. A clear mitogenic effect was observed only with concentrations higher than 10% with peak effects around 60%. Further increase in CDM concentrations resulted in an MI decrease, and at 100% CDM the effect was totally abolished. Tests addressed to identify the presence of hepatocyte growth factor (HGF) yielded negative results. In order to identify the mitogenic factor(s) involved, we tested CDM obtained after lipopolysaccharide (LPS) stimulation of N-11 cells. Comparison of the results obtained with untreated or LPS stimulated CDMs suggested that macrophage activation does not affect the release of hepatomitogenic activity. To further characterize this macrophage-derived activity, we checked whether CDM could interact with the mitogenic effects of epidermal growth factor (EGF). CDM (10 or 50%) showed no stimulatory effect to hepatocytes cultured in the presence of a maximally stimulatory concentration of EGF. Conversely, both CDM concentrations were able to increase the MI of hepatocyte cultures treated with a suboptimal dose of EGF. These results suggest that macrophages release factor(s) which interact, in hepatocytes, with the EGF signal transduction mechanisms, or with the EGF receptor itself.

[Epidemiology of male infertility]. / Epidemiologia dell'infertilità maschile.

OBJECTIVE: The aim of the present study has been to analyze the variation of the main parameters which define sperm quality in a group of men who were referred to our center of sterility during the last ten years of activity. MATERIAL AND METHODS: Over the last ten years, we have examined 3203 men ranging from 25 to 50 years of age. We have considered different parameters of evaluation, such as semen volume, motility, total motility count and morphology of spermatozoa; the same parameters have been considered for a group of 192 men accustomed to consuming coffee, alcohol and tobacco. RESULTS: We have observed no significative variations with respect to sperm volume; in spite of this result, we have registered a considerable reduction of the frequency of men with normal sperm quality, when we have considered parameter such as sperm motility. The evaluation of the total motility count has confirmed a progressive reduction during the considered period of time. This parameter has also shown the existence of a circannual rhythm for the production of spermatozoa, which results minimum during the winter and during the warmest months, while it improves during spring and autumn. During the considered period, the study of morphology has shown a progressive reduction of men with normal semen quality. CONCLUSION: The results of our epidemiologic study, conducted over a group of men who have been referred to our center of sterility during the last ten years, confirm that the group representative of Northern Italy has faced a decline of sperm quality.

Inositol 1,4,5-trisphosphate distribution in Lycopersicon esculentum Mill seedlings cultivated "in vitro" under different conditions.

Measurements of inositol 1,4,5-trisphosphate (Ins1,4,5-P3) in cotyledons, epicotyls and roots of tomato seedlings grown "in vitro" either in the light or in the dark indicated that higher concentrations of this signal-transducing molecule are contained in hypogeous vs. epigeous tissues. The same was observed in induced cotyledon explants grown in the light in the presence of growth regulators. Data concerning phosphatidylinositol metabolism in seedling roots are also reported. Taken together, our results may be helpful in understanding the role of the polyphosphoinositide signal system in plants.

[Evaluation of micronuclei in primary hepatocyte culture in rats treated with organophosphoric compounds]. / Valutazione dei micronuclei in colture primarie di epatociti di ratto dopo trattamento con composti organofosforici.

We have evaluated the genotoxic effect of the three organophosphorous (OPs) insecticides dimethoate (DIM), azinphosmethyl (AZN) and diazinon (DIA) or in combination, at a ratio frequently found in foodstuffs by residual analysis. We have carried out the micronucleus (MN) test on cultured rat hepatocytes which were induced to proliferate by epidermal growth factor (EGF), and treated with the insecticides for 48 hours. The doses tested were chosen on the basis of cytotoxicity tests such as Trypan Blue exclusion and mitotic index inhibition. The results showed that AZN was the most toxic and DIM the least toxic compound. None of the compounds tested showed a genotoxic effect when examined separately. Conversely, a significant increase in MN frequency was observed when the three OPs were used in combination at the lowest concentration used when each compound was tested alone.

Modification of inositol 1,4,5-trisphosphate concentration of human erythrocytes under "in vivo" physiological conditions.

The concentration of inositol 1,4,5-trisphosphate (InsP3) in erythrocytes from volunteers has been found to modify following strenuous physical exercise. The basal value was almost regained within some 75 min after the completion of the effort. The concurrent variations of pH and blood lactate have also been evaluated. Our results represent, to our knowledge, the first evidence of in vivo induced intraerythrocyte InsP3 modification. They reinforce the idea of the participation of its precursor phosphatidylinositol 4,5-bisphosphate (PtdIns4,5-P2) in red bood cell shape regulation by contributing to the interactions between membrane and cytoskeleton components.

Increase of micronucleus frequency in cultured rat hepatocytes treated in vitro with benomyl and pirimiphos-methyl separately and in mixture.

The pesticides benomyl, a benzimidazole fungicide, and pirimiphos-methyl, an organophosphorus insecticide, were tested separately and in combination at a ratio of 6:1, a mixture frequently found in foodstuffs by residual analysis, to determine their possible genotoxic action. The effect was measured by the micronucleus test carried out on cultured rat hepatocytes stimulated to proliferate by epidermal growth factor (EGF). Adult rat hepatocytes were exposed in vitro for 48 h to the substances at increasing non-cytotoxic doses, chosen on the basis of cytotoxicity tests such as LDH and Neutral red assays. Benomyl induced a significant dose-related increase in micronucleus frequency; in contrast, pirimiphos-methyl was not genotoxic at any dose tested. When the hepatocytes were exposed to the two pesticides together at increasing doses, an enhancement in micronucleus frequency similar to that of benomyl alone was found, indicating that at this ratio and non-cytotoxic doses (up to 25 micrograms/ml benomyl + 4.2 micrograms/ml pirimiphos-methyl) no interaction occurs.

Plasma tryptophan levels and plasma tryptophan/neutral amino acids ratio in patients with mood disorder, patients with obsessive-compulsive disorder, and normal subjects.

Fasting plasma tryptophan (TRP) levels and ratios of total plasma tryptophan to the sum of five large neutral amino acids (LNAAs)--tyrosine, phenylalanine, leucine, isoleucine, and valine--that compete with tryptophan for passage across the blood-brain barrier were found to be significantly lower in a group of 28 patients with major depression compared with 29 normal subjects and 21 patients with obsessive-compulsive disorder (OCD). The OCD group was divided in two subgroups: patients with OCD alone and patients with a co-diagnosis of major depression. Since it has been considered that these biological parameters reflect brain tryptophan and serotonin levels, our results suggest their importance in relation to the presence or absence of depressive symptoms. The values of the other LNAAs and their sum did not differ significantly among the groups.

Glucose 1,6-bisphosphate-overloaded erythrocytes: a strategy to investigate the metabolic role of the bisphosphate in red blood cells.

Human erythrocytes overloaded with glucose 1,6-bisphosphate were prepared in order to establish the metabolic significance of this phosphorylated sugar in the intact red cell. The intracellular glucose 1,6-bisphosphate concentration was increased six- and twofold over the normal level by encapsulating (i) the commercially available compound and (ii) the glucose 1,6-bisphosphate synthase obtained from rabbit skeletal muscle, respectively. In both experimental conditions, a reduction of glucose utilization by the loaded cells was observed after reequilibration to the steady state. At the steady state, the concentrations of the glycolytic intermediates and of the adenine nucleotides appeared substantially unmodified when compared with those of controls, with the exception of a 50% reduction of glucose and fructose 6-phosphate measured in erythrocytes encapsulated with exogenous glucose 1,6-bisphosphate. Under the considered experimental conditions, the elevated intracellular glucose 1,6-bisphosphate appears to display an inhibitory effect on hexokinase that overcomes the possible activation of phosphofructokinase or pyruvate kinase.

Human red blood cells as bioreactors for the inactivation of harmful xenobiotics.

Human red blood cells are able to inactivate lipophilic electrophiles by conjugation with reduced glutathione. This metabolic ability was found to be limited by the rate of permeation of the xenobiotic into erythrocytes and by the amount of available reduced glutathione. By a procedure of hypotonic dialysis, isotonic resealing and reannealing human red blood cells were overloaded with increasing amounts of reduced glutathione up to three- to fourfold the normal level without modification of their metabolic functions or of their energetic state. These overloaded erythrocytes were able to conjugate increasing amounts of xenobiotics and to export the resulting conjugates from the cells. These properties of glutathione overloaded erythrocytes are significant for the use of carrier erythrocytes in cases of acute intoxication by lipophilic electrophiles.

Cell age dependent decay of human erythrocytes glutathione S-transferase.

Human red blood cells contain both glutathione S-transferase sigma (GST sigma) and glutathione S-transferase rho (GST rho). While the first isozyme does not change in red blood cell fractions of different mean density (age), GST rho, the main isozyme, shows a pronounced cell age dependent decay. Ion-exchange chromatographic experiments show that GST rho consists of only one isozymic form in young erythrocytes but is present in two components, with different electric charge, in mature and old cells. The "secondary" GST rho isozyme is more heat stable than the "primary" GST rho isozyme with the result that the total GST activity shows an apparent increase in heat stability during cell aging due to the formation of "secondary" isozymes. The kinetic properties and specificity of this enzyme do not show appreciable modifications during cell ageing. The data reported in this paper suggest that red blood cell aging is associated with a reduced detoxifying ability due to GST rho decay.

Purine nucleoside phosphorylase from human erythrocytes: a kinetic study of the fully separated isoenzymes.

Human red cell lysates contain at least seven electrophoretically distinct isoenzymes of purine-nucleoside phosphorylase (PNPase); the proportion of more anodal bands increases as the erythrocyte ages, suggesting that the native enzyme is subjected to progressive post-translational modifications. The age dependent electrophoretic changes observed in the hemolysate are associated with the downward curvature of the Lineweaver-Burk double reciprocal plot at high inosine-substrate concentrations unlike the single-banded PNPase from tissue cultures of rapidly dividing cells. Thanks to the high resolution power of the ion-exchange HPLC technique utilized we have been able to fully separate all the seven isoenzymes and correlate structural to functional modifications in PNPase from human erythrocytes. Our results indicate that the downward curvature of Lineweaver-Burk plot is not due to a mixture of isoforms with low and high Km for inosine but that the allosteric activation by the inosine substrate is the direct consequence of structural modification(s) on the "primary" form of the enzyme.

In vitro genotoxicity of dimethyl terephthalate.

Dimethyl terephthalate (DMTP), the para configuration of dimethyl phthalate, is one of the basic monomers used in the synthesis of polyethylene terephthalate (PET) plastics. Human exposure to DMTP may primarily occur during the manufacture of PET fibers and films. The mutagenic potential of dimethyl terephthalate was evaluated using a battery of in vitro short-term tests: the Ames test; DNA single-strand break assays in CO60 cells and in primary rat hepatocytes; UDS in HeLa cells; chromosome aberration and micronucleus assays in human peripheral blood lymphocytes; selective DNA amplification in CO60 and in Syrian hamster embryo cells. The results of this battery of in vitro assays clearly show that DMTP is nongenotoxic. By contrast, other authors have found DMTP to be an in vivo clastogenic compound and suggested that the mechanisms involved in these in vivo effects seem to have nothing in common with genotoxicity and are still unknown.

Specificity of glucose 1,6-bisphosphate synthesis in rabbit skeletal muscle.

1. To compare glucose 1,6-bisphosphate synthesis in different types of cells, we partially purified (2000-fold) a glycerate 1,3 P2-dependent glucose 1,6-bisphosphate synthase from rabbit skeletal muscle. 2. In agreement with the results reported by others for mouse brain and pig skeletal muscle, the enzyme can be separated from bulk phosphoglucomutase (PGM) activity by DEAE-cellulose chromatography of crude cellular extract. This cannot be achieved on human hemolysates where glycerate 1,3-P2-dependent glucose 1,2-bisphosphate synthesis is displayed only by multifunctional PGM2 isoenzymes. 3. The Km values for glycerate 1,3-P2 (0.50 microM), glucose 1-phosphate (90 microM), Mg2+ (0.22 mM), and also pH optimum (7.8) and mol. wt (70,000) of the rabbit skeletal muscle enzyme are similar to those of the enzymes from mouse brain and human red blood cells, but they differ from those reported for the pig skeletal muscle enzyme.