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.

Cell age dependent decay of human erythrocytes glucose-6-phosphate isomerase.

Glucose-6-phosphate isomerase shows a biphasic decay pattern during red blood cell aging, which is very fast during the first part of cell's life span in circulation. This decay is not due to accumulation of inactive enzyme molecules, as shown by immunological studies, but is accompanied by the formation of secondary isozymes (i.e., chemically modified forms). Electrophoretic and ion-exchange chromatographic experiments show that glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) consists of only one isozymic form in young erythrocytes but is present in two components, with different electric charge, in mature and old cells. This secondary isozyme is more stable to heat treatment and is inactivated by IgG anti-glucose-6-phosphate isomerase with a lower affinity than the native isozyme. In vitro incubation of homogeneous human glucose-6-phosphate isomerase under conditions known to produce enzyme deamination does not reproduce the isozymic pattern found in erythrocytes, suggesting that one or more mechanisms other than those previously described to explain glucose-6-phosphate isomerase microheterogeneity occur in the human erythrocyte.

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.

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.

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.

Relationships between the age-dependent decay of glucose-1,6-bisphosphate synthesis, phosphoribomutase and phosphoglucomutase in human red cells.

In human red blood cells phosphoglucomutase exists in multiple molecular forms with different isoelectric points determined by two distinct loci called PGM1 and PGM2. With regard to the phosphoglucomutase PGM1 and PGM2 isoenzymes, the latter appear to be more important in erythrocyte metabolism owing to their ability to mutate ribose monophosphates and synthetize glucose-1,6-bisphosphate. In this paper we show that, beside undergoing age-related postranslational modifications, both phosphoglucomutase PGM1 and PGM2 forms decrease their activities as the mean cell age increases. Under the experimental conditions used to separate erythrocytes by age the comparison of the younger erythrocytes with the older shows that total phosphoglucomutase, phosphoribomutase and glucose-1,6-bisphosphate synthetic activities decay by 55%, 26% and 28%, respectively. We consider that these results substantiate the multifunctionality of PGM2 isoenzymes. Furthermore we discuss the role of these forms in the age-related decay of erythrocyte metabolism.

The age-dependent metabolic decline of the red blood cell.

The oxidative and non-oxidative utilization of glucose was evaluated in human erythrocytes of different ages, separated by density gradient ultracentrifugation. Young red blood cells are able to metabolize 2.5 times more glucose than old ones; on the other hand, the amount of glucose utilized via the hexose monophosphate shunt does not show any age dependence. Glucose metabolism evaluated during in vivo ageing of a rabbit red cell population shows results very similar to those obtained for human cells. Metabolic stimulation of glucose utilization by high phosphate in both young and old human red cells increases glucose utilization by 40%. In the same way young and old erythrocytes were able to increase the amount of glucose metabolized via the hexose monophosphate shunt when an oxidative stimulus (methylene blue) was introduced. Human erythrocytes of different age possess similar abilities to transport glucose so that an age-dependent defect in glucose transport can be excluded. The ATP content of human and rabbit red blood cells, as a function of cell age, follows the decrease in glucose metabolized via the Embden-Meyerhof pathway. Reduced glutathione, on the other hand, after a small decrease associated with the "maturation" of reticulocytes into red cells, remains constant like the rate of the hexose monophosphate shunt.

Human erythrocyte phosphoglucomutase: comparison of the kinetic properties of PGM1 and PGM2 isoenzymes.

Kinetic properties of PGM1 and PGM2 phosphoglucomutase "primary" isoenzymes from human erythrocytes were studied. The two enzyme forms share a "ping-pong" kinetic mechanism and show similar Km for substrate (glucose 1-P) and cofactor (glucose 1,6-P2). Micromolar concentrations of fructose 1,6-P2 and glycerate 2,3-P2 inhibit both PGM1 and PGM2 isoenzymes to a similar extent. The sole PGM2 form is affected by ribose monophosphates (ribose 1-P and ribose 5-P) that act as mutase inhibitors vs. glucose 1,6-P2 and as apparent activators vs. glucose 1-P. The interaction between PGM2 isoenzyme and ribose monophosphates is discussed in the light of the ability of this form to also display phosphoribomutase activity.

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.

Glucose utilization in human erythrocytes during physical exercise.

Red blood cells obtained from well-trained athletes consumed glucose at a higher rate than those from sedentary subjects when incubated in vitro in a glucose containing medium at 37 degrees C. Similar results were obtained when fructose was substituted for glucose, but not when galactose was the monosaccharide. The utilization of glucose by erythrocytes can occur both via the Embden-Meyerhof and Pentose phosphate pathways (PPP). In the present experiments, there were no differences in the activities of select enzymes in these pathways or in metabolic intermediates of the erythrocytes. An exception was an elevated 2,3-diphosphoglycerate (2,3DPG) in the erythrocytes of the athletes. Additional experiments are needed to identify changes in the red blood cells of the athletes which produce the increased consumption of glucose.

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.

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.

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.

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.

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.

Factors influencing the metabolic pathways of glucose in human erythrocytes.

The metabolic effect of a lactate or pyruvate load and methylene blue (Mb) on hexose monophosphate shunt (HMPS) and Embden-Meyerhof pathway (EMP) were investigated in intact erythrocytes in the presence of glucose. Pyruvate enhances glucose consumption and 14CO2 production, lactate reduces 14CO2 production but glucose consumption is unchanged. These effects are dependent on the lactate and pyruvate concentrations in the buffer. A pyruvate load does not results to influence the net production of pyruvate, which is conversely markedly increased by Mb. The lactate load raises slightly the total pyruvate level but its rate of production is unchanged, with respect to that of the control. A considerable effect on the intracellular to extracellular pyruvate ratio is observed in these different conditions, as well as modifications in the levels of inorganic phosphate (Pi).

Isoelectric points and charge-dependent separation of erythrocyte phosphoglucomutase isoenzymes (PGM1 and PGM2).

Phosphoglucomutase can bind both negative and positive ions so that it may change its net electric charge according to the buffer species of the medium. For this reason the knowledge of the pIs of the erythrocyte phosphoglucomutase isoenzymes is not sufficient to forecast their separability by procedures based on charge separations such as ion exchange chromatography. In this paper we indicate the condition to obtain a satisfactory separation of the main erythrocyte phosphoglucomutase isoenzymes by DEAE-cellulose column chromatography. The pI values of the isolated isoenzymes are also reported and compared to those measured by others on whole hemolysates.

The relevance of glucose 1,6-bisphosphate formation and degradation to human red blood cell metabolism.

In human erythrocytes, in the absence of specific enzymes, G1,6P2 synthesis and degradation are carried out by phosphoglucomutase PGM2 isoenzymes. The results presented, obtained by using partially purified preparations of these enzyme forms, suggest that erythrocyte G1,6P2 may play a crucial role in the physiological interconversion of several important sugar monophosphates.

[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.

[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.