Evaluation of reference values of standard semen parameters in fertile Egyptian men.

The reference values of human semen, published in the WHO's latest edition in 2010, were lower than those previously reported. The objective of this study was to evaluate reference values of standard semen parameters in fertile Egyptian men. This cross-sectional study included 240 fertile men. Men were considered fertile when their wives had recent spontaneous pregnancies with time to pregnancy (TTP) ≤12 months. The mean age of fertile men was 33.8 ± 0.5 years (range 20-55 years). The 5th percentiles (95% confidence interval) of macroscopic semen parameters were 1.5 ml for volume and 7.2 for pH. The 5th percentiles of microscopic parameters were 15 million/ml for sperm concentration, 30 million per ejaculate for total sperm count, 50% for total motility, 40% for progressive motility, 62% for vitality, 4% for normal sperm forms and 0.1 million/ml for seminal leucocyte counts. In conclusion, fertile Egyptian men had higher reference values of sperm total motility, progressive motility and vitality, and lower reference values for total sperm counts as compared to those determined by the latest edition of the WHO laboratory manual in 2010. Other semen parameters were identical to those defined by the WHO 2010 manual.

Association between promoter methylation of MLH1 and MSH2 and reactive oxygen species in oligozoospermic men-A pilot study.

MLH1 and MSH2 are important genes for DNA mismatch repair and crossing over during meiosis and are implicated in male infertility. Therefore, the methylation patterns of the DNA mismatch repair genes MLH1 and MSH2 in oligozoospermic males were investigated. Ten oligozoospermic patients and 29 normozoospermic donors were analysed. Methylation profiles of the MLH1 and MSH2 promotors were analysed. In addition, sperm motility and seminal reactive oxygen species (ROS) were recorded. Receiver operating characteristic (ROC) analysis was conducted to determine the accuracy of the DNA methylation status of MLH1 and MSH2 to distinguish between oligozoospermic and normozoospermic men. In oligozoospermic men, MLH1 was significantly (p = .0013) more methylated compared to normozoospermic men. Additionally, there was a significant positive association (r = .384; p = .0159) between seminal ROS levels and MLH1 methylation. Contrary, no association between MSH2 methylation and oligozoospermia was found. ROC curve analysis for methylation status of MLH1 was significant (p = .0275) with an area under the curve of 61.1%, a sensitivity of 22.2% and a specificity of 100.0%. This pilot study indicates oligozoospermic patients have more methylation of MLH1 than normozoospermic patients. Whether hypermethylation of the MLH1 promoter plays a role in repairing relevant mismatches of sperm DNA strands in idiopathic oligozoospermia warrants further investigation.

Phytoandrogenic properties of Eurycoma longifolia as natural alternative to testosterone replacement therapy.

The testosterone deficiency syndrome (TDS) is characterised by numerous symptoms, including low libido, increased fat mass, fatigue, erectile dysfunction or osteoporosis, and up to 80% of men will experience some kind of ageing males' symptoms. This is caused by the age-depending decline in serum testosterone levels with concentrations being about 40-50% lower in men older than 60 years compared with young men. This significant decline in testosterone levels is further closely linked with medical conditions such as obesity, metabolic syndrome, diabetes or hypertension. The conventional way of treating TDS is the testosterone replacement therapy (TRT), for which preparations are on the market. Apart from the beneficial effects of TRT, significant adverse side effects have been described, and prostate cancer (PCa) as absolute contraindication is debated. Eurycoma longifolia (Tongkat Ali; TA) is natural alternative to TRT and has been shown to restore serum testosterone levels, thus significantly improving sexual health. This includes significant positive effects on bone health and physical condition of patients. In addition, a significant antihyperglycaemic effect and cytotoxicity against PCas cells has been shown. Thus far, at therapeutic concentrations, no significant side effects of the treatment were obvious. Therefore, TA might be a safe alternative to TRT.

Effect of the metabolic syndrome on male reproductive function: a case-controlled pilot study.

The metabolic syndrome (MetS) is a constellation of various risk factors. This study aimed to investigate the effect of MetS on testosterone and progesterone, and semen parameters, in a case-controlled pilot study. Male patients (n = 54) had body mass index, waist-to-hip ratio (WHR) and blood pressure recorded. Blood was analysed for HDL cholesterol, triglycerides and glucose. Saliva was assayed for free testosterone and free progesterone. Ejaculates were analysed for volume, sperm concentration, total sperm count, motility, vitality, mitochondrial membrane potential (MMP), DNA fragmentation and leucocyte concentration. Participants were divided into the control group (n = 28) and the MetS group (n = 26). Differences were found between the groups for body mass index, WHR, blood pressure, high-density lipoprotein (HDL), triglycerides and glucose. The MetS group showed significant reductions in sperm concentration (P = 0.0026), total sperm count (P = 0.0034), total motility (P = 0.0291), sperm vitality (P = 0.002), MMP (P = 0.0039), free testosterone (P = 0.0093) and free progesterone (P = 0.0130), while values for DNA fragmentation increased (P = 0.0287). Results indicate that patients with MetS have compromised sperm parameters in the absence of leucocytospermia. A reduction in free progesterone suggests that steroidogenesis cascades may be compromised. It is hypothesised that a systemic pro-inflammatory state with oxidative stress associated with MetS may provide a novel explanation.

Typha capensis (Rohrb.)N.E.Br. (bulrush) extract scavenges free radicals, inhibits collagenase activity and affects human sperm motility and mitochondrial membrane potential in vitro: a pilot study.

The biodiversity in South Africa provides more than 30,000 higher plants, of which more than 3000 are used by traditional healers to treat diseases. Typha capensis (bulrush) is one of the medicinal plants used in South Africa to treat male fertility problems. Considering that South African traditional healers have been recognised by Law and the health benefits of T. capensis have not been scientifically investigated yet, this study aimed at investigating the in vitro effects of aqueous extracts from this plant on male reproductive functions. Both leaves and rhizomes of T. capensis were dried, infused with distilled water and freeze-dried. Motile sperm from 50 men were isolated by swim-up and incubated with 1 µg ml(-1) aqueous extract of Typha rhizome for 1 h at 37 °C. Vitality, motility, sperm production of reactive oxygen species and mitochondrial membrane potential were analysed in the test sample, a control and in the pellet from the swim-up. Results showed that the rhizome extract had significant (P < 0.0001) negative effects on all parameters. The extracts from the leaves and rhizomes revealed dose-dependent inhibitory activity for collagenase and free radical formation. No inhibitory activity for elastase was found. The inhibitory activity for collagenase might indicate possible anti-cancer effects.

Serological evidence of alpha herpesvirus infection in sooty mangabeys.

Contact between sooty mangabeys (SMs) and a pigtailed macaque prompted the serological screening of SMs for evidence of infection with B virus. Serological tests detected SM antibodies that reacted with B virus polypeptides. Additional testing was performed with sera from SMs with no previous contact with macaques. Results from these tests indicated that 56% (33/59) of the SMs had antibodies that reacted with B virus and SA8. SM antibodies also reacted with herpesvirus papio 2 and to a lesser extent with human alpha herpesviruses (HSV-1 and HSV-2). There was an age-related increase in the presence of these antibodies in SMs that was consistent with the serological pattern of reactivity observed in other nonhuman primate species infected with alpha herpesviruses. These data suggest that SMs may be a host for a herpesvirus that is antigenically similar to those viruses present in other Old World nonhuman primates.

Cellular origins of ultraviolet radiation-induced corneal tumours in the grey, short-tailed South American opossum (Monodelphis domestica).

Corneal tumours were induced in almost 100% of grey, short-tailed South American opossums (Monodelphis domestica) exposed three times weekly to ultraviolet radiation (UVR) for periods of a year or more. Five tumours, representing the morphological spectrum of UVR-induced corneal tumours (two fibrosarcomas, one malignant fibrous histiocytoma, one putative haemangiosarcoma, and one squamous cell carcinoma overlying a sarcoma), were assayed immunohistochemically for reactivity with antibodies against the intermediate filaments vimentin, smooth muscle actin (alpha isoform), muscle-specific actins (alpha and gamma isoforms), desmin and cytokeratin, and with antibodies against the vascular endothelial marker von Willebrand factor. The squamous cell carcinoma was cytokeratin-positive. Other tumours were cytokeratin-negative and vimentin-positive. Three tumours had scattered individual cells and groups of cells immunoreactive with antibodies against smooth muscle actin and muscle-specific actins; two tumours (a fibrosarcoma and the malignant fibrous histiocytoma) had small numbers of desmin-positive cells. The putative haemangiosarcoma contained two populations of neoplastic cells, von Willebrand factor-positive vascular endothelial cells and smooth muscle actin-positive spindle cells. It was concluded (1) that UVR-induced corneal tumours may be composed of cells derived from resident epithelial cells, immigrant vascular endothelial cells, or fibroblast-like cells of unknown origin, and (2) that such tumours may contain more than one neoplastic cell type.

Advancement in biochemical assays in andrology.

Determination of markers of sperm function, accessory sex gland secretion and silent male genital tract inflammation is of considerable diagnostic value in the evaluation of male infertility. The introduction of biochemical tests into the analysis of male factor has the advantage that standardized assays with a coefficient of variation characteristic of clinical chemistry are performed, in contrast to biological test systems with a large variability. Biochemical parameters may be used in clinical practice to evaluate the sperm fertilizing capacity (acrosin, aniline blue, ROS), to characterize male accessory sex gland secretions (fructose, alpha-glucosidase, PSA), and to identify men with silent genital tract inflammation (elastase, C'3 complement component, coeruloplasmin, IgA, IgG, ROS).

Scavenging effect of N-acetyl-L-cysteine against reactive oxygen species in human semen: a possible therapeutic modality for male factor infertility?

A new approach to reduce the level of reactive oxygen species (ROS) in human semen by using N-acetyl-L-cysteine (NAC) was evaluated. Semen samples were incubated with or without NAC (1.0 mg ml-1) at room temperature. The chemiluminescent signal of the oxidation of luminol was detected by means of an MTP reader after 0, 20, 40, 60 and 120 min, respectively, using 200 microM luminol. In addition, the dose-dependent action of NAC (0.1, 1.0 and 5.0 mg ml-1) and the influence of NAC on functional sperm parameters (motility and acrosome reaction) were studied. ROS levels decreased significantly after 20 min incubation with NAC. This reduction was greater in the high ROS group (> 30000 counts/10(7) viable sperm at t = 0) than in the low ROS group (< 30000). In addition, a marked dose-dependence of NAC was observed. Concerning sperm function, total sperm motility improved after incubation with NAC, but no significant change was observed with respect to the acrosome reaction. NAC (at concentrations of 1.0 mg ml-1) significantly reduced ROS in human semen and showed the possibility of improving impaired sperm function. After further testing NAC might be useful for the treatment of male infertility patients.

[Intracavitary afterloading therapy as a new technique of boost irradiation in anal canal carcinoma]. / Intrakavitäre Afterloading-Therapie als neue Technik der Boost-Bestrahlung bei Analkanalkarzinomen.

PURPOSE: There are different techniques of boost irradiation in the treatment of patients with anal carcinoma. A new system of applicators is presented, which can be used for an intracavitary afterloading therapy. MATERIAL AND METHODS: Three different applicators are available, the first with a central catheter (K1), a second with 5 semicircular fixed catheters (K2) and an eccentric shield, a third with 8 circular fixed catheters and a central shield (K3). RESULTS: The adequate choice of applicator and catheters takes into consideration the individual localisation and extension of anal carcinoma in planning therapy. Thus, in circular growing tumors, an irradiation of the whole circumference of the anal canal is possible. In non-circular growing tumors, the dose applied in the non-affected part of the anal canal can be reduced to a quarter of the dose applied at the tumor. CONCLUSION: The new system of intracavitary afterloading therapy is a good alternative to previous techniques of boost irradiation in the treatment of anal carcinoma. By means of this technique, irradiation can be highly individualized, the tumor better included and non-affected sections of the anal canal saved.

Genetic and environmental contributions to cardiovascular risk factors in Mexican Americans. The San Antonio Family Heart Study.

BACKGROUND: The familial aggregation of coronary heart disease can be in large part accounted for by a clustering of cardiovascular disease risk factors. To elucidate the determinants of cardiovascular disease, many epidemiological studies have focused on the behavioral and lifestyle determinants of these risk factors, whereas others have examined whether specific candidate genes influence quantitative variation in these phenotypes. METHODS AND RESULTS: Among Mexican Americans from San Antonio (Tex), we quantified the relative contributions of both genetic and environmental influences to a large panel of cardiovascular risk factors, including serum levels of lipids, lipoproteins, glucose, hormones, adiposity, and blood pressure. Members of 42 extended families were studied, including 1236 first-, second-, and third-degree relatives of randomly ascertained probands and their spouses. In addition to the phenotypic assessments, information was obtained regarding usual dietary and physical activity patterns, medication use, smoking habits, alcohol consumption, and other lifestyle behaviors and medical factors. Maximum likelihood methods were used to partition the variance of each phenotype into components attributable to the measured covariates, additive genetic effects (heritability), household effects, and an unmeasured environmental residual. For the lipid and lipoprotein phenotypes, age, gender, and other environmental covariates accounted in general for < 15% of the total phenotypic variance, whereas genes accounted for 30% to 45% of the phenotypic variation. Similarly, genes accounted for 15% to 30% of the phenotypic variation in measures of glucose, hormones, adiposity, and blood pressure. CONCLUSIONS: These results highlight the importance of considering genetic factors in studies of risk factors for cardiovascular disease.

The monoclonal antibody GZS-1 detects a maturation-associated antigen of human spermatozoa that is also present on the surface of human mononuclear blood cells.

A monoclonal antibody (GZS-1) has been generated by fusion of mouse myeloma cells with spleen cells from BALB/c mice immunised with human sperm cells. The antibody was determined to be an IgG1. The corresponding antigen is present on the whole surface of ejaculated human spermatozoa. It is not detectable on spermatozoa of other mammalian species (rabbit, cat, dog, sheep, boar, bull, horse). In human male genital organs, immunostaining with GZS-1 is observed on sperm cells in the epididymis and the ductus deferens together with the lining epithelium of those organs. No reactivity of sperm cells or germ cell precursors in the testis has been detected. Functional tests using the antibody show a strong inhibitory effect of human sperm in the hamster egg penetration assay. Furthermore, the GZS-1 antigen is detectable on the surface of human lymphocytes and monocytes by immunogold electron microscopy and FACS analysis. By Western blotting of human sperm and seminal plasma performed under reducing conditions immunostaining was detected at 21-25, 31, 51-54, and 62 kDa. The reaction with human lymphocytes shows one major band at 62 kDa and additional bands at 31 and 54 kDa. The results suggest that the monoclonal antibody GZS-1 may recognise an antigen which is secreted from the epithelial cells of the epididymis and binds to ejaculated spermatozoa as a sperm coating antigen. This component may be involved in the capacitation of the sperm and the acrosome reaction. Molecules that are expressed both on sperm and on immunocompetent cells may be relevant for the regulation of immunological processes or for the development of the related immunological tolerance of sperm in the female reproductive tract.

[Parenteral S-adenosylmethionine compared to placebos in the treatment of alcoholic liver diseases]. / SAMe parenteral en el tratamiento de la hepatopatía alcohólica comparativo contra placebo.

The improvements in the knowledge of the action of ethanol over the hepatic cell, its direct action over the cell, and the intracytoplasmatic structures membranes, point out the possibilities of use of sulfo-adenosil-L-metionina (SAMe); as an util drug inn the treatment of the altered metilation reactions, that take place in those membranes, facilitating their physiological functions. The primary end point in this study was to demonstrate the therapeutic worth os SAMe, by parenteral route in 45 patients with alcoholic liver disease, which were determined by clinical laboratory and hepatic function test, label qith 32 points or more of the discriminatory function index. Divided into two groups, placebo-SAMe, randomized, double blind. As well as total plasmatic and reduced glutation and lipoperoxidation index, indirect form as malondehaldehyde. Were determined at the first visit anf after 8 and 15 days of treatment. Comparing the results of both groups there were a significative favorable results for the group treatment with SAMe and this confirms the utility of this drug in the treatment of patients with alcoholic liver disease with a discriminatory function index (Maddrey index), of 32 points or more.

Further indications of the multicomponent nature of the acrosome reaction-inducing substance of human follicular fluid.

Human follicular fluid (hFF), which has been treated with either unspecific proteases or dextran-coated charcoal (DCC) to remove proteins and/or steroids, cannot successfully induce the acrosome reaction (AR). After the removal of steroids, AR-inducing activity can be restored to hFF by supplementation with exogenous progesterone, but only in the presence of intact protein. Gel filtration experiments with 3H-progesterone-labelled hFF showed elution of the radioactive signal in the high molecular weight range, corresponding to bound progesterone. AR-inducing activity was seen in exactly the same fraction. Based on these results, the acrosome reaction-inducing substance (ARIS) appears to be a complex of progesterone and a progesterone-binding protein, which was shown to be identical with the plasma protein corticosteroid-binding globulin (CBG) by immunological techniques. AR induction was only observed in the presence of both CBG and progesterone, suggesting a combined effect of the two components.

An efficient method for routine Epstein-Barr virus immortalization of human B lymphocytes.

A variety of methods exist for the immortalization of B lymphocytes by Epstein-Barr virus due to the simplicity of such techniques to establish cell lines with stable genomic DNA. Two different methods for immortalizing lymphoblastoid cell lines were compared for differences in techniques and materials, time between initiation and immortalization, and success rate of immortalization. An incubation period in Epstein-Barr virus and the use of conditioned media improved immortalization efficiency from 86 to 98% and decreased the time (usually weeks) from culture initiation to cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in non-insulin-dependent diabetes mellitus.

Selective capacity of glass-wool filtration for the separation of human spermatozoa with condensed chromatin: a possible therapeutic modality for male-factor cases?

PURPOSE: The aim of this study was to evaluate chromatin condensation of human spermatozoa following swim-up compared to glass-wool separation. Semen aliquots from men attending an andrological outpatient clinic were processed by means of a swim-up procedure and glass-wool filtration. Chromatin condensation was recorded using aniline blue staining and results were reported according to color intensity of stained sperm heads. Morphometric measurements of sperm heads were performed on stained sperm samples. RESULTS: Glass-wool filtration resulted (i) in a significantly higher total motile sperm count (P < 0.0005) compared to swim-up and (ii) in a significantly higher percentage of normal chromatin-condensed spermatozoa compared to the ejaculate (P < 0.01). CONCLUSION: In contrast, comparing swim-up to the ejaculate, the percentage of matured nuclei (unstained spermatozoa) retrieved following swim-up was significantly lower (P < 0.005). Glass-wool filtration separates human spermatozoa according to motility and size of the sperm head. The size of the sperm head closely correlated with the chromatin condensation quality.

Biochemical and immunological characterization of the acrosome reaction-inducing substance (ARIS) of hFF.

Gel filtration experiments with 3H-progesterone labeled hFF demonstrated that the AR-inducing activity of hFF might be mediated by a progesterone-binding protein. Further immunological investigations added evidence in support to ARIS being identical with the SERPIN CBG. Moreover, only the protein-progesterone-complex was able to induce AR. It is suggested that the CBG-progesterone-complex is proteolytically cleaved at the plasma membrane of spermatozoa, releasing a high local concentration of progesterone which leads to induction of the AR.

Correlated expression of atrial myosin heavy chain and regulatory light chain isoforms with pressure overload hypertrophy in the non-human primate.

OBJECTIVE: The aim was to determine the extent to which myosin heavy chain and light chain isoform transitions in atrial myocardium are coordinately regulated under pathological conditions in tissue from normal baboons, hypertensive baboons with myocardial hypertrophy, and baboons in which hypertrophy had regressed. METHODS: Quantitative distributions of myosin heavy chain (MHC) and regulatory myosin light chain (MLC2) isoforms in atrial myocardium from 35 adult baboons were determined by electrophoresis under denaturing conditions and laser densitometry. RESULTS: A significant association was observed between the ratios of MHC and MLC2 isoforms in atrial myocardium (r = 0.73, p < 0.001, n = 69). Expressions of alpha MHC and atrial MLC2 (ALC2) isoforms were correlated in atrial myocardium, as were those of beta MHC and ventricular MLC2 (VLC2) isoforms. In a subset of baboons with experimentally induced renal hypertension (n = 12) both beta MHC and VLC2 isoforms were found at higher levels in left atria than were present in normotensive baboons (p = 0.006, n = 15). Left atria from hypertensive baboons with regressed LVH contained intermediate levels of both beta MHC and VLC2 isoforms. CONCLUSIONS: There is tight coupling between the expression of myosin subunit isoforms under pathological conditions from a primate species closely related to humans. The data suggest that the synthesis of these subunits of myosin may be coordinated at the molecular level.

Ultrastructure, protein synthesis and secretion of day-6 rabbit blastocysts cultured in a chemically defined, protein-free medium.

Day-6 rabbit blastocysts were cultured in Ham's F10 medium supplemented with polyvinylpyrrolidone as a macromolecular component, for 4 to 12 h. The integrity of the blastocyst cells was demonstrated by electron microscopy. Expansion and biosynthesis of proteins and of DNA were studied after culturing in the presence of 35S-methionine and 3H-thymidine. Polyvinylpyrrolidone did not interfere with the subsequent protein analysis, which was performed by two dimensional gel electrophoresis followed by silver staining and fluorography. More than 600 labelled proteins were found in the blastocyst tissue, many of them were also present in the blastocyst fluid and in the blastocyst coverings. Several proteins seemed to be produced for incorporation into the blastocyst coverings; others, only detected in the culture medium, might have been synthesized for secretion into the environment.