Showing papers in "International Journal of Andrology in 2011"

Type 2 diabetes mellitus and testosterone: a meta-analysis study.

Abstract: Several studies suggest that type 2 diabetes mellitus (T2DM) is often associated with male hypogonadism. Despite the well-known link, the role of testosterone replacement therapy (TRT) in T2DM has not been completely clarified. The aim of the present study was to analyse systematically the relationship between androgen levels and T2DM by reviewing and meta-analysing available prospective and cross-sectional studies. In addition, a specific meta-analysis on the metabolic effects of TRT in available randomized clinical trials (RCTs) was performed. An extensive Medline search was performed including the following words: 'testosterone', 'type 2 diabetes mellitus' and 'males'. Of 742 retrieved articles, 37 were included in the study. In particular 28, 5 and 3 were cross-sectional, longitudinal and interventional studies, respectively. A further unpublished RCT was retrieved from http://www.clinicaltrials.gov. T2DM patients showed significantly lower testosterone plasma levels in comparison with non-diabetic individuals. Similar results were obtained when T2DM subjects with and without erectile dysfunction were analysed separately. Meta-regression analysis demonstrated that ageing reduced, while obesity increased, these differences. However, in a multiple regression model, after adjusting for age and body mass index (BMI), T2DM was still associated with lower total testosterone (TT) levels (adjusted r = -0.568; p < 0.0001). Analysis of longitudinal studies demonstrated that baseline TT was significantly lower among patients with incident diabetes in comparison with controls (HR = -2.08[-3.57;-0.59]; p < 0.001). Combining the results of RCTs, TRT was associated with a significant reduction in fasting plasma glucose, HbA1c, fat mass and triglycerides. Conversely, no significant difference was observed for total and high-density lipoprotein cholesterol, blood pressure and BMI. The meta-analysis of the available cross-sectional data suggests that T2DM can be considered independently associated with male hypogonadism. Although only few RCTs have been reported, TRT seems to improve glycometabolic control as well as fat mass in T2DM subjects.

The effect of paternal diet-induced obesity on sperm function and fertilization in a mouse model.

Abstract: Although obvious effects of obesity on female reproduction and oocytes are emerging, the effects on male fertility and sperm quality are less clear with studies reporting conflicting results. We hypothesize that male obesity affects sperm function and physiology probably as a result of elevated oxidative stress in spermatozoa and therefore elevated levels of sperm DNA damage and loss of function. Six-week-old C57/Bl6 male mice (n = 36) were randomly allocated to two groups: group 1 (n = 18) received a control diet, whereas group 2 (n = 18) received a high-fat diet (HFD). At the completion of a 9-week period, mice were sacrificed and spermatozoa were obtained. Sperm motility, concentration, intracellular reactive oxygen species (ROS) production and sperm DNA damage were measured. The ability of the sperm to undergo capacitation, acrosome reaction, sperm binding and ability to fertilize an oocyte were also assessed. The percentage of motile spermatozoa was decreased in the HFD group compared with controls (36 ± 2% vs. 44 ± 4%; p < 0.05). Intracellular ROS was elevated (692 ± 83 vs. 409 ± 22 units; p < 0.01) in the HFD group compared with controls. Sperm DNA damage was also increased (1.64 ± 0.6% vs. 0.17 ± 0.06%; p < 0.05) in the HFD group compared with the control group. Furthermore, the percentage of non-capacitated sperm was significantly lower compared with controls (12.34% vs. 21.06%; p < 0.01). The number of sperm bound to each oocyte was significantly lower (41.14 ± 2.5 vs. 58.39 ± 2.4; p < 0.01) in the HFD group compared with that in controls and resulted in significantly lower fertilization rates (25.9% vs. 43.9%; p < 0.01). This report provides evidence that obesity may induce oxidative stress and sperm DNA damage as well as decreased fertilizing ability. This is important as DNA damage in the sperm as a result of oxidative stress has been linked to poor reproductive outcomes.

The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology.

Abstract: The purpose of this study was to evaluate the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay as a method for assessing DNA damage in human spermatozoa. The conventional assay was shown to be insensitive and unresponsive to the DNA fragmentation induced in human and mouse spermatozoa on exposure to Fenton reagents (H₂O₂ and Fe(2+) ). However, both time- and dose-dependent responses could be readily detected if the chromatin was exposed to 2 mm dithiothreitol (DTT) for 45 min prior to fixation. This modified version of the assay significantly enhanced the TUNEL signals generated by subpopulations of spermatozoa isolated on discontinuous Percoll gradients as well as the responses triggered by reagents (arachidonic acid and menadione) that are known to stimulate superoxide anion production by human spermatozoa. DTT exposure also improved the signals detected with chromomycin A₃ (CMA₃), a probe designed to determine the efficacy of chromatin protamination, and enhanced the correlation observed between this criterion of sperm quality and the TUNEL assay. Finally, the output of the TUNEL assay was found to be highly correlated with sperm vitality. The TUNEL methodology was therefore further refined to incorporate a vital stain that covalently bound to intracellular amine groups in non-viable cells. This tag remained associated with the spermatozoa during fixation and processing for the TUNEL assay so that ultimately, both DNA integrity and vitality could be simultaneously assessed in the same flow cytometry assay. The methods described in this article are simple and robust and should facilitate research into the causes of DNA damage in human spermatozoa.

Male accessory gland infection and sperm parameters (review).

Abstract: Summary Male accessory gland infection (MAGI) has been identified among those diagnostic categories which have a negative impact on the reproductive function and fertility in males (Rowe et al., World Health Organization Manual for the Standardised Investigation and Diagnosis of the Infertile Couple, Cambridge University Press, Cambridge, 1993). MAGI is a hypernym which groups the following different clinical categories: prostatitis, prostate-vesiculitis and prostate-vesiculo-epididymitis. Some of the characteristics they share are: common diseases, mainly have a chronic course, rarely cause obstruction of the seminal pathways, can have an unpredictable intracanicular spread to one or more sexual accessory glands of the reproductive tract, as well as to one or both sides. In this review, we show that all components involving the inflammatory response (from the agents which first trigger it to each component of the inflammatory response dynamic) can deteriorate conventional and/or non-conventional sperm parameters arising from one or more of the following mechanisms: altered secretory function of the epididymis, seminal vesicles, and prostate which reduce the antioxidant properties or scavenging role of the seminal plasma; deterioration of spermatogenesis; and (unilateral or bilateral) organic or functional sub-obstruction of the seminal tract.

Recent adverse trends in semen quality and testis cancer incidence among Finnish men

Abstract: Impaired semen quality and testicular cancer may be linked through a testicular dysgenesis syndrome of foetal origin. The incidence of testis cancer has been shown to increase among Finnish men, whereas there is no recent publication describing temporal trends in semen quality. Therefore, we carried out a prospective semen quality study and a registry study of testis cancer incidence among Finnish men to explore recent trends. A total of 858 men were investigated in the semen quality study during 1998–2006. Median sperm concentrations were 67 (95% CI 57–80) million/mL, 60 (51–71) and 48 (39–60) for birth cohorts 1979–81, 1982–83 and 1987; total sperm counts 227 (189–272) million, 202 (170–240) and 165 (132–207); total number of morphologically normal spermatozoa 18 (14–23) million, 15 (12–19) and 11 (8–15). Men aged 10–59 years at the time of diagnosis with testicular cancer during 1954–2008 were included in the registry study, which confirmed the increasing incidence of testicular cancer in recent cohorts. These simultaneous and rapidly occurring adverse trends suggest that the underlying causes are environmental and, as such, preventable. Our findings necessitate not only further surveillance of male reproductive health but also research to detect and remove the underlying factors.

Expression and interdependencies of pluripotency factors LIN28, OCT3/4, NANOG and SOX2 in human testicular germ cells and tumours of the testis.

Abstract: OCT3/4, NANOG, SOX2 and, most recently, LIN28 have been identified as key regulators of pluripotency in mammalian embryonic and induced stem cells, and are proven to be crucial for generation of the mouse germ-cell lineage. These factors are a hallmark of certain histological types of germ-cell tumours (GCTs). Here, we report novel information on the temporal and spatial expression pattern of LIN28 during normal human male germ-cell development as well as various types of GCTs. To investigate LIN28 expression, immunohistochemical analyses and quantitative proximity ligation assay-based TaqMan protein assays were applied on snap-frozen and formalin-fixed, paraffin-embedded samples as well as representative cell lines. LIN28 was found in primordial germ cells, gonocytes and pre-spermatogonia, in contrast to OCT3/4 and NANOG, which were found only in the first two stages. LIN28 was also found in all precursor lesions (carcinoma in situ and gonadoblastoma) of type II GCTs, as well as the invasive components seminoma and the non-seminomatous elements embryonal carcinoma and yolk sac tumour. Choriocarcinoma showed a heterogeneous pattern, while teratomas and spermatocytic seminomas (type III GCTs) were negative. This expression pattern suggests that LIN28 is associated with malignant behaviour of type II GCTs. Cell line experiments involving siRNA knockdown of LIN28, OCT3/4 and SOX2 showed that LIN28 plays a role in the maintenance of the undifferentiated state of both seminoma and embryonal carcinoma, closely linked to, and likely upstream of OCT3/4 and NANOG. In conclusion, LIN28 regulates the differentiation status of seminoma and embryonal carcinoma and is likely to play a related role in normal human germ-cell development.

Premature and Delayed Ejaculation: Two Ends of a Single Continuum Influenced by Hormonal Milieu

Abstract: Although it is well established that all the aspects of male reproduction are hormonally regulated, the endocrine control of the ejaculatory reflex is still not completely clarified. Sex steroids, thyroid and pituitary hormones (oxytocin and prolactin) have been proposed to control the ejaculatory process at various levels; however, only a few reports are currently available. The aim of this study was to evaluate the contribution of testosterone, thyrotropin (TSH) and prolactin (PRL) in the pathogenesis of ejaculatory dysfunction in a large series of subjects consulting for sexual dysfunction. Among the 2652 patients studied, 674 (25.2%) and 194 (7.3%) reported premature and delayed ejaculation (PE and DE), respectively. Categorizing ejaculatory difficulties on an eight-point scale starting from severe PE and ending with anejaculation (0 = severe PE, 1 = moderate PE, 2 = mild PE, 3 = no difficulties, 4 = mild DE, 5 = moderate DE, 6 = severe DE and 7 = anejaculation), PRL as well as TSH levels progressively increased from patients with severe PE towards those with anejaculation. Conversely, the opposite was observed for testosterone levels. All of these associations were confirmed after adjustment for age (adjusted r = 0.050, 0.053 and -0.038 for PRL, TSH and testosterone, respectively; all p < 0.05). When all hormonal parameters were introduced in the same regression model, adjusting for age, ΣMHQ (an index of general psychopathology) and use of selective serotonin reuptake inhibitor antidepressants, they were independently associated with ejaculatory problems (adjusted r = 0.056, 0.047 and -0.059 for PRL, TSH and testosterone, respectively; all p < 0.05). This study indicates endocrine system is involved in the control of ejaculatory function and that PRL, TSH and testosterone play an independent role.

Comparative proteome and lipid profiles of bovine epididymosomes collected in the intraluminal compartment of the caput and cauda epididymidis

Abstract: During the epididymal maturation, spermatozoa interact with different populations of epididymosomes and sequentially acquire some epididymosome-associated proteins critical to sperm functions. Although very few proteins associated with epididymosomes have been identified, the physiological importance of these vesicles in the sperm maturation remains unclear. To document these relevant issues, lipid and protein analysis of epididymosomes from caput and cauda epididymal fluids was determined. Lipid analysis revealed a particular composition of specific phospholipids in these vesicles; the levels of phosphatidyl-ethanolamine, phosphatidyl-inositol and phosphatidyl-choline being higher in caput epididymosomes. From the 555 and 438 proteins identified in caput- and cauda-derived epididymosomes, respectively, 231 proteins were identified in both types of epididymosome. Proteins exclusively identified in caput and cauda epididymosomes are mainly enzymes and transporter molecules. The presence of several glycan-modifying enzymes is the hallmark of the caput epididymosomes proteome. Among the common proteins in both types of epididymosome, a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion were identified. Together, these data suggest that epididymosome-associated proteins are involved in various molecular functions suggesting that during the epididymal transit, spermatozoa interact with different populations of epididymosomes, which could modify the male gamete in a sequential manner.

Clinical experience with azoospermia: aetiology and chances for spermatozoa detection upon biopsy.

Abstract: The clinical workup of the infertile male with azoospermia aims at determining the aetiology and estimating the chances of finding spermatozoa by testicular sperm extraction (TESE). To establish prognostic criteria, 1583 consecutive patients with azoospermia consulting the Centre of Reproductive Medicine and Andrology, Munster, a tertiary referral centre, between 1976 and 2009 comprising 9.8% of all patients providing a semen sample were included in this retrospective analysis. The frequencies of diagnoses were as follows: 21% genetic causes (14% Klinefelter syndrome, 1% other chromosomal aberrations, 2% Y-chromosomal microdeletions, 1% hypogonadotropic hypogonadism, 3% congenital bilateral absence of the vas deferens), 31% current or former maldescended testes, varicocele, urogenital infections, 15% malignancies, 11% obstructions, 7% endocrine or other chronic diseases and 12% idiopathic azoospermia. Receiver-operating characteristic curves for chances of finding spermatozoa by testicular biopsy were calculated for testicular volume, serum follicle-stimulating hormone (FSH) and the seminal markers α-glucosidase, fructose and zinc where these data were available (N=283). Histograms of the seminal markers comparing data from men with obstructive azoospermia and normozoospermia visualize their discriminating power. Evidence-based threshold values for high chances of positive testicular biopsy serving as surrogate marker for TESE were derived from the subgroup of men with obstructive azoospermia for testicular volume (≥21mL), FSH (≤10U/L) and seminal α-glucosidase (≤18mU/ejaculate). Fructose and zinc could not predict the chances of finding spermatozoa upon biopsy. Based on these three parameters, positive biopsy and presumably TESE success can be quickly and reliably estimated in everyday practice with the colour-coded figures constructed from these data. As a seminal α-glucosidase reference limit of 18mU/ejaculate can also be used to diagnose congenital bilateral absence of the vas deferens, α-glucosidase (rather than seminal fructose) should be determined as part of the clinical routine when counselling patients before testicular biopsy.

Relative importance of prenatal and postnatal androgen action in determining growth of the penis and anogenital distance in the rat before, during and after puberty.

Abstract: Experimental animal studies show that measurement of anogenital distance (AGD) and/or penis length may provide lifelong 'read-outs' of foetal androgen exposure during the masculinization programming window (MPW). However, variation in postnatal androgen exposure may complicate interpretation of such measurements. This is important to clarify if such measurements are to be applied to humans. The present aim was to evaluate effects of prenatal and/or postnatal manipulation of androgen production/action on growth of AGD and the penis in rats. Pregnant rats were treated daily before (e13.5-e21.5) and after birth (postnatal days 1-15) with either vehicle, 500 mg/kg di(n-butyl) phthalate (DBP) or 100 mg/kg flutamide (postnatal only) in prenatal + postnatal treatment combinations (N = 6 treatment combinations); DBP impairs androgen production whereas flutamide impairs androgen action. Male offspring were killed on postnatal day 8 (prepuberty), 25 (early puberty) or 90 (adulthood) when AGD was measured, the penis dissected out and its weight and length measured; plasma testosterone and ventral prostate weight were measured at day 90 to assess endogenous androgen exposure. In controls, penis length, girth and AGD increased 2.2-, 5.3-and 5.9-fold respectively from day 8 to day 90. Significant inhibition of penis growth and final length and girth was induced by treatments that inhibited postnatal androgen action. Conversely, growth and ultimate (adult) AGD was inhibited by prenatal inhibition of androgen production whereas postnatal androgen inhibition had negligible effect. Nevertheless, AGD and penis length were highly correlated at every age (R(2) > 0.33; p < 0.0001). However, altered endogenous androgen exposure may confound interpretation of changes in adults exposed prenatally/postnatally to DBP/flutamide. We conclude that AGD provides a lifelong guide to prenatal androgen exposure (in the MPW) whereas penis size reflects both prenatal + postnatal androgen exposure. At the group treatment level, prepubertal measurement of either AGD or penis size accurately predicts their size in adulthood.

Effect of microsurgical varicocelectomy on human sperm chromatin and DNA integrity: a prospective trial.

Abstract: Summary There is evidence from retrospective studies that varicocelectomy can improve sperm DNA damage in infertile men with a clinical varicocele. The objective of this prospective study was to examine further the effect of varicocelectomy on sperm chromatin and DNA integrity. We evaluated a consecutive series of infertile men (n = 25) who underwent microsurgical varicocelectomy for treatment of clinical varicocele. We examined conventional sperm parameters and sperm chromatin structure assay parameters (percentage DFI – DNA fragmentation index and percentage HDS – high DNA stainability, an index of chromatin compaction) before and 4 and 6 months after microsurgical varicocelectomy. Sperm DNA integrity improved significantly after surgery (percentage DFI decreased from 18 ± 11% before surgery to 10 ± 5%, and 7 ± 3%, at 4 and 6 months after surgery respectively). Sperm chromatin compaction also improved significantly after surgery (percentage HDS decreased from 11 ± 7% before surgery to 8 ± 6%, and 7 ± 5%, at 4 and 6 months after surgery, respectively). Sperm concentration and progressive motility improved after surgery, although the differences were not statistically significant when compared with that before surgery. The data show that varicocelectomy is associated with an improvement in sperm DNA integrity and chromatin compaction. These findings support the concept that correction of a varicocele can improve spermatogenesis, particularly spermiogenesis (the stage in spermatogenesis where compaction and stability of the sperm DNA and chromatin occur).

The effect of pulsed 900‐MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa

Abstract: Several recent studies have indicated that radiofrequency electromagnetic fields (RF-EMF) have an adverse effect on human sperm quality, which could translate into an effect on fertilization potential. This study evaluated the effect of RF-EMF on sperm-specific characteristics to assess the fertilizing competence of sperm. Highly motile human spermatozoa were exposed for 1 h to 900-MHz mobile phone radiation at a specific absorption rate of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed using computer-assisted sperm analysis. Significant reduction in sperm head area (9.2 ± 0.7 μm² vs. 18.8 ± 1.4 μm²) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) was reported among exposed sperm compared with unexposed controls. Sperm-zona binding was assessed directly after exposure using the hemizona assay. The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p < 0.05), respectively. This study concludes that although RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition, a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilization potential.

Influence of 5α-dihydrotestosterone and 17β-estradiol on human Sertoli cells metabolism.

Abstract: Sertoli cells metabolize glucose, converting it to lactate that is used by developing germ cells for their energy metabolism. Androgens and oestrogens have metabolic roles that reach far beyond reproductive processes. So, the main purpose of this study was to examine the effect of sex steroid hormones on metabolite secretion/consumption in human Sertoli cells. Human Sertoli cell-enriched primary cultures were maintained in a defined medium for 50 h and glucose, pyruvate, lactate and alanine variations were determined using (1) H-NMR spectra analysis, in the absence or presence of 100 nm 17β-estradiol (E(2) ) or 100 nm 5α-dihydrotestosterone (DHT). The mRNA expression levels of glucose transporters, lactate dehydrogenase and monocarboxylate transporters were also determined using semi-quantitative RT-PCR. Cells cultured in the absence (control) or presence of E(2) consumed the same amounts of glucose at similar rates during the 50 h. During the first 15 h of treatment with DHT, glucose consumption and glucose consumption rate were significantly higher. Nevertheless, DHT-treated cells secreted a significantly lower amount of lactate than control and E(2) -treated cells. Such a decrease was concomitant with a significant decrease in lactate dehydrogenase A mRNA levels after 50 h treatment in DHT-treated groups. Finally, alanine production was significantly increased in E(2) -treated cells after 25 h treatment, which indicated a lower redox/higher oxidative state for the cells on those conditions. These results support the existence of a relationship between sex steroid hormones action and energy metabolism, providing the first assessment of androgens and oestrogens as metabolic modulators of human Sertoli cells.

Human testicular insulin‐like factor 3: in relation to development, reproductive hormones and andrological disorders

Abstract: Knockout of the gene encoding insulin-like factor 3 (INSL3) results in cryptorchidism in mice due to disruption of the transabdominal phase of testicular descent This finding was essential for understanding the complete course of testis descensus, and wound up years of speculations regarding the endocrine regulation of this process INSL3 is, along with testosterone, a major secretory product of testicular Leydig cells In addition to its crucial function in testicular descent, INSL3 is suggested to play a paracrine role in germ cell survival and an endocrine role in bone metabolism INSL3 is produced in human prenatal and neonatal, and in adult Leydig cells to various extents, and is in a developmental context regulated like testosterone, with production during second trimester, an early postnatal peak and increasing secretion during puberty, resulting in high adult serum levels INSL3 production is entirely dependent on the state of Leydig cell differentiation, and is stimulated by the long-term trophic effects mediated by luteinizing hormone (LH) Once differentiated, Leydig cells apparently express INSL3 in a constitutive manner, and the hormone is thereby insensitive to the acute, steroidogenic effects of LH, which for example is an important factor in the regulation of testosterone Clinically, serum INSL3 levels can turn out to be a usable tool to monitor basal Leydig cell function in patients with various disorders affecting Leydig cell function According to animal studies, foetal INSL3 production is, directly or indirectly, sensitive to oestrogenic or anti-androgenic compounds This provides important insight into the mechanism by which maternal exposure to endocrine disrupters can result in cryptorchidism in the next generation Conclusively, INSL3 is an interesting testicular hormone with potential clinical value as a marker for Leydig cell function It should be considered on a par with testosterone in the evaluation of testicular function and the consequences of Leydig cell dysfunction

Prostasomes: inhibitors of capacitation and modulators of cellular signalling in human sperm.

Abstract: Summary Seminal fluid inhibits sperm capacitation mainly because of its high cholesterol content. Prostasomes are the main source of cholesterol in seminal fluid. They are known to have numerous protective properties and are able to transfer proteins and lipids to spermatozoa, but their impact on capacitation and acrosome reaction (AR) is not yet well understood. The aim of this study was to determine the effects of prostasomes on human sperm capacitation and AR. After 80% Percoll selection, freshly ejaculated human spermatozoa were incubated for 3 h under capacitating conditions with prostasomes, phosphodiesterase inhibitor 3-iso-butyl-methylxantine (IBMX), or a combination of prostasomes and IBMX. Physiological concentration of prostasomes significantly decreased tyrosine phosphorylation levels of human sperm capacitation markers P110 and P80 (p < 0.01), and the proportions of capacitated (p < 0.05) and acrosome-reacted spermatozoa (p < 0.05). Prostasomes significantly increased the proportion of spermatozoa that did not incorporate propidium iodide and significantly attenuated the effect of IBMX on P110 tyrosine phosphorylation. Prostasomes had no effect on the pHi increase associated with capacitation. They significantly increased intracellular cAMP concentration ([cAMP]i) and, when prostasomes and IBMX were present together, [cAMP]i was further increased. To our knowledge, this is the first study to show clearly that prostasomes inhibit capacitation and spontaneous AR.

The response to FSH treatment in oligozoospermic men depends on FSH receptor gene polymorphisms.

Abstract: Summary In the last years, follice-stimulating hormone (FSH) receptor (FSHR) gene polymorphisms have been studied as potential risk factors for spermatogenetic failure. In this study, we have evaluated the response of FSH treatment in terms of sperm production on the basis of Ala307Thr-Asn680Ser polymorphisms in the FSHR gene in a group of oligozoospermic subjects with hypospermatogenesis and normal FSH levels. Patients were randomized into two groups: 70 treated with recombinant FSH (150 IU thrice per week for 3 months) and 35 without treatment. After 3 months of treatment, we observed significant increase in total sperm count, sperm concentration, forward motility, percentage of normal morphology forms and total motile sperm. When 70 treated subjects were subdivided based on FSHR genotype, only subjects with at least one serine in position 680 showed a statistically significant increase in these sperm parameters, whereas subjects with homozygote Thr307-Asn680 showed no difference in any seminal parameters evaluated. Non-treated subjects showed no differences in any parameter evaluated. This study suggests that the analysis of this gene represents a valid pharmacogenetic approach to the treatment of male infertility, confirming also the importance of strict criteria for the selection of patients to be treated with FSH.

Testosterone modulates gene expression pathways regulating nutrient accumulation, glucose metabolism and protein turnover in mouse skeletal muscle.

Abstract: Testosterone regulates energy metabolism and skeletal muscle mass in males, but the molecular mechanisms are not fully understood This study investigated the response of skeletal muscle to castration and testosterone replacement in 8-week-old male mice Using microarray analyses of mRNA levels in gastrocnemius muscle, 91 genes were found to be negatively regulated by testosterone and 68 genes were positively regulated The mRNA levels of the insulin signalling suppressor molecule Grb10 and the glycogen synthesis inhibitors, protein phosphatase inhibitor-1 and phosphorylase kinase-γ, were negatively regulated by testosterone The insulin-sensitive glucose and amino acid transporters, Glut3 and SAT2, the lipodystrophy gene, Lpin1 and protein targeting to glycogen were positively regulated These changes would be expected to increase nutrient availability and sensing within skeletal muscle, increase metabolic rate and carbohydrate utilization and promote glycogen accumulation The observed positive regulation of atrogin-1 (Fbxo32) by testosterone could be explained by the phosphorylation of Akt and Foxo3a, as determined by Western blotting Testosterone prevented the castration-induced increase in interleukin-1α, the decrease in interferon-γ and the atrophy of the levator ani muscle, which were all correlated with testosterone-regulated gene expression These findings identify specific mechanisms by which testosterone may regulate skeletal muscle glucose and protein metabolism

Early onset of primary hypogonadism revealed by serum anti-Mullerian hormone determination during infancy and childhood in trisomy 21

Abstract: Summary Male patients with an extra sex chromosome or autosome are expected to present primary hypogonadism at puberty owing to meiotic germ-cell failure. Scarce information is available on trisomy 21, a frequent autosomal aneuploidy. Our objective was to assess whether trisomy 21 presents with pubertal-onset, germ-cell specific, primary hypogonadism in males, or whether the hypogonadism is established earlier and affects other testicular cell populations. We assessed the functional status of the pituitary-testicular axis, especially Sertoli cell function, in 117 boys with trisomy 21 (ages: 2 months–20 year). To compare with an adequate control population, we established reference levels for serum anti-Mullerian hormone (AMH) in 421 normal males, from birth to adulthood, using a recently developed ultrasensitive assay. In trisomy 21, AMH was lower than normal, indicating Sertoli cell dysfunction, from early infancy, independently of the existence of cryptorchidism. The overall prevalence rate of AMH below the 3rd percentile was 64.3% in infants with trisomy 21. Follicle-stimulating hormone was elevated in patients <6 months and after pubertal onset. Testosterone was within the normal range, but luteinizing hormone was elevated in most patients <6 months and after pubertal onset, indicating a mild Leydig cell dysfunction. We conclude that in trisomy 21, primary hypogonadism involves a combined dysfunction of Sertoli and Leydig cells, which can be observed independently of cryptorchidism soon after birth, thus prompting the search for new hypotheses to explain the pathophysiology of gonadal dysfunction in autosomal trisomy.

Dissecting the molecular pathways of (testicular) germ cell tumour pathogenesis; from initiation to treatment-resistance

Abstract: Human type II germ cell tumours (GCTs) originate from an embryonic germ cell, either as a primordial germ cell or gonocyte. This start determines the biological as well as clinical characteristics of this type of cancer, amongst others their totipotency as well as their overall (exceptional) sensitivity to DNA damaging agents. The histology of the precursor lesion, either carcinoma in situ or gonadoblastoma, depends on the level of testicularization (i.e. testis formation) of the gonad. The impact of either intrinsic (genetic) - and environmental factors involved in the pathogenesis is demonstrated by disorders of sex development as well as testicular dysgenesis syndrome as risk factors, including cryptorchidism, hypospadias and disturbed fertility as parameters. This knowledge allows identification of individuals at risk for development of this type of cancer, being a population of interest for screening. Factors known to regulate pluripotency during embryogenesis are proven to be of diagnostic value for type II GCTs, including OCT3/4, even applicable for non-invasive screening. In addition, presence of stem cell factor, also known as KITLG, allows distinction between delayed matured germ cells and the earliest stages of malignant transformation. This is of special interest because of the identified association between development of type II GCTs of the testis and a limited number of single nucleotide polymorphisms, including some likely related to KITL. Transition from the precursor lesion to an invasive cancer is associated with gain of the short arm of chromosome 12, in which multiple genes might be involved, including KRAS2 and possibly NANOG (pseudogenes). While most precursor lesions will progress to an invasive cancer, only a limited number of cancers will develop treatment resistance. Putative explanatory mechanisms are identified, including presence of microsatellite instability, BRAF mutations, apoptosis suppression and p21 sub-cellular localization. It remains to be investigated how these different pathways integrate to each other and how informative they are at the patient-individual level. Further understanding will allow development of more targeted treatment, which will benefit quality of life of these young cancer patients.

Scrotal heat stress causes a transient alteration in tight junctions and induction of TGF-β expression

Abstract: Summary Specialized junctions, which occur at sites of Sertoli–Sertoli and Sertoli–germ cell contact of seminiferous epithelium, play pivotal roles in spermatogenesis. Slight increase in scrotal temperature can induce oligospermia or azoospermia via increasing germ cell apoptosis. In this study, we demonstrated that the expression of tight junction (TJ) components, such as occludin, claudin-3 and zonula occludens-1 (ZO-1), was reduced 24–48 h after a single mild scrotal heat exposure (43 °C for 30 min), whereas mRNA levels of claudin-11 were increased. Moreover, the protein localization of occludin and ZO-1 was lost from the blood–testis barrier (BTB) site, whereas claudin-11 immunostaining became diffuse and cytoplasmic 2 days following heat exposure. Electron microscopic analysis showed that 2 days after the heat treatment, the intercellular space between the two adjacent Sertoli cells was expanded, coupled with defragmentation of actin bundles and the endoplasmic reticulum. In addition, the TJ permeability increased significantly 2 days after the heat exposure and recovered approximately 10 days later. Heat-induced reversible BTB disruption was associated with a transient induction of transforming growth factor (TGF)-β2, -3 and p38 mitogen-activated protein kinase activation. However, the TGF-β antagonist only partially prevented the heat-induced BTB disruption. In conclusion, the expression of TJ-associated molecules and BTB were reversibly perturbed after mild testicular hyperthermia, and the induction of TGF-β expression may be partially involved in heat-induced BTB damage.

Associations between urinary metabolites of di(2-ethylhexyl) phthalate and reproductive hormones in fertile men

Abstract: Widely used man-made chemicals, including phthalates, can induce hormonal alterations through a variety of cellular and molecular mechanisms. A number of rodent and observational studies have consistently demonstrated the anti-androgenic effect of several phthalates. However, there are only limited data on the relationship between exposure to these chemicals and reproductive hormone levels in men. All men (n = 425) were partners of pregnant women who participated in the Study for Future Families in five US cities and provided urine and serum samples on the same day. Eleven phthalate metabolites were measured in urine and serum samples were analysed for reproductive hormones, including follicle-stimulating hormone, luteinizing hormone, testosterone, inhibin B and oestradiol and sex hormone-binding globulin (SHBG). Pearson correlations and parametric tests were used for unadjusted analyses, and multiple linear regression analysis was performed controlling for appropriate covariates. We observed weak or no associations with urinary phthalates other than di(2-ethylhexyl) phthalate (DEHP). All measures of testosterone [total, calculated free testosterone and the free androgen index (FAI)] were inversely correlated with the urinary concentrations of four DEHP metabolites. After adjustment by appropriate covariates, there was no longer an association between urinary DEHP metabolite concentrations and total testosterone levels; however, FAI was significantly associated with the urinary concentrations of several DEHP metabolites. SHBG was positively related to the urinary concentrations of mono(2-ethylhexyl) phthalate, but not with other DEHP metabolites, an association that was attenuated after adjustment. Our results suggest that DEHP exposure of fertile men is associated with minor alterations of markers of free testosterone.

Pubertal development timing in urban Chinese boys

Abstract: We describe current pubertal development in healthy urban Chinese boys. A cross-sectional study of the pubertal development of 18,807 urban Chinese boys aged from 3.50 to 18.49years was conducted between 2003 and 2005. Testicular volume was evaluated with a Prader orchidometer. Pubic hair development was assessed according to the Tanner method. Data on spermarche were collected using the status quo method. Probit analysis was used to calculate the median age and 95% CI at different stages of testicular development, pubic hair development and spermarche. By age 9, 12.99% of the boys had a testicular volume of 4mL or greater. The median age of onset of puberty defined as the age at attainment of testicular volume of 4mL or greater was 10.55 (95% CI 10.27-10.79) years. The median age for onset of pubic hair development (PH(2) ) and spermarche was 12.78 (95%CI 12.67-12.89) years and 14.05 (95%CI 13.80-14.32) years, respectively. Pubertal onset in urban Chinese boys is earlier than currently used clinical norms but their pubic hair development occurs relatively late in comparison with the reported data from numerous other countries. There is also evidence of a secular trend towards an earlier age of spermarche since 1979 in Chinese urban boys.

Dietary antioxidants improve arteriogenic erectile dysfunction

Abstract: Most cases of erectile dysfunction (ED) are associated with oxidative stress risk factors such as diabetes mellitus, smoking, hypercholesterolaemia and hypertension. Our goal was to search for markers of oxidative stress in arteriogenic ED and examine the protective role of dietary antioxidants. Atherosclerosis-induced ED was developed in rabbits by balloon de-endothelialization of the iliac arteries. Ballooned and age-matched control animals were assigned into subgroups receiving pomegranate extract antioxidants in drinking water or tap water as placebo. After 8 weeks, penile blood flow and erectile activity were recorded. Erectile tissue relaxation, oxidative products, oxidative stress-responsive genes and structure were examined using organ bath, enzyme immunoassay, quantitative real-time polymerase chain reaction and transmission electron microscopy, respectively. Arterial ballooning caused diffused atherosclerosis, decreased intracavernosal blood flow and led to ED. Impairment of endothelium-dependent relaxation, diffused fibrosis, increased oxidative products, upregulation of superoxide dismutase (SOD) and aldose reductase (AR) gene expression, mitochondrial and endothelial structural damage and increased caveolae were evident in erectile tissues from atherosclerotic animals receiving placebo. Upregulation of antioxidant enzymes SOD and AR failed to protect ischaemic erectile tissue from oxidative injury. Pomegranate extract significantly improved intracavernosal blood flow, erectile activity, smooth muscle relaxation and fibrosis of the atherosclerotic group in comparison with the atherosclerotic group receiving placebo, but did not normalize them to the age-matched control levels. Pomegranate extract appeared more effective in diminishing oxidative products, preventing SOD and AR gene upregulation, and protecting mitochondrial, endothelial and caveolae structural integrity of the atherosclerotic group. Our data suggest the presence of oxidative stress in ED and a more efficient action of antioxidants on molecular and ultrastructural alterations than on distinct functional deficit and structural damage in the ischaemic penis.

Prospective study on the prevalence and associated risk factors of cryptorchidism in 6246 newborn boys from Nice area, France.

Abstract: To assess the incidence and risk factors of cryptorchidism in Nice area. A 3-year prospective study was conducted at two maternity wards involving neonatal screening of boys born ≥34weeks of amenorrhoea. Methodology was strict with examination at birth, 3 and 12months by the same paediatrician. Two strictly matched controls were included for each case. Information on child and parents (medical history, pregnancy, lifestyle) was recorded using medical chart and self-administered questionnaires. A total of 102 of 6246 boys were born with cryptorchidism (prevalence 1.6%, 95 included). Half of them were still cryptorchid at three and 12months with, however, 10% of secondary re-ascent (recurrent cryptorchidism) at 12months, justifying long-term follow-up. Cryptorchidism at birth was associated with instrumental delivery, inguinal hernia and urogenital malformations, particularly micropenis and paternal history of cryptorchidism. Our results suggest that maternal exposure to anti-rust or phthalates could be a risk factor, whereas eating fruits daily seemed somewhat protective. Prevalence of cryptorchidism in our area is on the lower bracket compared with other countries, and is associated with both familial and environmental risk factors.

Genome-wide association studies provide new insights into the genetic basis of testicular germ-cell tumour.

Abstract: Testicular germ-cell tumour (TGCT) is the most common cancer in young men, and genetic epidemiological studies suggest that the disease has a strong genetic basis. Until 2009, very little of this genetic component had been explained. Genome-wide association studies have since identified eight SNPs at six loci which together account for approximately 15% of the genetic risk of TGCT and offer novel biological insights into testicular germ-cell oncogenesis. In this review, we summarize the genetic epidemiology of TGCT, detail the contribution genome-wide association studies have made to our understanding of the genetic basis of TGCT and reflect on how future technological advances may assist in revealing the remaining genetic factors underlying TGCT susceptibility.

Organochlorine compounds and testicular dysgenesis syndrome: human data.

Abstract: Cryptorchidism, hypospadias, subfertility, and testicular germ-cell tumor have been suggested to comprise a testicular dysgenesis syndrome (TDS) based on the premise that each may derive from perturbations of embryonal programming and gonadal development during fetal life. Endocrine-disrupting chemicals have been hypothesized to be associated with these disorders given the importance of sex steroid hormones in urogenital development and homeostasis. Organochlorines are one such set of compounds which are defined as containing between one and ten covalently bonded chlorine atoms. These compounds are persistent pollutants with long half-lives, accumulate in adipose tissue when ingested, bioaccumulate and biomagnify, and have complex and variable toxicological profiles. Examples of organochlorines include dichlorodiphenyltrichloroethane (DDT) and its metabolites, polychlorinated biphenyls (PCBs) and chlordane. In this comprehensive review of human epidemiologic studies which have tested for associations between organochlorines and facets of TDS, we find evidence for associations between the exposures p,p′-DDE, cis-nonachlor, and trans-nonachlor with TGCT. The sum of the evidence from human epidemiologic studies does not indicate any association between specific organochlorines studied and cryptorchidism, hypospadias, or fertility. Many other endocrine-disrupting chemicals, including additional organochlorines, have yet to be assessed in relation to disorders associated with TDS, yet study of such chemicals has strong scientific merit given the relevance of such hypotheses to urogenital development.

Immunodeviation towards a Th17 immune response associated with testicular damage in azoospermic men.

Abstract: Infection and inflammation of the male reproductive tract are thought to be a primary aetiological factor of male infertility. Furthermore, several studies suggest that T lymphocytes are critically involved as regulator in the pathogenesis of male infertility under these conditions and are thought to induce autoimmune orchitis. In this context of autoimmunity the recently described T helper (Th) 17 subset has been suggested to play an essential role so that the aim of this study was to investigate the expression and characteristics of Th17 cells as well as the presence of Th17 inducing antigen presenting cells (APCs) in azoospermic testis with chronic inflammation (ATCI) compared with normal spermatogenesis. By stereological analysis, we detected base line expression of Th17 cells in Con. However, increased expression intensity and number of Th17 cells and their cytokines [interleukin (IL)-17A, IL-21, IL-22] and a decreased level of Foxp3(+) and interferon-γ(+) cells could be demonstrated in ATCI. Moreover, along with these data, increased numbers of Th17-inducing IL-23 producing CD11c(+) and CD68(+) APCs could be detected in ATCI. From these data, a picture emerges that Th17 cells orchestrated by IL-23 producing APCs are critically involved in chronic inflammation in ATCI.

Feelings of loss and uneasiness or shame after removal of a testicle by orchidectomy: a population-based long-term follow-up of testicular cancer survivors

Abstract: Few data illustrate the man's reaction to orchidectomy. We investigated long-lasting feelings of loss and uneasiness or shame about the body after removal of a testicle by orchidectomy. We identified 1173 eligible men diagnosed with non-seminomatous testicular cancer treated according to the national cancer-care programmes Swedish-Norwegian Testicular Cancer Group I-IV between 1981 and 2004. We asked the survivors about feelings of loss and uneasiness or shame after having had a testicle removed by orchidectomy. We obtained information from 960 (82%) testicular cancer survivors. We found that 32% of these men miss or previously missed their removed testicle(s) and that 26% have or previously had feelings of uneasiness or shame about their body because of the removed testicle(s). Men who had never been offered a prosthesis reported feelings of loss [relative risk (RR): 2.0; 95% confidence interval (CI): 1.3-3.0] and uneasiness or shame (RR: 2.0; 95% CI: 1.3-3.2) to a higher extent than those who had been offered, but rejected a prosthesis. An orchidectomy may result in long-lasting feelings of loss and uneasiness or shame in some men; offering a testicular prosthesis may hinder this experience.

Human spermatogonial stem cells: a possible origin for spermatocytic seminoma.

Abstract: In mammals, spermatogenesis is maintained throughout life by a small subpopulation of type A spermatogonia called spermatogonial stem cells (SSCs). In rodents, SSCs, or Asingle spermatogonia, form the self-renewing population. SSCs can also divide into Apaired (Apr) spermatogonia that are predestined to differentiate. Apaired spermatogonia produce chains of Aaligned (Aal) spermatogonia that divide to form A1 to A4, then type B spermatogonia. Type B spermatogonia will divide into primary spermatocytes that undergo meiosis. In human, there are only two different types of A spermatogonia, the Adark and Apale spermatogonia. The Adark spermatogonia are considered reserve stem cells, whereas the Apale spermatogonia are the self-renewing stem cells. There is only one generation of type B spermatogonia before differentiation into spermatocytes, which makes human spermatogenesis less efficient than in rodents. Although the biology of human SSCs is not well known, a panel of phenotypic markers has recently emerged that is remarkably similar to the list of markers expressed in mice. One such marker, the orphan receptor GPR125, is a plasma membrane protein that can be used to isolate human SSCs. Human SSCs proliferate in culture in response to growth factors such as GDNF, which is essential for SSC self-renewal in mice and triggers the same signalling pathways in both species. Therefore, despite differences in the spermatogonial differentiation scheme, both species use the same genes and proteins to maintain the pool of self-renewing SSCs within their niche. Spermatocytic seminomas are mainly found in the testes of older men, and they rarely metastasize. It is believed that these tumours originate from a post-natal germ cell. Because these lesions can express markers specific for meiotic prophase, they might originate from a primary spermatocyte. However, morphological appearance and overall immunohistochemical profile of these tumours indicate that the cell of origin could also be a spermatogonial stem cell.

Genotype and phenotype in Klinefelter syndrome – impact of androgen receptor polymorphism and skewed X inactivation

Abstract: The phenotypic variation of Klinefelter syndrome (KS) is wide and may by caused by various genetic and epigenetic effects. Skewed inactivation of the supra-numerical X chromosome and polymorphism in the androgen receptor (AR) have been suggested as plausible causes. We wanted to describe X-chromosome inactivation patterns and the AR polymorphism and correlate these to clinical findings in KS in a cross-sectional study. To that end, we studied 70 KS patients enrolled from fertility clinics and endocrine clinics and 70 age-matched control subjects. The main outcome was X-chromosome inactivation pattern (skewX), AR polymorphism (CAGn - repeat length) and correlation to anthropometrical, hormonal, metabolic and bone-related variables. Forty-six of 70 KS men were heterozygous for CAGn. The shortest and the longest alleles were equally frequent inactivated and the mean CAGn of the two alleles did not differ significantly from the CAGn from either KS men, homozygous for the CAGn, or from the control subjects (22 vs. 23 vs. 21). SkewX was found in 12 of the 46 informative KS men (26%). In KS, height and arm span correlated positively to CAGn, whereas total cholesterol and haematocrit correlated negatively to CAGn. In controls, bone mineral density at the spine and hip correlated positively with CAGn, whereas adiponectin correlated negatively with CAGn. SkewX did not correlate to any of the investigated parameters. We conclude that CAGn polymorphism in AR explain some of the phenotypic variation in KS, whereas skewed X-chromosome inactivation did not. The impact of CAGn on final height may be caused by later reactivation of the pituitary-gonadal axis.