Parameters of the Mouse Embryo Assay that affect detection of peroxides in mineral oil.

RESEARCH QUESTION: Can culture conditions influence the sensitivity of a Mouse Embryo Assay and its potential to detect peroxide-related toxicity in mineral oil samples? DESIGN: Protein type and concentration, embryo density and culture dish design were selected as the variables in the culture system with the potential to influence the assay's sensitivity. Fresh 1-cell mouse embryos were cultured under mineral oil samples with known peroxide concentrations. Protein type (human serum albumin [HSA] + α/ß-Globulins versus HSA versus bovine serum albumin [BSA]), concentration (5 mg/ml versus 0.5 mg/ml), embryo density (25 versus 3 µl/embryo) and culture dish (Petri versus micro-well dish) were adjusted to define the culture conditions with the highest sensitivity. RESULTS: High concentrations of peroxides can be easily detected by current quality control standards. However, for oil samples with a lower concentration of peroxides, supplementing the culture medium with 5 mg/ml of HSA + alpha/beta-globulins or with HSA resulted in an increased detection of embryo toxicity compared with when BSA was used as the protein supplement. The sensitivity of the assay was greatly reduced when embryos were cultured in groups and when certain micro-well dishes were used. CONCLUSIONS: Current quality control protocols may not be sensitive enough to identify low concentrations of peroxides, which, if undetected, can increase over time and become potentially harmful during gamete and embryo culture. The different parameters established in this study allow the sensitivity of the Mouse Embryo Assays to be optimized to specifically detect peroxides in mineral oil samples prior to their release into the market and their broad use in human IVF.

Genetic variations in the androgen receptor are associated with steroid concentrations and anthropometrics but not with muscle mass in healthy young men.

OBJECTIVE: The relationship between serum testosterone (T) levels, muscle mass and muscle force in eugonadal men is incompletely understood. As polymorphisms in the androgen receptor (AR) gene cause differences in androgen sensitivity, no straightforward correlation can be observed between the interindividual variation in T levels and different phenotypes. Therefore, we aim to investigate the relationship between genetic variations in the AR, circulating androgens and muscle mass and function in young healthy male siblings. DESIGN: 677 men (25-45 years) were recruited in a cross-sectional, population-based sibling pair study. METHODS: Relations between genetic variation in the AR gene (CAGn, GGNn, SNPs), sex steroid levels (by LC-MS/MS), body composition (by DXA), muscle cross-sectional area (CSA) (by pQCT), muscle force (isokinetic peak torque, grip strength) and anthropometrics were studied using linear mixed-effect modelling. RESULTS: Muscle mass and force were highly heritable and related to age, physical activity, body composition and anthropometrics. Total T (TT) and free T (FT) levels were positively related to muscle CSA, whereas estradiol (E2) and free E2 (FE2) concentrations were negatively associated with muscle force. Subjects with longer CAG repeat length had higher circulating TT, FT, and higher E2 and FE2 concentrations. Weak associations with TT and FT were found for the rs5965433 and rs5919392 SNP in the AR, whereas no association between GGN repeat polymorphism and T concentrations were found. Arm span and 2D:4D finger length ratio were inversely associated, whereas muscle mass and force were not associated with the number of CAG repeats. CONCLUSIONS: Age, physical activity, body composition, sex steroid levels and anthropometrics are determinants of muscle mass and function in young men. Although the number of CAG repeats of the AR are related to sex steroid levels and anthropometrics, we have no evidence that these variations in the AR gene also affect muscle mass or function.

An Improved Neutral a-Glucosidase Assay for Assessment of Epididymal Function-Validation and Comparison to the WHO Method.

Neutral a-glucosidase (NAG) activity in human seminal plasma is an important indicator for epididymis functionality. In the present study, the classic World Health Organization (WHO) method has been adapted to enhance assay robustness. Changes include modified enzyme reaction buffer composition and usage of an alternative enzyme inhibitor for background correction (glucose instead of castanospermine). Both methods have been tested in parallel on 144 semen samples, obtained from 94 patients/donors and 50 vasectomized men (negative control), respectively. Passing-Bablok regression analysis demonstrated equal assay performance. In terms of assay validation, analytical specificity, detection limit, measuring range, precision, and cut-off values have been calculated. These data confirm that the adapted method is a reliable, improved tool for NAG analysis in human semen.

Health promotion intervention in mental health care: design and baseline findings of a cluster preference randomized controlled trial.

BACKGROUND: Growing attention is given to the effects of health promotion programs targeting physical activity and healthy eating in individuals with mental disorders. The design of evaluation studies of public health interventions poses several problems and the current literature appears to provide only limited evidence on the effectiveness of such programs. The aim of the study is to examine the effectiveness and cost-effectiveness of a health promotion intervention targeting physical activity and healthy eating in individuals with mental disorders living in sheltered housing. In this paper, the design of the study and baseline findings are described. METHODS/DESIGN: The design consists of a cluster preference randomized controlled trial. All sheltered housing organisations in the Flanders region (Belgium) were asked if they were interested to participate in the study and if they were having a preference to serve as intervention or control group. Those without a preference were randomly assigned to the intervention or control group. Individuals in the intervention group receive a 10-week health promotion intervention above their treatment as usual. Outcome assessments occur at baseline, at 10 and at 36 weeks. The primary outcomes include body weight, Body Mass Index, waist circumference, and fat mass. Secondary outcomes consist of physical activity levels, eating habits, health-related quality of life and psychiatric symptom severity. Cost-effectiveness of the intervention will be examined by calculating the Cost-Effectiveness ratio and through economic modeling.Twenty-five sheltered housing organisations agreed to participate. On the individual level 324 patients were willing to participate, including 225 individuals in the intervention group and 99 individuals in the control group. At baseline, no statistical significant differences between the two groups were found for the primary outcome variables. DISCUSSION: This is the first trial evaluating both the effectiveness and cost-effectiveness of a health promotion intervention targeting physical activity and healthy eating in mental health care using a cluster preference randomized controlled design. The baseline characteristics already demonstrate the unhealthy condition of the study population. TRIAL REGISTRATION: This study is registered at clinicaltrials.gov - NCT 01336946.

Sex hormone-binding globulin as an independent determinant of cortical bone status in men at the age of peak bone mass.

CONTEXT: Sex steroids are important determinants of the skeletal development, growth, and maintenance after achievement of peak bone mass. A large fraction of these hormones are bound by SHBG, and previous studies have shown that SHBG could be a determinant of bone characteristics. OBJECTIVE: We investigated associations of serum SHBG levels with cortical and trabecular bone characteristics in young healthy men. DESIGN AND SETTINGS: A total of 677 healthy male siblings aged 25-45 yr were recruited in a cross-sectional, population-based study. MAIN OUTCOMES: Areal bone parameters were assessed using dual-energy x-ray absorptiometry. Cortical bone parameters at the tibia and radius and trabecular vBMD at the radius were assessed using peripheral quantitative computed tomography. Serum testosterone, estradiol, and SHBG levels were measured using immunoassays. RESULTS: Regression models including age, height, and weight showed that SHBG levels were positively associated with bone area at the hip and the whole body, but not with areal bone mineral density (BMD). Higher SHBG levels were associated with a larger cortical bone area and periosteal and endosteal circumferences at both the tibia and the radius, whereas trabecular volumetric BMD at the radius was negatively associated with SHBG levels. Associations persisted after adjustment for (free) sex steroid levels. No associations were found with cortical volumetric BMD or cortical thickness. CONCLUSION: In this population of healthy adult men at the age of peak bone mass, SHBG levels were positively associated with cortical bone size, independently from sex-steroid levels. This suggests a possible independent role of SHBG in the determination of adult bone size.

Birth weight in relation to sex steroid status and body composition in young healthy male siblings.

CONTEXT: Sex steroid concentrations have a strong genetic determination, but environmental factors and body composition play an important role. From studies in children with intrauterine growth restriction, low birth weight has been associated with altered gonadotropin concentrations. OBJECTIVE: We aim to investigate sex steroid concentrations in healthy young brothers in relation to birth weight (normal gestational age), body composition, and parental steroid concentrations. DESIGN AND SETTING: We conducted a cross-sectional, population-based sibling pair study with inclusion of parental data. PARTICIPANTS: A total of 677 men (25-45 yr old) were included in this study, with 296 independent pairs of brothers and 122 fathers. MAIN OUTCOMES: We measured testosterone, estradiol, leptin, adiponectin, IGF-I (immunoassays), and free steroid hormones (calculated) in relation to birth weight and changes in body composition (dual-energy x-ray absorptiometry). RESULTS: Birth weight was associated with serum testosterone (P = 0.0004) and SHBG (P = 0.0001), independent from weight, age, or fat mass, whereas no association with (free) estradiol, LH, or FSH was found. Paternal testosterone (P = 0.02), estradiol (P = 0.04), and SHBG (P = 0.0004) were associated with the respective sex steroid concentrations in the brothers. Weight increase (population rank) during life, was associated with lower testosterone (-15%; P < 0.001), independent from current weight and with higher free estradiol concentrations (+8%; P = 0.002), whereas weight decrease was associated with higher testosterone (+13%; P < 0.001). CONCLUSION: Birth weight and paternal steroid concentrations are associated with testosterone concentrations, independent from adult weight. These findings support the concept of in utero programming across the range of birth weight.

Early smoking is associated with peak bone mass and prevalent fractures in young, healthy men.

Smoking is associated with lower areal bone mineral density (aBMD) and higher fracture risk, although most evidence has been derived from studies in elderly subjects. This study investigates smoking habits in relation to areal and volumetric bone parameters and fracture prevalence in young, healthy males at peak bone mass. Healthy male siblings (n = 677) at the age of peak bone mass (25 to 45 years) were recruited in a cross-sectional population-based study. Trabecular and cortical bone parameters of the radius and cortical bone parameters of the tibia were assessed using peripheral quantitative computed tomography (pQCT). Areal bone mass was determined using dual energy X-ray absorptiometry (DXA). Sex steroids and bone markers were determined using immunoassays. Prevalent fractures and smoking habits were assessed using questionnaires. Self-reported fractures were more prevalent in the current and early smokers than in the never smokers (p < .05), with a fracture prevalence odds ratio for early smokers of 1.96 (95% confidence interval 1.18-3.24) after adjustment for age, weight, educational level, and alcohol use and exclusion of childhood fractures. Current smoking was associated with a larger endosteal circumference (beta = 0.027 +/- 0.009, p = .016) and a decreased cortical thickness (beta = -0.034 +/- 0.01, p = .020) at the tibia. In particular, early smokers (< or =16 years) had a high fracture risk and lower areal BMD, together with a lower cortical bone area at the tibia and lower trabecular and cortical bone density at the radius. An interaction between free estradiol and current smoking was observed in statistical models predicting cortical area and thickness (beta = 0.29 +/- 0.11, p = .01). In conclusion, smoking at a young age is associated with unfavorable bone geometry and density and is associated with increased fracture prevalence, providing arguments for a disturbed acquisition of peak bone mass during puberty by smoking, possibly owing to an interaction with sex steroid action.

Small effect of the androgen receptor gene GGN repeat polymorphism on serum testosterone levels in healthy men.

OBJECTIVE: The human androgen receptor (AR) contains a polyglutamine and a polyglycine stretch which are highly polymorphic and are coded respectively by a CAG and GGN repeat in exon 1 of the AR gene. Although the in vitro studies indicated a possible effect of the GGN repeat polymorphism on the AR gene transcription and clinical observations suggest that it might modulate the androgen action, its functional significance remains unclear. We wanted to assess whether the GGN repeat affects the serum testosterone levels in healthy men, which is the expected outcome through feedback regulation if it influences androgen action as has been shown to be the case for the CAG repeat. DESIGN AND PATIENTS: A population based cross-sectional cohort study including 1476 healthy young, middle-aged, and elderly men. MEASUREMENT: Testosterone and LH levels were determined by immunoassay; free testosterone (FT) levels were calculated. Genotyping of the GGN repeat was performed using the sequencing technique. RESULTS: The GGN repeat number was significantly associated with circulating testosterone and FT levels (P=0.017 and P=0.013 respectively). However, taking into account that age, body mass index, and CAG are already in the regression model, the GGN repeat could explain only a small part of the variation of both testosterone and FT. CONCLUSION: To our knowledge, this study is the first to demonstrate a significant positive association between the GGN repeat and androgen levels in a large cohort of healthy men. Although the present study thus adds credence to the view that the polyglycine tract in the AR can modulate AR action, this effect appears to be only small so that its clinical relevance remains questionable.

Fat mass is negatively associated with cortical bone size in young healthy male siblings.

CONTEXT: Body weight has been associated with bone mass and bone size through shared genetic determination and environmental influences. Whereas lean mass exerts a positive influence on bone size, the relationship between fat and bone remains unclear. OBJECTIVE: The objective of the present study was to investigate the individual influence of fat mass and lean mass on volumetric bone density and size in young healthy male siblings at age of peak bone mass. DESIGN: This was a cross-sectional, population-based sibling pair study. PARTICIPANTS: A total of 677 men (25-45 yr) were included in this study with 296 independent pairs of brothers. MAIN OUTCOME MEASURES: Areal and volumetric bone parameters were determined using dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). Body composition was determined by DXA. Sex steroids, leptin, and adiponectin were determined by immunoassay. RESULTS: Total and regional fat mass were found to be inversely associated with areal bone mass and bone size, independent from lean mass (radius periosteal circumference beta: -0.29 +/- 0.04; P < 0.001). Lean mass was positively associated with bone size but inversely with cortical density at both tibia and radius (P < 0.01). The negative association between total fat mass and bone size was independent from sex steroid concentrations. Leptin but not adiponectin was inversely associated with bone size, but this was no longer significant after adjustment for body fat. CONCLUSIONS: Increased fat mass is associated with smaller bone size, challenging the view of a high bone mass index as a protective factor for osteoporosis, whereas lean mass was a consistent positive determinant of bone size.

Serum estradiol is associated with volumetric BMD and modulates the impact of physical activity on bone size at the age of peak bone mass: a study in healthy male siblings.

This study investigates determinants of peak bone mass (PBM) in healthy men, focusing on effects and interactions of parameters reflecting mechanical loading and sex steroids. Healthy male siblings (n = 677; 25-45 yr) were recruited in a cross-sectional, population-based study. Physical activity score was assessed by a self-reported questionnaire. Cross-sectional muscle area (CSMA) and bone parameters of radius (4% and 66% site) and tibia (66% site) were assessed using pQCT. Peak torque of biceps and quadriceps muscles was assessed by isokinetic dynamometry. Serum testosterone (T) and estradiol (E(2)) levels were measured using immunoassays; free hormone fractions were calculated. Relations between indices of bone strength, CSMA, muscle strength, and sex steroids were studied using linear mixed-effects modeling. Physical activity, CSMA, and muscle strength were positively associated with indices of bone strength, except for volumetric BMD (vBMD). After controlling for age, weight, and height, free E(2) levels were positively associated with trabecular and cortical vBMD, negatively associated with endosteal circumference at the radius, and positively associated with cortical vBMD at the tibia. In addition, positive interactions between physical activity and serum E(2) concentrations were observed for bone size at the tibia. No associations between free T levels and pQCT bone parameters were found. In this population of healthy men at the age of PBM, parameters reflecting mechanical loading are confirmed as important determinants of bone size. E(2), but not T, levels are positively associated with vBMD and modulate the impact of physical activity on bone size at the tibia.

Polymorphisms of the SHBG gene contribute to the interindividual variation of sex steroid hormone blood levels in young, middle-aged and elderly men.

OBJECTIVE: In men there is a large interindividual variation of SHBG levels and consequently of testosterone (T) and E(2) levels. Family and twin studies suggested a strong genetic contribution, besides metabolic and hormonal influences. The aim of this study was to examine the influence of a missense mutation in exon 8 (Asp327Asn) and a (TAAAA)(n)-repeat in the promoter region of the SHBG gene, on SHBG and sex steroid serum concentrations in a population of healthy men. DESIGN: SHBG and hormone levels were measured in 1485 men, contributed by three independent cohort studies and representing three different age groups (young, middle-aged and elderly men). The number of TAAAA-repeats was determined by fragment-analysis; carriers of the Asn(327)-allele were identified using restriction fragment length polymorphism analysis. RESULTS: In the different age groups, carriers of six TAAAA-repeats presented with higher SHBG (young 19%, middle-aged 20% and elderly 26%; P < 0.001) and T (young 9%, middle-aged 22% and elderly 21%; P < 0.05) levels compared to non-carriers. For free T, a modest increase was found for carriers in the middle-aged group, but not for the young and elderly group. E(2) and free E(2) did not differ between carriers and non-carriers in the different age-groups. The Asn(327)-allele was associated with higher mean SHBG (14.20%, P < 0.001) and T levels (7.33%; P = 0.01) in the middle-aged group only. CONCLUSIONS: Our findings show that and the (TAAAA)(n)-repeat and the Asp327Asn polymorphism contribute to the genetically determined interindividual variation in total serum T levels in healthy men through variation in SHBG concentrations.

Heritability of blood concentrations of sex-steroids in relation to body composition in young adult male siblings.

OBJECTIVE: Sex steroid concentrations in men are related to body composition and both are determined by genetic and environmental factors. This study investigates heritability estimates of sex steroid serum concentrations and body composition as well as the genetic and environmental components of their interrelation. PATIENTS: Six hundred and seventy-four men (25-45 years) were included in this study with 274 independent pairs of brothers. MEASUREMENTS: Body composition and regional fat mass estimates were determined using dual-energy X-ray absorptiometry. Serum testosterone (T), SHBG, oestradiol (E(2)) and LH levels were determined by immunoassay; free T and E(2) levels were calculated. RESULTS: Both sex steroid hormone concentrations and indices of body composition exhibited significant heritability estimates. Among sex steroid hormones, T had the highest heritability (h(2) = 0.65), followed by free T (h(2) = 0.54). A heritability of 0.73 was observed for SHBG; a heritability estimate of 0.83 was obtained for body weight. Significant genetic correlations were found between whole body fat mass and serum T (rho(G) = -0.46), free T (rho(G) = -0.27) and SHBG (rho(G) = -0.48) concentrations. No genetic relationship was observed between total (F) E(2) or LH concentrations, respectively, and body composition. CONCLUSION: Both sex steroid serum levels and body composition are under strong genetic control. Their interrelation is in part underlied by a genetic correlation, indicative of the action of shared genes.

Estrogen receptor signaling is an unstable feature of the gonadotropic LbetaT2 cell line.

The murine, gonadotropic LbetaT2 cell line was assessed as a potential in vitro model to analyze estrogen receptor (ER)-mediated regulation of luteinizing hormone (LH) synthesis and secretion. In agreement with limited literature data, repeated exposure to (sub) physiological concentrations of gonadotropin-releasing hormone enhanced LHbeta-subunit gene expression, being the rate-limiting step of LH synthesis, and the corresponding LH secretory response. However, in the same subclone of the LbetaT2 cell line, we observed that LH production was not affected following exposure to E(2), which is in contrast to previously reported weak or modest effects. One explanation may be the absence of measurable ERalpha protein expression on the one hand and impaired ER signal transduction on the other. Furthermore, an alternative ERalpha mRNA splicing variant was detected in the LbetaT2 cell line, which (theoretically) encodes for a protein that may alter ERalpha transcriptional activity, depending on the cellular context. The studied LbetaT2 subclone did not show a generalized impairment of nuclear receptor function, as we observed androgen- and glucocorticoid-induced gene transcription, together with enhanced LH secretory response following dexamethasone treatment. Since its development, the gonadotropic LbetaT2 cell line served as a reference model to study gonadotroph-specific effects because of its mature properties. Nevertheless, this cell line does not seem to be a suitable in vitro model for the study of estrogenic regulatory effects at the level of the pituitary gonadotrophs in view of the unstable nature of ER signaling in LbetaT2 cells.

Adenoviral gene transfer of ABIN-1 protects mice from TNF/galactosamine-induced acute liver failure and lethality.

Tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a central role in acute and chronic hepatitis B and C infection and alcoholic liver disease as well as fulminant liver failure. TNF-induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. The transcription factor NF-kappaB is believed to mediate at least part of the proinflammatory effects of TNF, and is therefore a favorite drug target. However, NF-kappaB also suppresses TNF-mediated hepatocyte apoptosis, implicating a potential cytotoxic effect of NF-kappaB inhibitors in the liver. This dual function of NF-kappaB emphasizes the need for therapeutics that can inhibit both TNF-induced NF-kappaB activation and cell death. Here we describe that adenoviral expression of the NF-kappaB inhibitory protein ABIN-1, but not an IkappaBalpha superrepressor (IkappaBalpha(s)), completely prevents lethality in the TNF/D-(+)-galactosamine-induced model of liver failure. Protection was associated with a significant decrease in TNF-induced leukocyte infiltration as well as hepatocyte apoptosis. The differential effects of ABIN-1 and IkappaBalpha(s) suggest a role for an NF-kappaB independent function of ABIN-1. Indeed, ABIN-1 was found to prevent not only NF-kappaB activation, but also apoptosis of cultured hepatocytes in response to TNF, explaining its protective effect against TNF-induced liver failure. In conclusion, ABIN-1 has a dual NF-kappaB inhibitory and anti-apoptotic activity in the liver, which might be of considerable interest for the treatment of inflammatory liver diseases.