AIMTo perform screening, related to A-kinase anchoring proteins 4 (AKAP4) and tubulin proteins, in spermatozoa with absent or severely reduced motility in order to detect the status of the fibrous sheath and the axonemal structure.

METHODSAn immunocytochemical study of tubulin, used as a positive control, and AKAP4 was carried out to detect the presence and the distribution of these proteins in different sperm samples. The morphological characteristics of sperm were studied by transmission electron microscope (TEM) and the results were elaborated using a formula reported in previous studies. PCR was carried out on DNA extracted from peripheral blood lymphocytes to analyse partial sequences of the Akap4 and Akap3 genes.

RESULTSImmunolabelling of tubulin and AKAP4 showed different patterns, which led us to divide the patients into groups. In group I, the absence of AKAP4 and tubulin was revealed, although these patients did not show alterations in the Akap4/Akap3 binding site. TEM evaluation highlighted that a high presence of necrosis was associated with total sperm immotility. In group II, a regular AKAP4 and tubulin signal was present, although motility was reduced and TEM analysis revealed the presence of immaturity. In group III, in which a weak AKAP4 label associated with normal tubulin staining and reduced motility was observed, a severe disorganization of the fibrous sheath was highlighted by TEM.

CONCLUSIONWhile the role of AKAP4 in sperm motility is unclear, absent or weak AKAP4-labelling seems to be associated with absent or weak sperm motility.

A Kinase Anchor Proteins ; Humans ; Infertility, Male ; genetics ; physiopathology ; Male ; Microscopy, Electron ; Polymerase Chain Reaction ; Protein Precursors ; genetics ; metabolism ; Semen ; physiology ; Sperm Motility ; Spermatozoa ; physiology ; ultrastructure ; Tubulin ; genetics ; metabolism

We analysed ejaculated spermatozoa from five infertile men with different balanced reciprocal translocations to contribute to the study of meiotic segregation of chromosomes 18, X and Y and also to evaluate sperm morphology by transmission electron microscopy (TEM) analysis. Conventional lymphocyte karyotype analyses highlighted different reciprocal balanced translocations: t(12;13), t(4;9), t(X;8), t(8;10) and t(3;16). Semen analysis was performed by light and TEM. Fluorescence in situ hybridization was performed directly on sperm nuclei using centromeric probes for chromosomes 18, X and Y. The carriers of the balanced reciprocal translocations considered in the present study showed a very similar pattern of sperm pathologies: diffused presence of apoptosis and immaturity. All patients showed meiotic segregation derangements, highlighted by the presence of sperm diploidies and sex chromosome disomies particularly related to the failure of the first meiotic division. However, an increased incidence of chromosome 18 aneuploidy was detected in spermatozoa from t(X;8) and t(8;10) carriers. We have also reported values from sex chromosomes such as t(X;8), although the X chromosome was involved in translocation. Since patients with reciprocal translocations and spermatogenetic impairment are candidates for intracytoplasmic sperm injection cycles, the study of sperm parameters, and particularly of the level of aneuploidy rates, would provide better information for couples at risk and would contribute to the data in the literature for a better understanding of the effects of chromosomal rearrangement on the whole meiotic process and, in particular, on chromosomes not involved in translocation.
Adult ; Aneuploidy ; Apoptosis ; Chromosome Banding ; Chromosomes, Human, X ; Chromosomes, Human, Y ; Humans ; In Situ Hybridization, Fluorescence ; Infertility, Male ; genetics ; pathology ; Lymphocytes ; physiology ; Male ; Microscopy, Electron, Transmission ; Spermatozoa ; pathology ; ultrastructure ; Translocation, Genetic

Objective: Objective of this review is to summarize the currently available information on possible chondroprotective effects of mineral waters or mineral components on chondrocyte or cartilage cultures. Methodology: We conducted a search of the literature by PubMed and Scopus (the period examined was 1980-2013) using the terms “chondrocyte” and/or “cartilage” in combination with ”mineral water”, “hydrogen sulphide”, “sulphur hydrogen”. Results: A chondroprotective role of mineral water or mineral components was demonstrated by some pilot studies in chondrocyte cultures. Burguera1) studied the activity of hydrogen sulphide (H2S) in human osteoarthritic (OA) chondrocytes stimulated with Interleukin (IL)-1β. They analyzed the effects of different concentrations of a fast (NaHS) or a slow (GYY4137) release H2S donor demonstrating a significant reduction of Nitric Oxide (NO), Prostaglandin(PG)-E2, and Reactive Oxygen Species (ROS) levels in culture medium and of inducible Nitric Oxide Synthase (iNOS) gene expression, induced by IL-1β. These data were confirmed by Li2) in normal human chondrocytes stimulated by Lipopolysaccharide(LPS). GYY4137 decreased LPS-induced production of NO, PGE2, Tumor Necrosis Factor (TNF)-α and IL-6, reduced the levels and catalytic activity of iNOS and of Cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-kB activation. Furthermore GYY4137 showed a strong inhibition on oxidative stress-induced cell death3). The incubation of chondrocytes cell line C-28/I2 with another H2S donor, Natrium Hydrogen Sulphide (NaHS), proved that constitutive as well as IL-1β-induced IL-6 and IL-8 expression was partially and transiently blocked by the NaHS4). Fioravanti studied the chondroprotective role of highly mineralized water, strongly acidic sulfate, rich in calcium, magnesium and iron [Vetriolo’s thermal water (VW)] in human OA chondrocytes cultivated with or without IL-1β5). For this purpose 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%) or in part (50% or 25%) substituted with VW. The results showed that VW alone at 25% or 50% concentration did not affect the viability of cultured chondrocytes, and determined a significant survival recovery rate in cultures stimulated with IL-1β. NO levels were low both in DW-DMEM cultures and in those reconstituted with 25% or 50% of VW, and were significantly increased by IL-1β. VW at 25% or 50% concentration significantly reduced the NO production induced by IL-1β. The data of NO levels were confirmed by the immunocytochemistry assay for iNOS. Furthermore, the authors demonstrated a protective effect of VW at 25% or 50% concentration on IL-1β-induced apoptosis. Conclusions: Presented data are stimulating, but we don’t ignore the existence of a complex series of problems and limitations. One of the critical points is the controversial problem of the absorption of the minerals dissolved in mineral waters, furthermore, extrapolation of in vitro results to in vivo should be undertaken with caution.   Further studies are needed in vitro to confirm these preliminary findings.

Objective: To assess both the short-term and the long-term effectiveness of spa therapy in patients with primary knee osteoarthritis (OA) in a prospective, randomized, single blind, controlled trial. Materials and Methods: 103 outpatients with OA of the knee according to the ACR criteria (1) were enrolled. Patients were randomized 1 : 1 and allocated into two groups: 53 patients (Group A) received in addition to usual treatment (exercise, NSAIDs and/or analgesics, established SYSADOAs) a combination of daily local mud-packs applied on both knees for 20 min at an initial temperature of 45°C and bicarbonate-sulphate-calcic mineral bath water at 38°C for 15 min, from the spa centre of Chianciano Terme (Siena, Italy) for 12 applications carried out over a period of 2 weeks. 50 patients (Group B, controls) continued routine ambulatory care. Clinical assessments were performed at basal time after 2 weeks, after 3, 6, 9 and 12 months - end of the study. The primary outcome criteria were the change from baseline to month 12 in WOMAC - Total Pain Score (W-TPS) (range 0-20)* and in WOMAC - Total Physical Function Score (W-TPFS) (range 0-68)* scored by a 5-point Likert scale. A set of secondary outcomes was also assessed such as WOMAC Total Stiffness Score (W-TSS), Physical Component Summary (PCS) and Mental Component Summary (MCS) of SF-12 (ranges 0-100) and consumption of analgesic medications (paracetamol and/or NSAIDs). Results: Ten patients (9.5%) withdrew from the study: 2 in the spa-group and 8 (16%) in the control group. The assessment of pain showed a very significant improvement (p<0.001) in patients treated with mud-packs until 6 months and a less significant reduction (p<0.05) after 9 and 12 months. The control group showed a significant improvement after 2 weeks and 3 months, however this improvement is less expressed than in group A.   The differences between the two groups were significant already from 2 weeks and lasted during the follow-up.   A similar trend was observed for the WOMAC - Physical Function in the group A, group B showed a significant worsening after 6 months persisting throughout the follow-up.   The results obtained from the quality of life, SF-12 survey showed a significant improvement (p<0.001) in Physical Component, persisting throughout the follow-up period in group A. No significant modifications were found in group B during the study period.   On the contrary, significant improvement of the Mental Component Summary of SF-12 was shown at the end of the therapy in group A, but no significant differences were observed in the other time of the follow-up.   These effect on pain and function were also confirmed by the observed reduction of symptomatic drugs consumption. Tolerability of spa therapy seemed to be good, with light and transitory side effects. Conclusions: In conclusion our results, in keeping with other studies (2, 3) confirm that the beneficial effects of mud-bath therapy in patients with knee OA last over time, with significant reduction on the painful symptomatology and a significant improvement on functional capacities and on quality of life. Spa therapy can represent a useful backup to pharmacological treatment of knee OA or a valid alternative for patients who do not tolerate pharmacological treatments.