Showing papers in "Journal of Andrology in 2015"

International patterns and trends in testicular cancer incidence, overall and by histologic subtype, 1973-2007

Abstract: Background Incidence rates of testicular cancer in Northern European and North American countries have been widely reported, whereas rates in other populations, such as Eastern Europe, Central/South America, Asia, and Africa, have been less frequently evaluated. We examined testicular cancer incidence rates overall and by histologic type by calendar time and birth cohort for selected global populations 1973-2007.

Dietary fatty acids affect semen quality: a review.

Abstract: Mammalian spermatozoa are characterized by a high proportion of polyunsaturated fatty acids (PUFA) which play a crucial role in fertilization. This review focuses on analysis of sperm fatty acid profiles and the effects of omega-3, saturated and trans dietary and sperm fatty acids on sperm parameters. Two major points have been pivotal points of investigation in the field of sperm fatty acid profiles: first, the comparison between fatty acid profiles of fertile and infertile men and second, the effect of dietary fatty acids on sperm fatty acid profiles as well as sperm quality and quantity. Docosahexaenoic acid (DHA, C22:6n-3), and palmitic acid (C16:0) are the predominant PUFA and saturated fatty acids, respectively, in human sperm cells. Higher levels of DHA are concentrated on the sperm's head or tail varying among different species. However, the human sperm head contains a higher concentration of DHA. Dietary fatty acids influence on sperm fatty acid profiles and it seems that sperm fatty acid profiles are most sensitive to dietary omega-3 PUFA. Although improvements in sperm parameters are a response to omega-3 sources after more than 4 weeks of supplementation in the male diet, time-dependent and dose-dependent responses may explain the failure in some experiments. In human spermatozoa, elevated saturated or trans fatty acid concentration and a low DHA level is a concern. The regulations of the sperm fatty acid mean melting point as well as expression regulation of peroxisome proliferator-activated receptor gamma (PPARG) alongside with spermatozoon assembly, anti-apoptosis effects, eicosanoid formation, and hormone activity are the putative key factors that induce a response by inclusion of omega-3 PUFA.

Recent trends in the incidence of testicular germ cell tumors in the United States

Abstract: Testicular germ cell tumors (TGCT), which comprise 98% of all testicular malignancies, are the most commonly occurring cancers among men between the ages of 15 and 44 years in the United States (U.S.). A prior report from our group found that while TGCT incidence among all U.S. men increased between 1973 and 2003, the rate of increase among black men was more pronounced starting in 1989–1993 than was the rate of increase among other men. In addition, TGCT incidence increased among Hispanic white men between 1992 and 2003. To determine whether these patterns have continued, in the current study we examined temporal trends in incidence through 2011. Between 1992 and 2011, 21,271 TGCTs (12,419 seminomas; 8,715 nonseminomas; 137 spermatocytic seminomas) were diagnosed among residents of the Surveillance, Epidemiology, and End Results (SEER) 13 registry areas. The incidence of TGCT was highest among non-Hispanic white men (6.97 per 100,000 man-years) followed by American Indian/Alaska Native (4.66), Hispanic white (4.11), Asian/Pacific Islander (1.95), and black (1.20) men. Non-Hispanic white men were more likely to present with smaller tumors (3.5 cm) and localized disease (72.6%) than were men of other races/ethnicities. Between 1992 and 2011, TGCT incidence increased significantly among Hispanic white (APC: 2.94, p<0.0001), black (APC: 1.67, p=0.03), non-Hispanic white (APC: 1.23, p<0.0001), and Asian/Pacific Islander (APC: 1.04, p=0.05) men. Incidence rates also increased, although not significantly, among American Indian/Alaska Native men (APC: 2.96, p=0.06). The increases were greater for nonseminoma than seminoma. In summary, while non-Hispanic white men in the U.S. continue to have the highest incidence of TGCT, they present at more favorable stages of disease and with smaller tumors than do other men. The increasing rates among non-white men, in conjunction with the larger proportion of non-localized stage disease, suggest an area where future research is warranted.

Sperm global DNA methylation level: association with semen parameters and genome integrity.

Abstract: Sperm DNA methylation abnormalities have been detected in oligozoospermic men. However, the association between sperm DNA methylation defects, sperm parameters and sperm DNA, and chromatin integrity remains poorly understood. This study was designed to clarify this issue. We recruited a cohort of 92 men (62 normozoospermic and 30 oligoasthenozoospermic) presenting for infertility evaluation during a 1-year period. Sperm global DNA methylation was evaluated by an ELISA-like method, DNA fragmentation was evaluated by flow cytometry-based terminal transferase dUTP nick end-labeling (TUNEL) assay (reported as DNA fragmentation index or DFI), and sperm denaturation was evaluated by aniline blue staining (reported as sperm denaturation index or SDI, a marker of chromatin compaction). We found a significant positive association between sperm global DNA methylation level and conventional sperm parameters (sperm concentration and motility), supported by the results of methylation analysis on H19-DMR. We also identified significant inverse relationships between sperm global DNA methylation, and, both DFI and SDI. However, sperm global DNA methylation level was not related to sperm vitality or morphology. Our findings suggest that global sperm DNA methylation levels are related to conventional sperm parameters, as well as, sperm chromatin and DNA integrity.

Age and markers of Leydig cell function, but not of Sertoli cell function predict the success of sperm retrieval in adolescents and adults with Klinefelter's syndrome.

Abstract: Summary Microsurgical testicular sperm extraction (mTESE), combined with intracytoplasmic sperm injection (ICSI) represents a chance for azoospermic men with Klinefelter′s syndrome (KS) to father children. The objective of this study was to identify predictive factors for the success of mTESE from adolescents and adults with KS. The clinical data of 50 late pubertal adolescents (13–19 years) and 85 adult patients (20–61 years) with non-mosaic KS, who underwent mTESE, were analysed with respect to factors, potentially predictive of active spermatogenesis; specifically a history of cryptorchidism, age, testicular volumes, serum levels of LH, FSH, testosterone (T) and estradiol at the time of surgery. Inhibin B, AMH and INSL3 were additionally analysed in the adolescents. A younger age and a near-compensated Leydig cell function were associated with higher success of sperm retrieval via mTESE: In adolescents ≥15–19 years, spermatozoa were retrieved in 45%, compared to 31% in adults; in adolescents aged 13–14 years, spermatozoa were collected in only 10%. Adolescents with an LH ≤17.5 U/L, along with a T level ≥7.5 nmol/L had the best success rate (54%), which fell to 44% with higher LH, whereas those with low T (<7.5 nmol/L), irrespective of LH had no sperm retrieval. In adults with T levels above and LH below these thresholds, the success rate was 51%, falling to 19%, if LH was higher. When T was lower than threshold, the rate was 17%. No association between testicular volumes, serum levels of FSH, Inhibin B, AMH, estradiol and mTESE success was found. A history of cryptorchidism was associated with lower retrieval rates. A window of opportunity for an approximate 50% chance to retrieve spermatozoa via mTESE exists for young, late pubertal KS patients between age 15 and young adulthood, when Leydig cell function is at its best. In these cases, referral to a centre of expertise should be considered.

Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus

Abstract: Summary Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) and poor (poor freezability ejaculates: PFE) freezability stallion ejaculates in other sperm parameters, including peroxide and superoxide levels, potential of mitochondrial membrane and nuclear integrity. With this purpose, a total of 24 stallion ejaculates were cryopreserved and classified into two groups (GFE vs. PFE), depending on their sperm membrane integrity and motility after freeze-thawing. From the total of 24 ejaculates, 13 were classified as GFE and the other 11 were classified as PFE. Apart from differences in sperm membrane permeability and lipid disorder after freeze-thawing, GFE presented significantly (p < 0.05) higher percentages of viable spermatozoa with high content of peroxides and of superoxides than PFE. In contrast, and despite cryopreservation of stallion spermatozoa increasing DNA fragmentation and disrupting disulphide bonds in sperm head proteins, no significant differences between GFE and PFE were seen. We can thus conclude that good and poor freezability stallion ejaculates differ in their reactive oxygen species levels after cryopreservation, but not in the damage extent on sperm nucleus.

HPV-DNA sperm infection and infertility: from a systematic literature review to a possible clinical management proposal.

Abstract: The objectives of this study were to investigate the implications of human papillomavirus (HPV) sperm infection on male fertility, impairment of sperm parameters, and possible alteration of sperm nuclear status and to identify a possible effective management of infertile men with HPV sperm infection. We employed a systematic review and clinical management proposal at the Centers for Reproductive and Health care for treating infertile male patients with HPV infection. Literature search was carried out in electronic databases in the last two decades. We focused our attention on: (i) HPV sperm prevalence (ii) HPV-related alteration of sperm parameters; (iii) molecular mechanisms of HPV semen infection and infertility. The main outcome measures were HPV prevalence in infertile male patients and semen parameters. The prevalence of HPV sperm infection ranges between 2 and 31% in men from general population and between 10 and 35.7% in men affected by unexplained infertility. The presence of HPV in semen is associated with an impairment of sperm motility and the presence of anti-sperm antibodies. The molecular mechanisms underlying impairment of sperm motility apparatus need further evaluations. A greater attention should be applied to assess HPV sperm infection, particularly in men undergoing assisted reproduction techniques cycle for male infertility or sperm banking. It would be useful to perform HPV test and fluorescent in situ hybridization analysis for HPV in semen from these patients both at first admission, to define the possible presence and localization of semen infection, and after 6 months, to assess the possible virus clearance retrieval on normal sperm parameters.

Cisplatin resistance in germ cell tumours: models and mechanisms.

Abstract: Recent years have led to a better understanding of the mechanisms underlying cisplatin response and resistance in germ cell tumours (GCT), and several promising targets have been identified. Two main mechanisms of the responsiveness to DNA damaging agents have been postulated. Firstly, GCT readily activate a DNA damage response, but show deficits in several damage repair pathways. In particular, they have been found to have defects in interstrand crosslink repair and in homologous recombination (HR). Secondly, GCT, especially embryonal carcinoma (EC) cells, show a hypersensitive apoptotic response to DNA damage, which activates p53, and leads to up-regulation of the pro-apoptotic factors Noxa, Puma and Fas in non-resistant EC. These cells fail to activate p21 which induces a G1/S arrest, but accumulate in G2/M phase. In the absence of functional p53, family members like p73 and GTAp63 might be important in initiating this response. Mechanisms involved in cisplatin resistance are as follows: down-regulation of Oct4 (e.g. as a result of hypoxia, treatment with retinoic acid or exposure to cisplatin) and failure to induce Puma and Noxa; changes in the expression levels of micro-RNAs such as miR-17/-106b, miR-302a, or miR-371 to -373; elevated levels of MDM2 and cytoplasmic translocation of p21 by phosphorylation; and activation of the PDGFRβ/PI3K/pAKT pathway. Several approaches to overcome resistance have been successfully examined in vitro and in vivo, including PARP inhibitors, especially in cells showing deficient HR-repair; stabilization of p53 using nutlin-3; inhibition of several components of the PI3K/pAKT pathway using small molecules; and DNA demethylation by 5-azacytidine or 5-aza-deoxy-cytidine, among others. Many of these substances deserve further exploration, alone or in combination with DNA damaging agents, and the most promising approaches should be taken forward to clinical testing. Targeted therapy based on mechanistic insights holds the promise to turn cisplatin-resistant GCT into a curable disease.

Mycoplasma and ureaplasma infection and male infertility: a systematic review and meta-analysis.

Abstract: The relationship between mycoplasma and ureaplasma infection and male infertility has been studied widely; however, results remain controversial. This meta-analysis investigated the association between genital ureaplasmas (Ureaplasma urealyticum, Ureaplasma parvum) and mycoplasmas (Mycoplasma hominis, Mycoplasma genitalium), and risk of male infertility. Differences in prevalence of ureaplasma and mycoplasma infection between China and the rest of the world were also compared. Study data were collected from PubMed, Embase and the China National Knowledge Infrastructure. Summary odds ratio (OR) with 95% confidence interval (CI) was applied to assess the relationship. Heterogeneity testing and publication bias testing were also performed. A total of 14 studies were used: five case-control studies with 611 infertile cases and 506 controls featuring U. urealyticum infection, and nine case-control studies with 2410 cases and 1223 controls concerning M. hominis infection. Two other infection (U. parvum and M. genitalium) were featured in five and three studies, respectively. The meta-analysis results indicated that U. parvum and M. genitalium are not associated with male infertility. However, a significant relationship existed between U. urealyticum and M. hominis and male infertility. Comparing the global average with China, a significantly higher positive rate of U. urealyticum, but a significantly lower positive rate of M. hominis, was observed in both the infertile and control groups in China.

MicroRNA miR‐371a‐3p in serum of patients with germ cell tumours: evaluations for establishing a serum biomarker

Abstract: Summary As only 60% of the patients with germ cell tumour (GCT) express the classical markers, new markers as for example microRNAs (miRNAs) are required. One promising candidate is miR-371a-3p, but data are sparse to date. We measured serum levels of miR-371a-3p in GCT patients, in controls, and in cases with other malignancies. We also assessed the expression in other body fluids and we looked to the decline of serum miR-371a-3p levels after treatment. miR-371a-3p levels were measured by quantitative polymerase chain reaction in serum samples of 25 GCT patients, 6 testicular intraepithelial neoplasia (TIN) patients, 20 healthy males and 24 non-testicular malignancies (NTMs). Testicular vein blood (TVB) was examined in five GCT patients and five controls. Five GCT patients had serial daily measurements after orchiectomy. Five seminal plasma samples, three urine specimens and one pleural effusion fluid were processed likewise. GCT patients had significantly higher miR-371a-3p serum levels than controls and NTMs. Serum levels of controls, TINs and NTMs were not significantly different. TVB samples of GCT patients had 65.4-fold higher serum levels than peripheral blood. Malignant pleural effusion fluid had extremely high levels of miR-371a-3p, seminal plasma had strongly elevated levels by comparison with serum levels of controls. In urine of GCT patients, no miR-371a-3p expression was detected. Daily measurements after orchiectomy in stage 1 patients revealed a decline by 95% within 24 h. Serum levels of miR-371a-3p appear to be a promising specific biomarker of GCTs as is suggested by high serum levels in GCT patients, the rapid return of elevated levels to normal range after treatment, the association of serum levels with tumour bulk, the non-expression in NTMs and the much higher levels of miR-371a-3p in TVB. This potential marker deserves further exploration in a large-scale clinical study.

Comparative analysis of boar seminal plasma proteome from different freezability ejaculates and identification of Fibronectin 1 as sperm freezability marker.

Abstract: Variation in boar sperm freezability (i.e. capacity to withstand cryopreservation) between ejaculates is a limitation largely reported in the literature. Prediction of sperm freezability and classification of boar ejaculates into good (GFEs) and poor freezability ejaculates (PFEs) before cryopreservation takes place may increase the use of frozen-thawed spermatozoa. While markers of boar sperm freezability have been found from sperm cell extracts, little attention has been paid to seminal plasma. On this basis, the present study compared the fresh seminal plasma proteome of 9 GFEs and 9 PFEs through two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography mass spectrometry (LC-MS/MS). The ejaculates were previously classified as GFE or PFE upon their sperm viability and progressive motility assessments at 30 and 240 min post thawing. From a total of 51 spots, four were found to significantly (p < 0.05) differ between GFEs and PFEs, and two were identified as fibronectin-1 (FN1) and glutathione peroxidase 5 (GPX5). These two potential markers were further studied by western blot and correlation analysis between protein relative abundances in fresh seminal plasma and regression factors from principal component analyses (PCA) run using post-thawing sperm quality parameters. Results confirmed that FN1 is a reliable marker of boar sperm freezability, because GFEs presented significantly (p < 0.05) higher FN1-amounts than PFEs and FN1 was found to be correlated with the first PCA component at 240 min post thawing. In contrast, GPX5 was not validated as a boar sperm freezability marker. We can thus conclude that levels of FN1 in fresh seminal plasma from boar semen may be used as a sperm freezability marker, thereby facilitating the use of frozen-thawed boar spermatozoa.

Designer steroids - over-the-counter supplements and their androgenic component: review of an increasing problem.

Abstract: Colloquially referred to by various misleading monikers ('pro-hormones', 'natural steroids', 'testosterone boosters', etc.) designer anabolic steroids have been popular now for over a decade as a way to achieve classic anabolic steroid-like results from products sold in the legal marketplace. Recent evidence suggests that anabolic steroid use may be the most common cause of hypogonadism in men of reproductive age. Despite recent regulatory efforts that have banned specific compounds, many anabolic-androgenic steroids (AAS) remain available in over-the-counter dietary supplements that are legally sold in the United States. Severe side effects including hepatotoxicity, cholestasis, renal failure, hypogonadism, gynecomastia, and infertility have been reported secondary to the use of these products. While some of these side effects may be reversible, more aggressive use may result in more permanent end-organ damage as has been previously described for the case of aggressive AAS users (Rahnema et al., Fertil Steril, 2014). Designer AAS remain easily available for purchase in over-the-counter bodybuilding supplements and these products appear to be increasingly popular, despite the known health risks associated with their use. We conducted a systematic search to identify the designer steroids that are most commonly sold in dietary supplements as of April 2014 and review what is known regarding their potency and toxicity. We propose that the impact of AAS use on the reproductive and hormonal health of men is underestimated in the literature owing to previous studies' failure to account for designer steroid use. Lastly, we make clinical recommendations to help physicians steer patients away from potentially harmful supplements, and summarize key regulatory obstacles that have allowed potent androgens to remain unregulated in the legal marketplace.

Surveillance vs. adjuvant therapy of clinical stage I testicular tumors - a review and the SWENOTECA experience

Abstract: Although clinical stage I (CS I) testicular cancer is highly curable, the optimal management is controversial. The aims of the Swedish and Norwegian Testicular Cancer Group (SWENOTECA) studies for CS I non-seminoma (NS) and seminoma (S) have been to reduce treatment intensity while maintaining high survival rates, reduce the number of patients needing salvage treatment and implement patient autonomy with regard to adjuvant treatment. During 1998-2010 NS CSI patients with lymphovascular invasion (LVI) of the primary tumor (high risk) were recommended bleomycin, etoposide, cisplatin (BEP)x1. During 2000-2006 S CS I patients had the option to choose surveillance or adjuvant radiotherapy (AR). In 2004, carboplatinx1 (AUC7) was added as a third treatment option. In 2007 a new risk-adapted treatment protocol for S CS I was initiated. Patients with two risk factors (tumor size>4cm, tumor growth in the rete testis) were recommended carboplatinx1 and patients with 0-1 risk factor were recommended surveillance. All patients were provided with oral and written information of possible management options and could choose the other alternative. The relapse rate for NS CS I with BEPx1 was 3.2% for high risk, and 1.6% for low-risk patients. Five-year cause-specific survival was 100%. For S CS I-patients treated before 2007, 14.3% on surveillance relapsed, 3.9% after carboplatin, and 0.8% after AR. Five-year cause-specific survival was 99.9%. For S CS I-patients treated from 2007, a relapse rate <3% was confirmed for patients without risk factors. SWENOTECA considers BEPx1 standard adjuvant treatment in NS CS I high-risk patients. Low-risk patients should have the opportunity to receive BEPx1 following thorough information regarding pros and cons. For S CS I patients without risk factors, adjuvant treatment is not necessary. For patients with risk factors, patient autonomy should be respected. (Less)

Myoinositol improves sperm parameters and serum reproductive hormones in patients with idiopathic infertility: a prospective double-blind randomized placebo-controlled study.

Abstract: Summary Male infertility is a multifactorial disorder that affects a significant percentage of couples. Its etiology and pathogenesis remain elusive in about one-third of the cases; this is referred to as idiopathic infertility. Inositols mediate the sperm processes involved into oocyte fertilization, such as penetration of the ovum cumulus oophorus, binding with the zona pellucida and the acrosome reaction. The aim of this double-blind, randomized, placebo-controlled trial was to evaluate the efficacy and safety of myoinositol (the most abundant form of inositols present in nature) treatment in men with idiopathic infertility. To accomplish this, we evaluated the effects of myoinositol on sperm parameters and reproductive hormones at baseline and after 3 months of treatment in men with idiopathic infertility. No adverse reaction was observed. Myoinositol significantly increased the percentage of acrosome-reacted spermatozoa, sperm concentration, and total count and progressive motility compared to placebo. In addition, myoinositol rebalanced serum luteinizing hormone, follicle-stimulating hormone, and inhibin B concentrations. The clinical improvement of idiopathic infertile patients should encourage myoinositol use for the treatment of this disorder, even though its detailed mechanisms at the testicular level remain still unclear.

Combination of vitamin E and clomiphene citrate in treating patients with idiopathic oligoasthenozoospermia: A prospective, randomized trial

Abstract: The most common cause of male infertility is idiopathic oligoasthenozoospermia. Empirical medical treatment for idiopathic male infertility is still a controversial issue. The aim of this study was to evaluate any possible effects of combining vitamin E as antioxidant and clomiphene citrate as antiestrogen on spermatozoa concentration and motility in comparison to give either of medications alone in patients with idiopathic oligoasthenozoospermia. This is a comparative prospective randomized study. Ninety patients with idiopathic oligoasthenozoospermia were randomized into equally three groups: Group A: received vitamin E (400 mg/day) for 6 months. Group B: received clomiphene citrate (25 mg daily) for 6 months. Group C: received combination of both drugs in the same doses for 6 months. All patients were subjected to the following: history taking, general and genital examination, semen analysis, serum FSH, total testosterone, and scrotal duplex. Semen examination was performed according to the guidelines of (WHO, 2010), at the start of treatment and was repeated after 3 months and after 6 months of treatment. Regarding vitamin E group, there was insignificant increase in mean sperm concentration after 6 months of treatment in comparison to baseline. On the other hand, there was a significant improvement of mean sperm concentration in the other two groups after 6 months of treatment, with more significance in combination therapy group (p = 0.001). The mean total sperm motility has improved in all patients groups, in comparison to baseline, with more significance in combination therapy group. In vitamin E group, it was 28.07 ± 9.65% (p = 0.000). For those in clomiphene citrate group, was 33.33 ± 14.10% (p = 0.003) and 40.50 ± 17.54% (p = 0.000) in combination therapy group. Combining antioxidant and anti-estrogen therapy is a valid option for the treatment of a selected group of men with unexplained isolated oligoasthenozoospermia.

How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view

Abstract: A viable spermatozoon is the prerequisite for initiating fertilization in intracytoplasmic sperm injection. Usually motility is the primary sign used to determine a sperm's viability. However, in every in vitro fertilization (IVF) laboratory cases are observed in which none or only few spermatozoa are motile. This can occur in treatment cycles involving ejaculated samples but is most common in cases where surgically extracted testicular spermatozoa are used. To aid in selection, several techniques have been developed to identify viable spermatozoa from the immotile fraction. Amongst the more commonly used approaches are (i) the hypo-osmotic swelling test (ii) chemical substances for induction of tail movements (iii) the sperm tail flexibility test and (iv) laser-assisted immotile sperm selection. All can be used routinely in an IVF laboratory with each having both strengths and weaknesses. It is the purpose of this short review to focus on the technical issues involved in the performance of each of these techniques and to highlight the advantages and disadvantages of each approach.

Involvement of seminal leukocytes, reactive oxygen species, and sperm mitochondrial membrane potential in the DNA damage of the human spermatozoa

Abstract: Summary Measurement of reactive oxygen species (ROS) producing leukocytes in semen has been a standard component of the semen analysis, but its true significance remains still unknown. In this study, we have correlated the number of seminal leukocytes to various semen parameters. We found a negative correlation between the leukocyte number and sperm concentration (rs = −0.22; p = 0.01) and motility (rs = −0.20; p = 0.02). In contrast, a positive correlation between the number of leukocytes and both seminal ROS (rs = 0.70, p < 0.001; n = 125) and the number of spermatozoa with DNA fragmentation (rs = 0.43, p = 0.032; n = 25) was found. However, only a trend of positive correlation between ROS and the number of spermatozoa with TUNEL-detected DNA fragmentation was observed. Moreover, this latter was not correlated with loss of sperm mitochondrial membrane potential (MMP) (10% vs 35%, rs = 0.25, p = 0.08; n = 50). Overall these results indicate that the presence of high number of leukocytes in the ejaculate negatively affects key semen parameters, as sperm concentration and motility, associated with infertility conditions. Moreover, they suggest that leukocytes are the major source of the seminal ROS and cause of sperm DNA fragmentation. However, the absence of a clear correlation between ROS and sperm DNA fragmentation, and spermatozoa with damaged DNA and MMP loss, suggest that ROS produced by leukocytes might be not the only cause of DNA damage in spermatozoa and that intrinsic mitochondrial-dependent apoptotic pathways might not have a major impact on sperm DNA fragmentation.

Adding liraglutide to lifestyle changes, metformin and testosterone therapy boosts erectile function in diabetic obese men with overt hypogonadism.

Abstract: The aim of this retrospective observational study was to evaluate whether adding liraglutide to lifestyle changes, metformin (Met) and testosterone replacement therapy (TRT), by means of improving weight and glycaemic control, could boost erectile function in type 2 diabetic obese men with overt hypogonadism and erectile dysfunction (ED) in a 'real-life setting'. Forty-three obese, diabetic and hypogonadal men (aged 45-59 years) were evaluated because of complaining about the recent onset of ED. They were subdivided into two groups according to whether hypogonadism occurred after puberty (G1; n = 30: 25 with dysfunctional hypogonadism and 5 with acquired hypogonadotropic hypogonadism) or before puberty (G2; n = 13: 10 with Klinefelter's syndrome and 3 with idiopathic hypogonadotropic hypogonadism). Both G1 and G2 patients were given a combination of testosterone (T) [testosterone undecanoate (TU) 1000 mg/every 12 weeks] and Met (2000-3000 mg/day) for 1 year. In the poor responders (N) to this therapy in terms of glycaemic target (G1N: n = 16; G2N: n = 10), liraglutide (L) (1.2 μg/day) was added for a second year, while the good responders (Y) to T + Met (G1Y: 14/30 and G2Y: 3/13) continued this two drugs regimen therapy for another year. All patients were asked to fill in the International Index of Erectile Function (IIEF 15) questionnaire before starting TU plus Met (T1) and after 12 months (T2) and 24 months (T3) of treatment. Patients underwent a clinical examination and a determination of serum sex hormone binding globulin (SHBG), total testosterone (T) and glycosylated haemoglobin (HbA1c) at T1, T2 and T3. At T2, each patient obtained an improvement of ED (p 7.5% (>58 mmol/mol)], while T turned out to be within the range of young men. L added to TU and Met regimen in G1N and G2N allowed these patients to reach not only the glycaemic target [HbA1c = <7.5% (<58 nmol/mol)] and a significant reduction in body weight (p < 0.01), but also a further increase in SHBG (p < 0.05) and T (p < 0.01) plasma levels as well as a significant increment of IIEF score (T3). Conversely, at T3 G1Y and G2Y, who received the combined therapy with TRT and Met for the second year, showed a partial failure of that treatment given that there was no improvement of the IIEF score and they showed a significant rise in serum HbA1c (p < 0.05) and weight (p < 0.04) compared with the assessments at T2. These results suggest that TRT could improve clinical and metabolic parameters in obese, type 2 diabetic men with ED and overt hypogonadism (independently of when T deficit occurred). Furthermore, in case of insufficient metabolic control the addition of L to TRT and Met regimen allows to achieve serum T levels in the range of healthy men, as well as to reach glycaemic target and to lower weight, leading to a considerable improvement of ED.

Intracellular calcium movements of boar spermatozoa during 'in vitro' capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model.

Abstract: This work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca(2+) signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca(2+) labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca(2+) signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca(2+) . The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca(2+) -induced peak, O2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.

Reproductive function in male patients with type 1 diabetes mellitus.

Abstract: This study was undertaken to evaluate conventional and some of the main bio-functional spermatozoa parameters, serum gonadal hormones and didymo-epididymal ultrasound features in patients with type 1 diabetes mellitus (DM1). DM1 affects an increasing number of men of reproductive age. Diabetes may affect male reproduction by acting on the hypothalamic-pituitary-testicular axis, causing sexual dysfunction or disrupting male accessory gland function. However, data on spermatozoa parameters and other aspects of the reproductive function in these patients are scanty. Thirty-two patients with DM1 [27.0 (25.0-30.0 years)] and 20 age-matched fertile healthy men [28.0 (27.25-30.75 years)] were enrolled. Patients with diabetic neuropathy, other endocrine disorders or conditions known to alter spermatozoa parameters were excluded. Each subject underwent semen analysis, blood withdrawal for fasting and post-prandial glycaemia, hormonal analysis and didymo-epididymal ultrasound evaluation before and after ejaculation. Patients with DM1 had a lower percentage of spermatozoa with progressive motility [10.0 (7.0-12.75) vs. 45.0 (42.0-47.75) %; p < 0.01] and a higher percentage of spermatozoa with abnormal mitochondrial function than controls [47.0 (43.0-55.0) vs. 2.0 (1.0-5.0) %; p < 0.01]. Patients also had greater post-ejaculatory diameters of cephalic [11.5 (10.2-13.6) vs. 6.0 (4.0-7.0) mm; p < 0.01] and caudal epididymis [5.5 (4.00-7.55) vs. 3.0 (2.0-4.0) mm; p < 0.01] compared to controls, suggesting a lack of the physiological post-ejaculation epididymal shrinkage. Correlation analysis suggested that progressive motility was associated with fasting glucose (r = -0.68; p < 0.01). The other parameters did not show any significant difference. Patients with DM1 had a lower percentage of spermatozoa with progressive motility, impaired mitochondrial function and epididymal post-ejaculatory dysfunction. These findings may explain why patients with DM1 experience fertility disturbance. Larger multi-centric studies are necessary to confirm these results.

Inhibin B, AMH, but not INSL3, IGF1 or DHEAS support differentiation between constitutional delay of growth and puberty and hypogonadotropic hypogonadism.

Abstract: In pre-pubertal boys ≥ 14 years, the differentiation between constitutional delay of growth and puberty (CDGP) and hypogonadotropic hypogonadism (HH) is challenging, as current diagnostic tools have limitations in sensitivity and specificity. The aim of this study was to assess the usefulness of markers of gonadal activity, growth axis activation and adrenarche in differentiation between pre-pubertal CDGP and HH. This retrospective study was carried out between 2006 and 2015 in an academic out-patient referral centre. The clinical data of 94 boys, aged 13.9-23.2 years and referred for "pubertal delay" were reviewed. Definite diagnoses were established on initial work-up and clinical follow-up: 24 boys were diagnosed with HH, 22 boys with CDGP, pre-pubertal (PP CDGP) at referral and 28 boys with CDGP, early pubertal at referral (EP CDGP), the latter serving as control group. Twenty patients were excluded from evaluation because of previous sex steroid treatment or associated chronic disease. Inhibin B and AMH were measured in all (n = 74); INSL3, IGF1, IGFBP3 and DHEAS in a subset of patients (n = 45) in serum of first presentation. Inhibin B and AMH were higher in boys with PP CDGP than in boys with HH: inhibin B: 87.6 ± 42.5 vs. 19.8 ± 13.9 pg/mL; p < 0.001; AMH: 44.9 ± 27.1 vs. 15.4 ± 8.3 ng/mL; p < 0.001. Receiver operating characteristics (ROC) for the diagnosis of PPCDGP vs. HH (inhibin B ≥ 28.5 pg/mL): sensitivity: 95%, specificity: 75%; AUC: 0.955. In combination with an AMH cut-off ≥20 ng/mL the specificity increased to 83%. INSL3, IGF1, IGFBP3 and DHEAS levels were not different. In boys with EP CDGP, inhibin B and IGF1 levels were highest (138.7 ± 59.9 pg/mL/289.7 ± 117 ng/mL), whereas AMH levels were lowest (11.7 ± 9.1 ng/mL). Sertoli cell markers are helpful for establishing a prognosis, whether a boy with pubertal delay will enter puberty spontaneously, whereas Leydig cell, growth and adrenal markers are not.

Identification of endogenous metabolites in human sperm cells using proton nuclear magnetic resonance (1H‐NMR) spectroscopy and gas chromatography‐mass spectrometry (GC‐MS)

Abstract: The objective of this study was to contribute to the first comprehensive metabolomic characterization of the human sperm cell through the application of two untargeted platforms based on proton nuclear magnetic resonance ((1) H-NMR) spectroscopy and gas chromatography coupled to mass spectrometry (GC-MS). Using these two complementary strategies, we were able to identify a total of 69 metabolites, of which 42 were identified using NMR, 27 using GC-MS and 4 by both techniques. The identity of some of these metabolites was further confirmed by two-dimensional (1) H-(1) H homonuclear correlation spectroscopy (COSY) and (1) H-(13) C heteronuclear single-quantum correlation (HSQC) spectroscopy. Most of the metabolites identified are reported here for the first time in mature human spermatozoa. The relationship between the metabolites identified and the previously reported sperm proteome was also explored. Interestingly, overrepresented pathways included not only the metabolism of carbohydrates, but also of lipids and lipoproteins. Of note, a large number of the metabolites identified belonged to the amino acids, peptides and analogues super class. The identification of this initial set of metabolites represents an important first step to further study their function in male gamete physiology and to explore potential reasons for dysfunction in future studies. We also demonstrate that the application of NMR and MS provides complementary results, thus constituting a promising strategy towards the completion of the human sperm cell metabolome.

Assessment of the optimal vitrification protocol for pre‐pubertal mice testes leading to successful in vitro production of flagellated spermatozoa

Abstract: Testicular tissue cryopreservation offers the hope of preserved future fertility to pre-pubertal boys with cancer before exposition to gonadotoxic treatments. The objective of this study was to compare controlled slow freezing (CSF) with five vitrification techniques for cryopreservation of murine pre-pubertal testicular tissue and to evaluate the best protocol that could provide a successful completion of spermatogenesis after in vitro maturation. Testicular tissue from 24 mice at 6.5 days post-partum (dpp) was used to compare several vitrification protocols with one another, as well as with a CSF protocol. Toxicity test using additional 12 mice was performed for all cryopreservation solutions. Fresh tissue (FT) from six mice was used as a control. Once the optimal vitrification protocol was selected [the modified solid surface vitrification No. 1 (mSSV1 )], testes from 18 mice were cultured in vitro for 30 days with (i) fresh, (ii) slow-frozen/thawed and (iii) vitrified/warmed tissues. Testes from six mice at 36.5 dpp were used as controls. At day 30 of in vitro culture, germ cells of the seminiferous tubules showed a high ability to proliferate and elongated spermatids were observed after both freezing techniques, confirming the successful completion of in vitro spermatogenesis. However, after mSSV1 , the morphological alterations and the percentage of pyknotic seminiferous tubules were lower than CSF (4.67 ± 0.53 vs. 10.1 ± 1.12 and 22.7 ± 2.83% vs. 37.3 ± 4.24% respectively). Moreover, the number of flagellated spermatozoa produced per mg of tissue was higher for mSSV1 than for CSF (35 ± 3 vs. 9 ± 4 cells), with amounts of secreted testosterone during the culture close to those of FT. The mSSV1 protocol resulted in success rates better than CSF in maintaining testicular tissue structure, tubular morphology and tissue functions not solely for immediate frozen/thawed tissues but also after a long-term in vitro culture.

Cell-specific ablation in the testis: what have we learned?

Abstract: Testicular development and function is the culmination of a complex process of autocrine, paracrine and endocrine interactions between multiple cell types. Dissecting this has classically involved the use of systemic treatments to perturb endocrine function, or more recently, transgenic models to knockout individual genes. However, targeting genes one at a time does not capture the more wide-ranging role of each cell type in its entirety. An often overlooked, but extremely powerful approach to elucidate cellular function is the use of cell ablation strategies, specifically removing one cellular population and examining the resultant impacts on development and function. Cell ablation studies reveal a more holistic overview of cell-cell interactions. This not only identifies important roles for the ablated cell type, which warrant further downstream study, but also, and importantly, reveals functions within the tissue that occur completely independently of the ablated cell type. To date, cell ablation studies in the testis have specifically removed germ cells, Leydig cells, macrophages and recently Sertoli cells. These studies have provided great leaps in understanding not possible via other approaches; as such, cell ablation represents an essential component in the researchers' tool-kit, and should be viewed as a complement to the more mainstream approaches to advancing our understanding of testis biology. In this review, we summarise the cell ablation models used in the testis, and discuss what each of these have taught us about testis development and function.

Identification of known and novel germ cell cancer‐specific (embryonic) miRs in serum by high‐throughput profiling

Abstract: microRNAs (miRs) are short non-coding RNA molecules (≈21 nucleotides) involved in regulation of translation. As such they are crucial for normal cell development and differentiation as well as cellular maintenance. Dysregulation of miRs has been reported in various diseases, including cancer. Interestingly, miRs can be informative as tumor classifiers and disease biomarkers. Recent studies demonstrated the presence of miRs in body fluids like serum, thus providing a putative non-invasive tool to study and monitor disease state. Earlier targeted studies by several independent groups identified specific embryonic miRs as characteristic for germ cell tumors (GCT) (miR-371-2-3 & miR-302/367 clusters). This study reports a high-throughput miR profiling (≈750 miRs) approach on serum from testicular germ cell tumor patients (14 seminoma and 10 non-seminoma) and controls (n = 11), aiming at independent identification of miRs as candidate biomarkers for testicular GCT. A magnetic bead capture system was used to isolate miRs from serum. Subsequently, the TaqMan Array Card 3.0 platform was used for profiling. The previously identified miRs 371 and 372 were confirmed to be specifically elevated in serum from germ cell tumor patients. In addition, several novels miRs were identified that were discriminative between germ cell cancer and controls: miR-511, -26b, -769, -23a, -106b, -365, -598, -340, and let-7a. In conclusion, this study validates the power of the embryonic miRs 371 and 372 in detecting malignant GCT (SE and NS) based on serum miR levels and identifies several potential novel miR targets.

Common variants identified in genome-wide association studies of testicular germ cell tumour: an update, biological insights and clinical application

Abstract: Testicular germ cell tumour (TGCT) is the most common cause of cancer in young men (aged 15-45years) in many populations. Multiple genome-wide association studies (GWAS) of TGCT have now been conducted, yielding over 25 disease-associated single-nucleotide polymorphism (SNP)s at 19 independent loci. The genes at these loci have provided rich biological and genetic insight into possible mechanisms underlying testicular germ cell oncogenesis. In this review, we summarize these mechanisms which can be grouped into five distinct categories: KIT/KITLG signalling, other pathways of male germ cell development/differentiation, telomerase function, microtubule assembly and DNA damage repair. The TGCT risk markers identified through GWAS include individual SNPs carrying per allele odds ratios (OR) in excess of 2.5. These ORs are among the highest reported in GWAS of any cancer type, hence suggesting a potential clinical utility in risk determination. Here, we present analysis of such an approach, using polygenic risk scores to calculate the combined effect of all risk loci on overall TGCT risk and discuss how a potential screening strategy may fit within a broader clinical context.

Low testosterone is associated with poor health status in men with human immunodeficiency virus infection: a retrospective study

Abstract: Men with human immunodeficiency virus (HIV) infection are often hypogonadal and develop several HIV-associated non-acquired immunodeficiency syndrome (AIDS) (HANA) conditions that impair overall health status. No studies explored the relationship between health status and serum testosterone (T) in HIV-infected men. This study aims to investigate the association between total serum T and HANA, multimorbidity, and frailty in a large cohort of 1359 HIV-infected men and to explore the relationship between patients' overall health status and serum T. Among biochemical and hormonal measurement performed the main are serum total T, free triiodothyronine (fT3), and luteinizing hormone. Other outcome measurements include anthropometry, assessment of comorbidities and disabilities, overall health status defined as the number of HANA and by the 38-item multimorbidity frailty index, anthropometry, and bone mineral density. The cumulative relative risk of comorbidities is increased in HIV-infected men with hypogonadism (p < 0.001) and hypogonadism is associated with several comorbidities. The prevalence of hypogonadism increases progressively with the increase of the number of comorbidities. Frailty index is inversely related to serum total T (age-adjusted r = 0.298, r(2) = 0.089, p < 0.0001). Serum fT3 levels are significantly lower in hypogonadal than eugonadal men (p = 0.022). This suggests that low serum T could be considered a sensitive marker of frailty and poor health status and that the latter might induce hypogonadism. The more HIV-infected men are frail the more they are hypogonadal. This suggests that hypogonadism might be a naturally occurring condition in unhealthy HIV-infected men and raises concern about the safety of T treatment. In conclusion, low serum T is associated with multimorbidity, HANA, and frailty in HIV-infected men and this association seems to be bidirectional. Given the wide attitude to offer T treatment to HIV-infected men, caution is needed when prescribing T to HIV-infected male patients, especially if the patient is unhealthy or frail.

Semen characteristics and malondialdehyde levels in men with different reproductive problems.

Abstract: The aim of this study was to assess the level of malondialdehyde (MDA) in the seminal plasma of infertile men and to highlight a relationship between the level of MDA and semen parameters. Eighty-one infertile patients were divided into groups according to their clinical diagnosis: genitourinary infections, varicocele and idiopathic infertility. Semen quality was assessed by light and transmission electron microscopy (TEM). TEM data were quantified with a mathematical formula able to obtain a fertility index and the percentage of sperm apoptosis, immaturity, and necrosis. Seminal MDA levels were determined by spectrofluorometry. Scrotal Eco-color Doppler was used to detect the varicocele. Infected patients had a positive bacteriological semen analysis. A control group consisted of 14 normospermic fertile men. Fertile group showed significantly increased values of sperm concentration, motility, and fertility index compared to infertile groups. In the infertile groups, sperm motility, concentration, apoptosis, and fertility index were not significantly different. In infection group, the percentage of necrosis was significantly higher than that observed in fertile men, varicocele, and idiopathic infertility groups (p < 0.001). MDA levels increased significantly in infection group in comparison with varicocele group (p < 0.01), idiopathic infertility group, and fertile men (p < 0.001) and in varicocele group compared to idiopathic infertility group (p < 0.001). In infection group, MDA levels positively correlated with sperm concentration (p < 0.01), fertility index (p < 0.05), and necrosis (p < 0.001), whereas a negative correlation was found with motility (p < 0.01). In varicocele group MDA levels correlated positively with necrosis and negatively with immaturity (p < 0.05). In fertile men and idiopathic infertility group, they did not show any correlation. In conclusion, we suggest that the evaluation of seminal MDA may be a good marker for understanding pathologies responsible of a sperm motility reduction such as urogenital infections or inflammatory status.

Biology of childhood germ cell tumours, focussing on the significance of microRNAs.

Abstract: Genomic and protein-coding transcriptomic data have suggested that germ cell tumours (GCTs) of childhood are biologically distinct from those of adulthood. Global messenger RNA profiles segregate malignant GCTs primarily by histology, but then also by age, with numerous transcripts showing age-related differential expression. Such differences are likely to account for the heterogeneous clinico-pathological behaviour of paediatric and adult malignant GCTs. In contrast, as global microRNA signatures of human tumours reflect their developmental lineage, we hypothesized that microRNA profiles would identify common biological abnormalities in all malignant GCTs owing to their presumed shared origin from primordial germ cells. MicroRNAs are short, non-protein-coding RNAs that regulate gene expression via translational repression and/or mRNA degradation. We showed that all malignant GCTs over-express the miR-371-373 and miR-302/367 clusters, regardless of patient age, histological subtype or anatomical tumour site. Furthermore, bioinformatic approaches and subsequent Gene Ontology analysis revealed that these two over-expressed microRNAs clusters co-ordinately down-regulated genes involved in biologically significant pathways in malignant GCTs. The translational potential of this finding has been demonstrated with the detection of elevated serum levels of miR-371-373 and miR-302/367 microRNAs at the time of malignant GCT diagnosis, with levels falling after treatment. The tumour-suppressor let-7 microRNA family has also been shown to be universally down-regulated in malignant GCTs, because of abundant expression of the regulatory gene LIN28. Low let-7 levels resulted in up-regulation of oncogenes including MYCN, AURKB and LIN28 itself, the latter through a direct feedback mechanism. Targeting LIN28, or restoring let-7 levels, both led to effective inhibition of this pathway. In summary, paediatric malignant GCTs show biological differences from their adult counterparts at a genomic and protein-coding transcriptome level, whereas they both display very similar microRNA expression profiles. These similarities and differences may be exploited for diagnostic and/or therapeutic purposes.

Effect of sodium fluoride on male mouse fertility.

Abstract: Sodium fluoride (NaF), an environmental pollutant, has been tested for its impact on fertility in several species of laboratory animals. A literature demonstrated that NaF adversely affects sperm motility, morphology, capacitation, and the acrosome reaction. However, the molecular mechanisms underlying these alterations have not yet been elucidated. Therefore, present study was designed to evaluate the regulatory pathways involved in the effect of NaF on sperm function and fertilization. In this in vitro study, mouse spermatozoa were incubated with a range of concentrations (2.5, 5, and 10 mm) of NaF for 90 min in media that support in vitro fertilization. Our results showed that NaF was associated with reduced intracellular ATP generation, motility, and motion kinematics. Likewise, short-term exposure of spermatozoa to NaF significantly reduced the intracellular calcium concentration, protein kinase-A activity, and tyrosine phosphorylation of sperm proteins, which were associated with a significant decrease in the rate of capacitation and the acrosome reaction. Finally, NaF significantly reduced the fertilization and blastocyst formation during early embryonic development. On the basis of these results, we propose that NaF reduces sperm motility, capacitation, and the acrosome reaction leading to poor fertilization and suppressed embryonic development.