Protective effect of Propolfenol® on induced oxidative stress in human spermatozoa.

The propolis extract was shown to possess the capacity to protect sperm membrane from the deleterious action of oxidative attack. Oxidative stress can induce propagation of a lipid peroxidation (LPO) chain reaction because spermatozoa contain high concentration of unsaturated fatty acids. This study aimed at evaluating in vitro the possible toxicity and/or the antioxidant properties of Propolfenol® in ejaculated human spermatozoa. A colorimetric assay determined the total flavonoid content by spectrophotometry and a high-performance liquid chromatography-diode array detection analysis the quantity of galangin, pinocembrin and caffeic acid phenylethilic ester (CAPE). Sperm parameters such as motility, vitality and DNA integrity were assessed utilising optical microscopy. The antioxidant properties Propolfenol® against LPO induced by tert-Butyl Hydroperoxide were evaluated using the C11-BODIPY581/591 probe. Chemical analysis of Propolfenol® revealed low quantities of galangin, pinocembrin and CAPE; cyclic voltammetry experiments showed that Propolfenol® may exert an antioxidant activity. A protective action of Propolfenol® (20 and 100 µg/ml) on induced LPO in human spermatozoa was detected. Propolfenol® may be proposed as the supplement in media for sperm preparation techniques or cryopreservation to counteract the increased presence of reactive oxygen species generated by these methods.

Can balneotherapy modify microRNA expression levels in osteoarthritis? A comparative study in patients with knee osteoarthritis.

The aim of this study was to evaluate the whole-blood levels of miR-155, miR-223, miR-181a, miR-146a, and miR-let-7e in patients with bilateral knee osteoarthritis (OA) after a cycle of mud-bath therapy (MBT). Thirty-two patients with knee OA defined by the ACR criteria were included. Twenty-one patients (MBT group) were daily treated with a combination of local mud-packs at 42 °C and baths in mineral water, at 37 °C for 15 min, for 12 applications over a period of 2 weeks, in addition to standard therapy; 11 patients (control group) continued their conventional treatment alone. Global pain score evaluated by visual analog scale (VAS), WOMAC subscores, and microRNA expression were evaluated at baseline and after 2 weeks. Peripheral whole blood was collected into PAXgene™ Blood RNA tubes, stored at - 80 °C, and total RNA was extracted. The expression of miR-155, miR-223, miR-181a, miR-146a, and miR-let-7e was determined by qRT-PCR. After MBT, we observed a statistically significant improvement of clinical parameters and a significant decrease of miR-155, miR-181a, miR-146a (p < 0.001), and miR-223 (p < 0.01) expression levels. No clinical and biochemical modifications were detected in the control group. No significant variations of miR-let-7e were shown in both groups after 2 weeks. In conclusion, MBT can modify the expression of miR-155, miR-181a, miR-146a, and miR-223, which are upregulated in OA. It could be due to the heat stress and the hydrostatic pressure, since some miRNAs were found to be temperature- and mechano-responsive. Further studies are needed to better explain the mechanism of action of MBT and the role of miRNAs in OA.

Sperm with fibrous sheath dysplasia and anomalies in head-neck junction: focus on centriole and centrin 1.

Spermatozoa with a rare combination of two monomorphic sperm defects, dysplasia of the fibrous sheath (DFS) and alterations in head-mid-piece junction were analysed. The main focus was to explore the status of the centriole, a key organisation during fertilisation, using the centrin 1, a calcium-binding protein linked to this structure. The sperm quality was examined by light, scanning and transmission electron microscopy (SEM, TEM); immunocytochemistry was performed for tubulin, A-kinase anchor protein 4 (AKAP4) and centrin 1. Spermatozoa showed DFS defect associated with anomalies in head-tail attachment detected by SEM and TEM. Immunolocalisation of tubulin, AKAP4 and centrin 1 confirmed these alterations. Centrin 1 was visible in 67% of spermatozoa (in only 13% centrin localised in a normal position); in the majority of sperm centrin 1's location was altered, sometimes bent; often four spots, indicating the presence of two implantation fossae, were detected. At the centriolar level, immunoreactive fragments, frequently invading the entire short and thick tail, were observed. Centrin 1 is an essential component of the spermatozoa connecting piece and plays a role in centrosome dynamics during sperm morphogenesis and in zygotes and early embryos during spindle assembly. It is important to shed light on these rare conditions in order to better manage the patients during assisted reproductive technology.

One-year follow-up of mud-bath therapy in patients with bilateral knee osteoarthritis: a randomized, single-blind controlled trial.

The objective of this prospective parallel randomized single-blind study was to assess that a cycle of mud-bath therapy (MBT) provides any benefits over usual treatment in patients with bilateral knee osteoarthritis (OA). Patients with symptomatic primary bilateral knee OA, according to ACR criteria, were included in the study and randomized to one of two groups: one group received a cycle of MBT at spa center of Chianciano Terme (Italy) in addition to the usual treatment, and one group continued their regular care routine alone. Clinical assessments were performed 7 days before enrollment (screening visit), at the time of enrollment (basal time), after 2 weeks, and after 3, 6, 9, and 12 months after the beginning of the study. All assessments were conducted by two researchers blinded to treatment allocation. The primary efficacy outcomes were the global pain score evaluated by Visual Analog Scale (VAS) and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) subscore for physical function (W-TPFS). Of the 235 patients screened, 103 met the inclusion criteria: 53 patients were included in the MBT group and 50 in the control group. In the group of patients treated with MBT, we observed a statistically significant (p < 0.001) reduction of VAS and W-TPFS score at the end of the treatment; this improvement was significant (p < 0.05) also at 3 months of follow-up. The control group did not show significant differences between baseline time and all other times. The differences between one group were significant for both primary parameters already from the 15th day and persisted up to the 9th month. This beneficial effect was confirmed by the significant reduction of symptomatic drug consumption. Tolerability of MBT seemed to be good, with light and transitory side effects. Our results confirm that a cycle of MBT added to usual treatment provides a beneficial effect on the painful symptoms and functional capacities in patients with knee OA that lasts over time. Mud-bath therapy can represent a useful backup to pharmacologic treatment of knee OA or a valid alternative for patients who do not tolerate pharmacological treatments.

The experience of West Nile virus integrated surveillance system in the Emilia-Romagna region: five years of implementation, Italy, 2009 to 2013.

Predicting West Nile virus (WNV) circulation and the risk of WNV epidemics is difficult due to complex interactions of multiple factors involved. Surveillance systems that timely detect virus activity in targeted areas, and allow evidence-based risk assessments may therefore be necessary. Since 2009, a system integrating environmental (mosquitoes and birds) and human surveillance has been implemented and progressively improved in the Emilia-Romagna region, Italy. The objective is to increase knowledge of WNV circulation and to reduce the probability of virus transmission via blood, tissue and organ donation. As of 2013, the system has shown highly satisfactory results in terms of early detection capacity (the environmental surveillance component allowed detection of WNV circulation 3­4 weeks before human cases of West Nile neuroinvasive disease (WNND) occurred), sensitivity (capacity to detect virus circulation even at the enzootic level) and area specificity (capacity to indicate the spatial distribution of the risk for WNND). Strong correlations were observed between the vector index values and the number of human WNND cases registered at the province level. Taking into consideration two scenarios of surveillance, the first with environmental surveillance and the second without, the total costs for the period from 2009 to 2013 were reduced when environmental surveillance was considered (EUR 2.093 million for the first scenario vs EUR 2.560 million for the second). Environmental surveillance helped to reduce costs by enabling a more targeted blood unit testing strategy. The inclusion of environmental surveillance also increased the efficiency of detecting infected blood units and further allowed evidence-based adoption of preventative public health measures.

Thermal water of Vetriolo, Trentino, inhibits the negative effect of interleukin-1β on nitric oxide production and apoptosis in human osteoarthritic chondrocyte.

The thermal water of Vetriolo in Trentino, Italy (VW) has been known over 150 years for its therapeutic properties in the treatment of osteoarthritis (OA). This is a highly mineralized water, strongly acidic sulfate, rich in calcium, magnesium and iron and used for balneotherapy after dilution. The aim of our study was to investigate the possible in vitro effects of the VW in human OA chondrocytes cultivated in the presence or in the absence of Interleukin-1 beta (IL-1beta). OA chondrocytes were cultivated in Deionized Water (DW) (DW-DMEM, controls), or in one of three different VW-DMEM media, in which DW had been totally (100 percent) or in part (25 or 50 percent) substituted with VW. All samples were analyzed before and after treatment with IL-1beta at a concentration of 5 ng/ml. After 48 h, we evaluated the cell viability, the release of nitric oxide (NO) in culture medium, the inducible nitric oxide synthase (iNOS) expression, and the percentage of apoptosis and necrosis. Finally, we carried out a morphological assessment using a transmission electron microscope (TEM). Our data showed that VW alone at 25 or 50 percent concentration did not affect the viability of cultured OA chondrocytes, and determined a significant survival recovery rate in cultures stimulated with IL-1beta. On the contrary, the VW alone at 100 percent of concentration reduced, in a significant (P less than 0.05) manner, the cells viability. NO levels were low both in DW-DMEM cultures and in those reconstituted with 25 or 50 percent of VW, and were significantly (P less than 0.05) increased in cultures with 100 percent of VW. VW at 25 or 50 percent concentration significantly (P less than 0.001) reduced the NO production induced by IL-1beta. The data of the NO levels were confirmed by the immunocytochemistry assay for iNOS. Our experiments confirmed the pro-apoptotic effect of IL-1beta and demonstrated a protective effect of VW at 25 or 50 percent concentration. These findings were confirmed by TEM. In conclusion, our study demonstrated that VW alone at 25 or 50 percent concentration modifies neither morphology nor NO production and neither iNOS expression nor apoptosis, but it inhibits the negative effects of IL-1beta in chondrocytes cultures.

TEM and FISH studies in sperm from men of couples with recurrent pregnancy loss.

The role of the male partner in recurrent pregnancy loss (RPL) is not clear. In this study, semen characteristics of 22 men whose partners had experienced RPL were examined by light microscopy. Sperm morphology was analysed by transmission electron microscopy (TEM) and the data were mathematically elaborated to obtain numerical indices expressing the status of an ejaculate: the fertility index and the percentage of apoptosis, necrosis and immaturity. Sperm apoptosis and necrosis were also evaluated by annexin V/propidium iodide assay. To explore the status of meiotic segregation, fluorescence in situ hybridisation (FISH) with probes for chromosomes 18, X and Y, was applied directly on sperm nuclei. Sperm characteristics from a group of men of proven fertility were used as controls. Among the considered sperm characteristics, apoptosis (P < 0.01), 1818YY diploidy (P < 0.05) and 18YY disomy (P < 0.01) scores were significantly higher in men with RPL compared with controls. Our study showed that some patients with normal semen parameters can present a slight increase in aneuploidy compared with controls, indicating a possible involvement of sperm in some cases of RPL. Chromosomal FISH analysis and chromatin tests of sperm could be included in RPL work-ups when no other cause has been detected.

Retrospective assessment of potential negative synergistic effects of varicocele and tobacco use on ultrastructural sperm morphology.

OBJECTIVES: To investigate, in a retrospective study, whether smoking cigarettes increases the effect of varicocele on sperm morphology. METHODS: The semen quality of 2 groups of patients with varicocele were compared, those who smoked (n = 121) and those who did not (n = 158). The semen parameters were evaluated, and sperm morphology was assessed using transmission electron microscopy and quantitatively elaborated (fertility index, immaturity, necrosis, and apoptosis percentages). RESULTS: In the smoker and nonsmoker varicocele-associated cases, sperm motility and the results from transmission electron microscopy analysis were significantly impaired compared with controls. However, a nonsignificant difference was detected when the semen parameters were compared. Subsequently, we divided the patients into 4 groups: mild (> or = 1 but < or = 10 cigarettes/d), moderate (>10 but <20 cigarettes/d), and heavy (> or = 20 cigarettes/d) smokers and a group of randomly chosen nonsmoker patients with varicocele. The sperm motility, sperm concentration, and fertility index decreased and the percentage of sperm pathologic features increased as the number of cigarettes smoked daily increased. CONCLUSIONS: A detrimental effect of cigarette smoking (>10 cigarettes/d) associated with varicocele on sperm motility and morphology was observed. Because much of reduced fecundity associated with smoking may be reversed within 1 year of cessation, as reported in published studies, effective interventions targeted at helping patients quit smoking should be addressed for the benefit of general health and fertility.

The "Brisighella Terme" Project: effectiveness of preventive medicine in thermal ambient. Hematochemical parameters.

BACKGROUND: The main aims of the Brisighella Terme Project are: 1. evaluation of feasibility and effectiveness of preventive medicine intervention in thermal ambient; 2. identification of subjects with cardiovascular risk factors; 3. to give patients informations on risk factor correction. The data of this work concern the hematochemical parameters: CT, HDL-CT, LDL-CT, TG, ApoA1, ApoB, fibrinogen, GOT, GPT, CPK, glucose, uric acid. METHODS: CT, HDL-CT (after precipitation of the non-HDL fractions), TG, glucose, uric acid are evaluated by enzymatic-colorimetric reactions; GOT, GPT, CK by enzymatic method. ApoA1, ApoB, fibrinogen by immunoturbidimetric methods. LDL-CT is calculated by the Friedewald formula. RESULTS: 390 subjects spontaneously adhered to the Brisighella Terme Project. Of these subjects 38% requested laboratory service and we observed a 47% increment, from 1995 to 1996, with regard to this request. Females showed higher mean values than males of these parameters: CT, LDL-CT, HDL-CT, ApoA1, ApoB, fibrinogen, CPK. 55% of females had LDL-CT > 159 mg/dl, values considered high risk for cardiovascular diseases. 36% of males presented HDL-CT < 40 mg/dl, with 2 cases < 25 mg/dl. TG values > 399 mg/dl were found only in males (2%). We identified new cases of hypertriglyceridaemia, hypercholesterolaemia and hypoHD Laemia; these metabolic pathologies had not yet been diagnosed, whereas the patients already knew they were affected by hyperglicaemia or hyperuricaemia. CONCLUSIONS: People's interest in the Brisighella Terme Project, new case identification, the possibility of providing correct information about risk factors and healthy life style confirm the feasibility and effectiveness of preventive medicine in a thermal ambient.

Cardiovascular disease and Lp(a) in the adult population and in the elderly: the Brisighella study.

OBJECTIVE: The aim of this study was to evaluate the distribution of the concentrations of lipoprotein(a) [Lp(a)] in a free-living population, that of Brisighella, and to study the degree of association with cardiovascular disease (CVD) and other associated risk factors. The Brisighella study is included in the framework of observational and interventional longitudinal studies; it began in 1972 to monitor the spontaneous trend of the risk factors for atherosclerosis and to evaluate the incidence of CVD in a rural population. METHODS: The studies were carried out on 1319 subjects, 627 males and 692 females, aged over 14 years, of which 134 men and 113 women were geriatric (age > 64 years); the data are relative to the control of the population in 1988. The following were evaluated for each subject: (a) weight and height; (b) hematological parameters; (c) clinical events; (d) presence of other concomitant diseases. For the dosage of the hematological parameters, enzymatic-colorimetric parameters were used (total and HDL cholesterol, triglycerides, glycemia and uremia), radial immunodiffusion and immunoturbidimetry (apoAI and B), ELISA-sandwich immunoenzymatic method (Lp(a)). All the methods used are standardized and internal and external laboratory quality control was carried out. The data collected were analyzed with the program STATGRAPHIC VERSION 6.0; the mean, the standard deviation and the median were calculated for all the variables. The frequency tables, distribution curves (approximation estimates with the chi 2 test), and single and multiple regression were also calculated. A value of p < 0.01 was taken as the level of significance. RESULTS: The distribution of Lp(a) in the control population and in subjects with CVD was substantially the same for both sexes; the differences between the mean levels of Lp(a) were not statistically significant (18.5 mg/dl vs 20.09 mg/dl for men and 19.98 mg/dl vs 22.78 mg/dl for women). The same also applies to the elderly population (18.81 mg/dl vs 23.31 in the men and 21.13 mg/dl vs 21.47 mg/dl in the women). No significant variations were observed in the mean values of Lp(a) even when other risk factors were taken into consideration, such as hypertension, obesity and diabetes. Finally, multiple regression analysis did not show any correlation between Lp(a) levels and those of the other hematological parameters. CONCLUSIONS: In this transversal study, we found no evidence to suggest that Lp(a) can be considered and independent and predictive risk factor for CVD. It would therefore seem that in the population of Brisighella the levels of Lp(a) are "causally" distributed, without any correlation with the presence of cardiovascular events or with hypertension, diabetes or obesity in both sexes.