Calcium/calmodulin-dependent protein kinase IV (CAMK4) is a multifunctional serine/threonine protein kinase, which plays an important role in the spermatogenesis by phosphorylating protamines. It has been shown to be involved in the regulation of human sperm motility. Moreover, the Camk4 knockout mice were infertile because of severely reduced sperm count and morphological abnormalities. As no study is available on the association of this gene with male infertility, we analysed all the exons of CAMK4 gene in ethnically matched 283 infertile and 268 fertile Indian men. We identified twenty nucleotide substitutions, of which twelve were novel. Of these novel variants, eight were exclusively detected in infertile men. Moreover, two infertile men-specific mutations were non-synonymous replacing amino acids at the highly conserved region. In silico analysis predicted both of these mutations as 'deleterious'. In addition to nucleotide substitutions, we identified five novel insertion-deletion mutations; of these, g.150264_66delGCG was exclusively found in two oligoasthenoteratozoospermic men. In silico analysis of infertile men exclusive mutations predicted that they can alter/diminish the potential binding sites of splicing factors, which may affect the mRNA splicing and protein translation. Our study suggests that the mutations in CAMK4 may lead to abnormal semen parameters.

Imbalance between the oestrogen and androgen levels in utero is hypothesized to influence testicular cancer (TC) risk. Thus, variation in genes involved in the action of sex hormones may contribute to variability of an individual's susceptibility to TC. Mutations in testosterone pathway genes may alter the level of testosterone in vivo and hypothetically the risk of developing TC. Luteinizing hormone receptor (LHR), 5α-reductase II (SRD5A2) and androgen receptor (AR) are key elements in androgen action. A case-control study comprising 651 TC cases and 313 controls in a Norwegian population was conducted for investigation of polymorphisms in the LHR, SRD5A and AR genes and their possible association with TC. A statistical significant difference was observed in patients being heterozygous for the LHR Asn312Ser polymorphism when comparing genotypes between all TC cases and controls (OR = 0.66, 95% CI = 0.48-0.89, p(adj) = 0.049). No statistically significant difference between the histological subtypes seminoma and non-seminoma was observed. Our results may suggest a possible association between genetic variation in the LHR gene and the risk of developing TC.

Premature ejaculation (PE) is one of the most prevalent male sexual dysfunctions. Selective resection of the dorsal nerve (SRDN) of penis has recently been used for the treatment of PE and has shown some efficacy. To further clarify the efficacy and safety of SRDN on PE, we performed a preliminary, randomized, placebo-controlled clinical observational study. Persons with the complaints of rapid ejaculation, asking for circumcision because of redundant foreskin, intravaginal ejaculation latency time (IELT) within 2 min, not responding to antidepressant medication or disliking oral medication were randomly enrolled in two groups. From April 2007 to August 2010, a total of 101 eligible persons were enrolled, 40 of them received SRDN which dorsal nerves of the penis were selectively resected, and those (n = 61) enrolled in the control group were circumcised only. IELT and the Brief Male Sexual Function Inventory (BMSFI) questionnaire were implemented pre- and post-operatively for the evaluation of the effect and safety of the surgery. There are no statistically significant differences in the baseline data including mean ages, mean IELTs, perceived control abilities and the BMSFI mean scores between the two groups. With regard to the post-operative data of the surgery, both IELTs and perceived control abilities were significantly increased after SRDN (1.1 ± 0.9 min vs. 3.8 ± 3.1 min for pre- and post-operative IELT, respectively, p < 0.01),whereas the post-operative results were not significantly improved for the control group (1.2 ± 0.7 min vs. 1.5 ± 1.1 min, p > 0.05). Also, there were no statistically significant differences both in BMSFI composite and subscale scores between the two groups after surgery. Hence, we conclude that SRDN is effective in delaying ejaculation and improving ejaculatory control, whereas erectile function is not affected. The results imply that SRDN may be an alternative method for the treatment of PE for some patients.

The effective separation of X- and Y-bearing chromosome spermatozoa has been a topic of major attraction to a number of scientific disciplines. Approaches have typically been based upon either the kinetic or the physical characteristics of spermatozoa. Much of the information available to date has either suggested conflicting evidence between different approaches or a lack of repeatability, while other robust and reproducible techniques require expensive equipment and are being questioned with relation to their safety in clinical applications. This study describes a safe and efficient method for the successful enrichment of the Y-bearing chromosome spermatozoa cells from their X counterparts in the human male using a simple approach based on centrifugation. Five donor candidates with normal semen profiles and proven fertility were recruited. In total, 20 semen specimens were processed using conventional swim-up. During each attempt, half of the swim-up product was subjected to the enrichment technique and the other half served as control. Parameters important for successfully skewing sex ratios included the relative centrifugal force, the duration of centrifugal separation and the number of centrifugation rounds. Assessment of samples following the separation technique was effected by a three-colour-labelled fluorescent in situ hybridization. More than 1000 spermatozoa for each donor specimen were assessed for the presence of an X or Y chromosome. The enrichment technique produced a significantly higher (p < 0.001) overall frequency of 85.5% for the Y-bearing chromosome spermatozoa in the experimental group (3606 X-bearing/21, 209 Y-bearing) compared with a frequency of 50.1% in the control group (11,801 X-bearing/11,269 Y-bearing), where there was no statistically significant difference in the number of either X- or Y-chromosome-bearing spermatozoa. In conclusion, successful skewing of human Y-bearing chromosome spermatozoa can be reproducibly achieved by the use of simple swim-up followed by a meticulous centrifugation protocol.

The role of thyroid hormones in the control of erectile functioning has been only superficially investigated. The aim of the present study was to investigate the association between thyroid and erectile function in two different cohorts of subjects. The first one derives from the European Male Ageing Study (EMAS study), a multicentre survey performed on a sample of 3369 community-dwelling men aged 40-79 years (mean 60 ± 11 years). The second cohort is a consecutive series of 3203 heterosexual male patients (mean age 51.8 ± 13.0 years) attending our Andrology and Sexual Medicine Outpatient Clinic for sexual dysfunction at the University of Florence (UNIFI study). In the EMAS study all subjects were tested for thyroid-stimulating hormone (TSH) and free thyroxine (FT4). Similarly, TSH levels were checked in all patients in the UNIFI study, while FT4 only when TSH resulted outside the reference range. Overt primary hyperthyroidism (reduced TSH and elevated FT4, according to the reference range) was found in 0.3 and 0.2% of EMAS and UNIFI study respectively. In both study cohorts, suppressed TSH levels were associated with erectile dysfunction (ED). Overt hyperthyroidism was associated with an increased risk of severe erectile dysfunction (ED, hazard ratio = 14 and 16 in the EMAS and UNIFI study, respectively; both p < 0.05), after adjusting for confounding factors. These associations were confirmed in nested case-control analyses, comparing subjects with overt hyperthyroidism to age, BMI, smoking status and testosterone-matched controls. Conversely, no association between primary hypothyroidism and ED was observed. In conclusion, erectile function should be evaluated in all individuals with hyperthyroidism. Conversely, assessment of thyroid function cannot be recommended as routine practice in all ED patients.

Spermatozoa contribute to approximately half of the genome of future progeny, and therefore, have a profound impact on embryo development post-fertilization. Sperm selection based on viability and normal morphology does not eliminate the chance for DNA damaged spermatozoa to be inseminated and may account for a considerable percentage of failed embryo development post-ICSI (Intra cytoplasmic sperm injection). Therefore, sperm selection based on functional sperm characteristics to preclude insemination of DNA damaged spermatozoa have paved the way for successful ICSI outcomes. In regard to this, different laboratories have introduced novel procedures to replace traditional or orthodox sperm selection methods. This review attempts to provide information on the scientific bases of each procedure, and pinpoint their advantages and disadvantages. In addition to data from our research, a systematic search on the literature, publications and presentations was carried out using such databases as PubMed and ISI-Web.

Haemorrhoids are associated with regional vascular abnormalities and rectal pain, which are hypothesized to increase the risk of erectile dysfunction (ED); however, few studies have investigated the association between ED and haemorrhoids. This case-control study aimed to estimate the association between haemorrhoids and ED by using a population-based data in Taiwan. We identified 6,310 patients with ED as cases and randomly selected 31,550 controls. Conditional logistic regression was performed to compute the odds ratio (OR) for having been previously diagnosed with haemorrhoids between cases and controls. The results show that haemorrhoids were found to be present among 1,572 (24.9%) cases and 4,491 (14.20%) controls. The OR for prior haemorrhoids among cases was 1.90 (95% CI = 1.78-2.03) when compared with controls after adjusting for monthly income, geographical location, hypertension, diabetes, coronary heart disease, hyperlipidemia, obesity and alcohol abuse/alcohol dependence syndrome. Younger cases demonstrated a higher risk for prior haemorrhoids when compared with controls. In particular, the adjusted OR among cases <30 years old was 3.71 (95% CI = 2.74-5.02) when compared with controls. We concluded that there was an association between ED and a prior diagnosis of haemorrhoids.

Progesterone has been identified to be one of the physiological regulators of sperm hyperactivation and acrosome reaction. However, the high sensitivity of human spermatozoa to progesterone implies that many may undergo premature hyperactivation and acrosome reaction thereby compromising their ability to fertilize. We hypothesized that if a spermatozoon has to preclude the occurrence of these events prematurely, there should be differential dose- and time-dependent effects on motility and acrosome reaction. We observed that low concentrations of progesterone (10 and 100 nm) induce sperm motility and activate tyrosine kinase; higher concentrations (1-10 μm) are required to induce extracellular signal regulated kinases 1/2 (Erk1/2), p90 ribosomal S6 kinase (p90RSK), p38 mitogen-activated protein kinase (p38MAPK), c-Jun N-terminal kinase (JNK1) and AKT phosphorylation, hyperactivation and acrosome reaction. The induction of acrosome reaction and tyrosine phosphorylation in response to higher concentration of progesterone is not absolutely dependent on activation of T-type voltage-gated Ca(2+) channel or CatSper as Mibefradil did not completely abrogate progesterone-mediated effects. These results imply that although the spermatozoa are sensitive to low concentrations of progesterone, they only activate motility and tyrosine kinase activation; higher concentrations are required to induce hyperactivation and acrosome reaction probably by activating multiple kinase pathways including the MAPK and AKT.

Testosterone (T) and vitamin D (VD) interact in androgen deficient men, however, this interaction and subsequent semen quality and bone mineral density (BMD) status is not clear in infertile men. Our objective was to investigate T, VD, semen quality, BMD and their relationships in Chinese infertile men. We conducted a cross-sectional study of 559 men aged 20-40 years, including 195 fertile men, 9 infertile men with known risk factors for osteoporosis (WR) and 355 infertile men without known risk factors for osteoporosis (WOR). WOR infertile men constituted 314 oligo-, astheno-, teratospermic or normospermic infertile men (OATN men) and 41 non-obstructive azoospermic men (NOA men). Differences of parameters were assessed, and the relationships were adjusted by multiple linear regression. WOR infertile men had significantly lower T, lumbar spine and total hip BMD than fertile men (all p < 0.05). Bioavailable T (Bio-T) and 25-hydroxyvitamin D [25(OH)D] were independent determinants of BMD in WOR infertile men (all p < 0.01) but not in fertile men. After stratifying Bio-T, WOR infertile men had lower BMD than fertile men (all p < 0.05) in low Bio-T subgroups (Bio-T ≤ 11.6 nmol/L), but not high Bio-T subgroups (Bio-T > 11.6 nmol/L). 25(OH)D was an independent determinant of sperm motility and morphology in WOR OATN men (all p < 0.05), with only borderline significance in fertile men(motility: p = 0.047; morphology: p = 0.056). T determined sperm concentration (square root) and morphology in WOR OATN men (all p < 0.001). No correlations between T and 25(OH)D were found in all groups. We suggest that infertile men have lower T and BMD than fertile men. 25(OH)D and T were associated with low BMD and poor semen quality in infertile men.

Testicular cancer is one of the most rapidly increasing tumour types but its aetiology is still largely unexplained. Cryptorchidism and familial testicular cancer, established risk factors, explain less than 10% of all cases. Among investigated post-natal factors, early puberty was suggested as a potential risk factor but the topic has been poorly investigated. We undertook a meta-analysis of the effect of age at puberty on testicular cancer risk, attempting at enhancing the homogeneity in the definition of the exposure among studies to obtain valid pooled estimates. Search strategies were conducted in PubMed on December 2011. All markers of puberty onset (age at voice change, age when started shaving and reported age at onset) were considered. We re-categorized age at puberty from all studies into a common three-level variable: younger than peers, same age as peers, older than peers. A total of 391 references were retrieved, of which 12 met the inclusion criteria. Later puberty appeared to be protective. In particular late vs. same age at start shaving gave an OR of 0.84 (95% CI: 0.75-0.95, five studies); late vs. same age at voice change gave an OR of 0.87 (95% CI: 0.75-1.01, five studies); and later age than peers at reported onset of puberty gave an OR of 0.81 (95% CI: 0.73-0.89, eight studies). Early puberty showed no effect on testicular cancer risk. This meta-analysis has found consistent evidence of a decreased risk of testicular cancer in association with later puberty, suggesting that post-natal factors may contribute to testicular cancer risk.

Chromosomal polymorphism has been reported to be associated with infertility, but its effect on IVF/ICSI-ET outcome is still controversial. To evaluate whether or not chromosomal polymorphism in men plays a role in spermatogenesis and the outcome of IVF/ICSI-ET, we retrospectively analysed 281 infertile couples. Measures included fertilization rate, implantation rate, pregnancy rate, clinical pregnancy rate, ongoing pregnancy rate, early miscarriage rate and preterm rate. Men with chromosomal polymorphism had significantly higher frequencies of severe oligozoospermia and azoospermia than those without (37.12% vs. 16.11%, p < 0.001; 27.27% vs. 10.74%, p < 0.001; respectively). Significantly, lower fertilization rate (68.02% vs. 78.00%, p < 0.001) and clinical pregnancy rate (45.00% vs. 66.67%, p = 0.031) were observed in polymorphism-carrying men with severe oligozoospermia compared with non-carriers with severe oligozoospermia. This suggests that chromosomal polymorphism has adverse effects on spermatogenesis, negatively influencing the outcome of IVF/ICSI-ET treatment. Polymorphic variations on the Y chromosome have been found to be the most prevalent polymorphism in infertile men, most frequently occurring in patients with severe oligozoospermia.

The histological classification of testicular germ cell tumours (TGCTs) to seminoma or non-seminomatous germ cell tumours is at present the main criterion for the clinical outcome and selection of the treatment strategy. In view of the need to identify novel prognostic biomarkers for TGCTs, we investigated the expression of the matrix metalloproteinases MMP-2 and MMP-9 in testicular tumour tissues and cell lines of both seminoma and non-seminoma origin. Immunohistochemistry and zymography analysis of tumoural tissues showed significantly higher levels of MMP-2 and MMP-9 compared with normal testis with the active forms detected only in the tumour tissues. Three cell lines representative of the different tumour types, JKT-1 seminoma, NCCIT teratocarcinoma and NTERA2/D1 embryonal carcinoma were also evaluated for their expression of these MMPs using qPCR and zymography and for their invasive properties. The more invasive non-seminomatous teratocarcinoma and embryonal cells expressed considerably more MMP-2 and MMP-9 compared with seminoma cells exhibiting lower invasiveness. Furthermore, an inverse relation was observed between invasiveness and the expression of endogenous inhibitors TIMP-1 and TIMP-2. The MMP inhibitor Marimastat inhibited invasion in all cell lines, the highest inhibition was observed in the more invasive NTERA2/D1 and NCCIT cells, which presented the highest ratio of MMP-2 and MMP-9 vs. TIMP-1 and TIMP-2. These results highlight the importance of MMP-2 and MMP-9 in the invasiveness of testicular tumours and suggest that their levels, vs. those of TIMP-1 and TIMP-2, may represent potential biomarkers for testicular malignancy.

The defence of the male reproductive tract against microorganisms is critical for fertilization. The prostate gland has been reported to express several molecules of the innate immune system. However, little information is available about how androgens may modulate host defences within the prostate. We therefore aimed to examine in the rat the expression of the TLR4 system, which is strongly involved in pathogen recognition, and the secretion of the antibacterial substances rBD-1 and SP-D after androgen withdrawal. Immunoblotting and immunocytochemical analysis revealed a time-dependent increase in these molecules after orchiectomy, with epithelial and stromal cells being an important source of prostatic host defence proteins. In view of this, we evaluated the potential improvement in antibacterial ability of the prostatic fluid from orchiectomized animals ex vivo. Only samples from rats at 5 days post-orchiectomy showed a slight inhibition of Escherichia coli growth. Finally, E. coli was inoculated into the ventral prostate of orchiectomized or control rats, with bacterial growth being counted at 5 days after infection. Animals with androgen depletion presented a lower bacterial count, and showed few histological signs of prostatic inflammation compared with controls. In vitro studies confirmed that isolated lipopolysaccharide (LPS)-treated prostatic cells in the absence of testosterone increased SP-D. Moreover, media from these cells showed a higher antimicrobial activity than supernatants from testosterone- and LPS-treated cells. Our findings indicate that testosterone maintains a reduced expression of key elements for innate immunity and diminishes the antibacterial ability of the rat prostate. These data may represent an important mechanism underlying the immunosuppressive activity of testosterone in the gland. However, this immunosuppressive function of androgens is understandable as a means of avoiding uncontrolled immune responses against the haploid male gamete in the reproductive tract.