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  • Trehalose sustains a higher post-thaw sperm motility than sucrose in vitrified human sperm
    One of the cryopreservation methods that best preserves sperm function is vitrification. However, comparative studies have not been performed to evaluate the effect of nonpermeable cryoprotectors on sperm function for prolonged periods of time post-devitrification. These times are necessary, especially in in vitro fertilisation and intrauterine insemination, for gamete interaction and then fertilisation to occur, while maintaining motility to arrive at the fertilisation site. In this study, sucrose (.25 m) and trehalose (.1 and .05 m) were compared in essential parameters like motility and plasma membrane integrity for 12 hr. Post-devitrification sperm motility using .1 m trehalose was 68.9%, higher than that obtained with .05 m trehalose (59.9%, p < .0081) and .25 m sucrose (57.9%, p < .0002). Similar results were obtained at 6 and 12 hr with .1 m trehalose (58.0% and 42.3% respectively) compared to .05 m trehalose (p < .0184 and p < .033) and .25 m sucrose (p < .0001 and p < .0012).There was no difference between .25 m sucrose and .05 m trehalose. Membrane integrity was best preserved at time 0 by .1 m trehalose (p < .05), but there was no significance at 6 and 12 hr compared to sucrose. Our results suggest that for assisted reproduction techniques that require motile spermatozoa for a longer period of time, use of .1 m trehalose is recommended in the sperm vitrification technique.
  • Testicular thermoregulation, scrotal surface temperature patterns and semen quality of water buffalo bulls reared in a tropical climate
    This study evaluated the capacity of thermoregulation and its consequences on the scrotal surface temperature patterns and semen quality of buffalo bulls raised in a wet tropical climate. Eleven water buffaloes were evaluated in the rainiest, in the transitional and in the less rainy season. Air temperature and humidity were consistently high, but the animals did not show thermal stress in any season. The scrotal temperature gradient of buffalo bulls using infrared thermography was described, and three parallel and decreasing thermal bands were characterised. Sperm quality (n = 176 ejaculates) was maintained in normal parameters over the periods. Pearson's coefficients showed that sperm volume and progressive motility were negatively correlated with ocular globe, epididymal tail and minimum scrotal temperatures (p < .01). Sperm membrane integrity was negatively influenced by increases in epididymal tail and minimum scrotal temperatures (p < .01). Ocular globe temperature also showed positive correlation with rectal, spermatic cord, and epididymal tail temperatures (p < .01). Therefore, even under high temperature and humidity, the thermoregulatory system was effective in preventing heat stress and the normality of scrotal surface temperatures, spermatogenesis and sperm maturation were maintained.
  • Sperm chromatin quality and DNA integrity in partial versus total globozoospermia
    Globozoospermia is a severe form of teratozoospermia with low incidence in infertile patients, considered as one of the important causes of male infertility. The objective was to investigate the chromatin/DNA integrity as well as apoptosis in ejaculated spermatozoa of cases with partial or total globozoospermia. Fifty-seven semen samples were divided into three groups of partial globozoospermia (n = 17), total globozoospermia (n = 10) and normozoospermia (control; n = 30). Sperm chromatin condensation, DNA integrity and apoptosis were assessed using cytochemical assays. The results showed significant differences in sperm parameters of count and motility between two case groups versus controls. The percentages of spermatozoa with abnormal chromatin packaging and protamine deficiency were significantly higher in total and partial globozoospermic men compared to normozoospermic samples. Also, the rates of TUNEL-positive spermatozoa were significantly increased in both globozoospermic cases with respect to the control (18.3 ± 10.1 and 12.3 ± 9.2 versus 5.9 ± 3 respectively). However, no significant differences were noticed between two subgroups of patients with regard to sperm DNA denaturation, DNA fragmentation and apoptosis. Abnormal chromatin packaging, DNA damage and apoptosis were significantly higher in cases than controls. The sperm chromatin/DNA anomalies may be considered as one of the main aetiology of ART failure in globozoospermic patients.
  • Cigarette smoking induces only marginal changes in sperm DNA methylation levels of patients undergoing intracytoplasmic sperm injection treatment
    DNA methylation plays important roles in genome stability and regulation of gene expression. This study was designed to determine the influence of cigarette smoking on sperm DNA methylation. From a genome-wide survey on sperm samples, differentially methylated target CpGs should be selected and subjected to local deep bisulphite sequencing. Obtained methylation data are compared to sperm parameters and (ICSI) outcome. Similar to pilot study, samples were subjected to Infinium 450K BeadChip arrays to identify alterations in sperm DNA methylation between smokers and nonsmokers males. Routine testing on a significantly altered CpG site was performed on more samples using local deep bisulphite sequencing. Of approximately 485,000 CpG sites analysed, only seven CpGs were found to show a significant DNA methylation difference of >20% with the top six CpGs overlapping common SNP sites. The remaining CpG site (cg19455396) is located in intron 12 of the TAP2 gene. The results of deep bisulphite sequencing showed only a tendency towards hypomethylation in the smoking group. This study could not detect biologically relevant CpG positions that are altered in sperm DNA methylation on the influence of cigarette smoking beyond individual-specific effects that may be caused by other environmental factors.
  • Comparison of the clinical efficacy and safety of the on-demand use of paroxetine, dapoxetine, sildenafil and combined dapoxetine with sildenafil in treatment of patients with premature ejaculation: A randomised placebo-controlled clinical trial
    The aim of the study was to compare the clinical efficacy and safety of the on-demand use of paroxetine, dapoxetine, sildenafil and combined dapoxetine with sildenafil in treatment of patients with premature ejaculation (PE). In a single-blind placebo-controlled clinical study, 150 PE patients without erectile dysfunction (ED) were included during the period of March 2015 to May 2016. Patients were randomly divided into five groups (30 patients each). On demand placebo, paroxetine (30 mg), dapoxetine (30 mg), sildenafil citrate (50 mg) and combined dapoxetine (30 mg) with sildenafil citrate (50 mg) were given for patients for 6 weeks in each group respectively. All patients were instructed to record intravaginal ejaculatory latency time (IELT) and evaluated with Premature Ejaculation Diagnostic Tool (PEDT) and the patient satisfaction score before and after treatment. The mean of IELT, satisfaction score and PEDT in all groups was significantly improved after treatment (p value = .001). Combined dapoxetine with sildenafil group had the best values of IELT, satisfaction scores and PEDT in comparison with other treatment groups (p value <.001). The combined dapoxetine with sildenafil therapy could significantly improve PE patients without ED as compared to paroxetine alone or dapoxetine alone or sildenafil alone with tolerated adverse effects.
  • ICSI outcome in patients with high DNA fragmentation: Testicular versus ejaculated spermatozoa
    Sperm DNA fragmentation (SDF) has emerged as an important biomarker in the assessment of male fertility potential with contradictory results regarding its effect on ICSI. The aim of this study was to evaluate intracytoplasmic sperm injection (ICSI) outcomes in male patients with high SDF using testicular versus ejaculated spermatozoa. This is a prospective study on 36 men with high-SDF levels who had a previous ICSI cycle from their ejaculates. A subsequent ICSI cycle was performed using spermatozoa retrieved through testicular sperm aspiration. Results of the prior ejaculate ICSI were compared with those of the TESA-ICSI. The mean (SD) SDF level was 56.36% (15.3%). Overall, there was no difference in the fertilization rate and embryo grading using ejaculate and testicular spermatozoa (46.4% vs. 47.8%, 50.2% vs. 53.4% respectively). However, clinical pregnancy was significantly higher in TESA group compared to ejaculated group (38.89% [14 of 36] vs. 13.8% [five of 36]). Moreover, 17 live births were documented in TESA group, and only three live births were documented in ejaculate group (p < .0001). We concluded that the use of testicular spermatozoa for ICSI significantly increases clinical pregnancy rate as well as live-birth rate in patients with high SDF.
  • Adverse effects of leptin on histone-to-protamine transition during spermatogenesis are prevented by melatonin in Sprague-Dawley rats
    This study examines the effect of melatonin on leptin-induced changes in transition of histone to protamine in adult rats during spermatogenesis. Twelve-week-old Sprague-Dawley rats were randomised into control, leptin-, leptin–melatonin-10-, leptin–melatonin-20- and melatonin-10-treated groups with six rats per group. Leptin was given via intraperitoneal injections (i.p.) daily for 42 days (60 μg/kg body weight). Rats in the leptin- and melatonin-treated groups were given either 10 or 20 mg day−1 kg−1 body weight of leptin in drinking water. Melatonin-10-treated group received only 10 mg of melatonin day−1 kg−1 body weight in drinking water for 42 days. Control rats received 0.1 ml of 0.9% saline. Upon completion of the treatment, sperm count, morphology and histone-to-protamine ratio were estimated. Gene expression of HAT, HDAC1, HDAC2, H2B, H2A, H1, PRM1, PRM2, TNP1 and TNP2 was determined. Data were analysed using ANOVA. Sperm count was significantly lower, whereas the fraction of spermatozoa with abnormal morphology, the ratio of histone-to-protamine transition and the expressions of HAT, HDAC1, HDAC2, H2B, H2A, H1, PRM1 were significantly higher in leptin-treated rats than those in controls or melatonin-treated rats. It appears that exogenous leptin administration adversely affects histone-to-protamine transition, which is prevented by concurrent administration of melatonin.
  • Effects of testosterone replacement therapy on metabolic syndrome among Japanese hypogonadal men: A subanalysis of a prospective randomised controlled trial (EARTH study)
    We investigated the effects of testosterone replacement therapy (TRT) on metabolic factors among hypogonadal men with a metabolic syndrome. From the study population of the EARTH study, which was a randomised controlled study in Japan, 65 hypogonadal patients with a metabolic syndrome, comprising the TRT group (n = 32) and controls (n = 33), were included in this study analysis. The TRT group was administered 250 mg of testosterone enanthate as an intramuscular injection every 4 weeks for 12 months. Waist circumference, body mass index, body fat volume and blood pressure were measured in all patients at baseline and at 12 months. In addition, blood biochemical data, including total cholesterol, triglyceride (TG), HDL cholesterol, fasting plasma glucose (FPG) and haemoglobin A1c (HbA1c) levels, were also evaluated. Changes in these categories from baseline to 12 months were compared between the TRT and control groups, with significant differences observed in waist circumference, body fat percentage, FPG, TG and HbA1c levels. No significant differences were observed in other parameters. TRT for 1 year was associated with improvements in some metabolic factors among Japanese men with hypogonadism and metabolic syndrome.
  • Can haematologic parameters be used for differential diagnosis of testicular torsion and epididymitis?
    A differential diagnosis of testicular torsion and epididymitis has serious importance for testicular health. In emergency conditions, if testicular torsion goes unnoticed and epididymo-orchitis is diagnosed, organ loss may occur. This study aimed to evaluate the usefulness of haematologic parameters for the diagnosis of both testicular torsion and epididymo-orchitis and for differential diagnosis of these two diseases. Patients were divided into three groups as those undergoing surgery for testicular torsion, those receiving medical treatment for epididymitis and a healthy control group. All patients had complete blood counts taken with determinations of mean platelet volume (MPV), platelet/lymphocyte ratio (PLR), neutrophil/lymphocyte ratio (NLR) and leucocyte counts. These were then compared between groups. Leucocyte, MPV and NLR values were higher in both the epididymitis and torsion groups compared to the controls (p < .001). Platelet counts and PLR were significantly higher in the epididymitis group compared to the other two groups (p < .001). Leucocyte, MPV and NLR values may be used in the diagnosis of epididymitis and testicular torsion. Platelet counts and PLR appear to be useful in differentiating epididymitis from testicular torsion. However, there is a need for prospective studies with larger numbers of patients.
  • Very late presentation of a disorder of sex development
    Disorders of sex development generally present in the neonatal period with ambiguity of external genitalia. We report a very old male patient presenting at 75 years because of panhypopituitarism and a large nonsecreting pituitary macroadenoma secondary to long-standing primary hypogonadism due to 46,XX sex reversal disorder now first diagnosed. Sex development disorders may go unrecognised for the entire life span, despite infertility and long-standing primary gonadic failure may lead to uncommon complications.
  • Inhibition of mTOR pathway decreases the expression of pre-meiotic and meiotic markers throughout postnatal development and in adult testes in mice
    Rapamycin (mTOR inhibitor) has been reported to have negative effect on human male gonadal function. Previously, we showed that mTOR signalling molecules are expressed during early spermatogenesis in mice. The objective of this study was to investigate the role of mTOR signalling in meiosis both during the first wave of spermatogenesis and also during adult spermatogenesis. Day 5 post-partum mice were administered rapamycin and retinoic acid (RA; a Stra8 activator), and expression of p-p70S6K and Stra8 proteins was evaluated. p-p70S6K and Stra8 protein expressions decreased in post-natal testes after rapamycin treatment. Stra8 protein expression increased after RA and rapamycin+RA administrations in post-natal testes. In adult mice, rapamycin was administrated for 1 or 4 weeks. Morphological analysis for testicular damage and TUNEL assay was performed. After rapamycin administration, germ cell loss increased in adult testes. Ultrastructural analysis revealed disorganised testicular morphology and vacuolisation. The number of apoptotic germ cells increased after 4 weeks rapamycin administration. Stra8 and Dmc1 expressions decreased in 4 weeks rapamycin group, whereas Sycp3 and VASA expression did not change. Our findings suggest that mTOR pathway has an important role in meiotic progress of male germ cells both during first wave of spermatogenesis and in adult mice.
  • Ethanol-induced mitophagy in rat Sertoli cells: Implications for male fertility
    Autophagy is a pro-survival mechanism involving lysosomal degradation of damaged cellular components following multiple forms of cellular stress. There is currently a lack of literature on the mechanism, and specifically on mitophagy (selective autophagy of damaged pro-apoptotic mitochondria) in Sertoli cells (SCs). Against such a background, the authors induced mitophagy in SCs of adult male rats using a single injection of ethanol (5 g/kg) and observed mitophagy in the SCs via transmission electron microscopy 24 hr later. In addition, we briefly discussed the possible clinical implications of enhanced autophagy and mitophagy in stressed SCs in our model and in other models of acute stress (e.g., heat and transplantation stress). Further studies on SC autophagy are required, as a full understanding of the molecular mechanisms controlling autophagy in stressed SCs may have therapeutic implications for infertility treatment.
  • Comparison of three different extenders on Murrah buffaloes (Bubalus bubalis) semen freezability
    The use of frozen semen for artificial insemination is the main approach utilised for the genetic improvement of most domesticated species. The advantages include lower transportation costs, continuous availability of semen, fewer occurrences of sexually transmitted diseases and the incorporation of desirable genes in a relatively short amount of time. Nevertheless, the use of frozen semen in buffalo herds remains limited due to the loss of sperm quality when buffalo semen is frozen. So, the goal of this study was to evaluate the pre- and post-cryopreservation quality of buffalo semen diluted in three distinct freezing media: Tris-egg yolk, Botu-bov® (BB) and ACP-111®. Thirty-two ejaculates from four bulls were analysed in terms of kinetics, morphology and sperm viability by epifluorescence microscope. Thawed samples were also evaluated for capacitation-like damage, DNA fragmentation and plasma and acrosomal membrane integrity using flow cytometry. The Tris-egg yolk and BB® extenders yielded better results than the ACP-111® extender for kinetics parameter (total motility, progressive motility and percentage of rapid cells). However, semen samples were similar for parameters evaluated by flow cytometry. Taken together, the data indicate that in comparison with Tris-egg yolk and BB extender, ACP-111® can also be used as an extender for buffalo semen cryopreservation.
  • Apoptotic sperm biomarkers and the correlation between conventional sperm parameters and clinical characteristics
    The principal aim of this retrospective study was to examine the relationship between sperm apoptotic biomarkers and the patient's biclinical characteristics, the conventional sperm parameters and the results of assisted reproductive technology. Sperm analysis, activated caspases, annexin V staining for phosphatidylserine (PS) externalisation and labelling assay for DNA fragmentation were assessed in 122 males of infertile couples. Fifty-seven couples were allocated to the natural conception group, and 65 couples underwent IVF or ICSI. Semen of IVF/ICSI patients showed a higher proportion of apoptotic spermatozoa in their spermatozoa when compared with a natural conception group (p < .05). Sperm apoptotic biomarkers correlated with age, FSH, and conventional sperm parameters. DNA fragmentation correlated positively with the percentage of semen having externalised PS (r = .78, p = 0) and activated caspases (r = .71, p = 0). Patients without clinical pregnancy had higher frequency of DNA fragmentation, externalised PS and activated caspases compared to patients with clinical pregnancy (p < .001). The best specificity and greater sensitivity were obtained with the test of the DNA fragmentation compared to the other biomarkers. Among the apoptotic biomarkers, only DNA fragmentation was found to predict natural or assisted pregnancy better than conventional sperm parameters.
  • Steroidogenesis decline accompanied with reduced antioxidation and endoplasmic reticulum stress in mice testes during ageing
    To gain an understanding of the mechanisms by which Leydig cell steroidogenic function degenerates with ageing, we explored steroidogenic gene expression in relation to antioxidation status and endoplasmic reticulum (ER) stress during the ageing of mice. Expression of StAR, P450scc and other steroidogenic enzymes decreased starting at middle age (12-month-old) compared to that of the young control (3-month-old) mice. The immunohistochemical staining intensity of 3β-HSD for Leydig cells was significantly weaker in the aged (24-month-old) group than that in the young control group. The number of Leydig cells showed no significant difference between the groups. A progressive reduction in antioxidants MnSOD and GPx4 was observed in the testicular tissue with down-regulated SIRT1 protein level in the middle-aged and aged (24-month-old) mice. The number of testicular macrophages was significantly higher in the aged group than that in the middle-aged and young mice. Age-associated up-regulation of ER stress markers such as GRP78 and Chop was observed. These results suggested that oxidative stress and ER stress might play a role in the deficit of Leydig cell steroidogenic function during ageing.
  • NGF promotes mitochondrial function by activating PGC-1α in TM4 Sertoli cells
    Nerve growth factor (NGF), which is required for the survival and differentiation of the nervous system, has been proved to play important roles in male reproductive physiology. Several studies have focused on the roles of NGF in the testes. However, no study has reported on the mechanism of paracrine and autocrine actions of NGF in Sertoli cells. This study showed that NGF stimulated mitochondrial activity and biogenesis in TM4 Sertoli cells. Moreover, our results demonstrated that peroxisome proliferator-activated receptor-gamma coactivator-1α is a possible downstream key target of the NGF signalling pathway. In a 3-nitropropionic acid cell model, NGF treatment attenuated mitochondrial activity defect and depolarisation. This NGF-triggered signalling may help in discovering new therapeutic targets for certain male infertility disorders.
  • Protective effect of Propolfenol® on induced oxidative stress in human spermatozoa
    The propolis extract was shown to possess the capacity to protect sperm membrane from the deleterious action of oxidative attack. Oxidative stress can induce propagation of a lipid peroxidation (LPO) chain reaction because spermatozoa contain high concentration of unsaturated fatty acids. This study aimed at evaluating in vitro the possible toxicity and/or the antioxidant properties of Propolfenol® in ejaculated human spermatozoa. A colorimetric assay determined the total flavonoid content by spectrophotometry and a high-performance liquid chromatography–diode array detection analysis the quantity of galangin, pinocembrin and caffeic acid phenylethilic ester (CAPE). Sperm parameters such as motility, vitality and DNA integrity were assessed utilising optical microscopy. The antioxidant properties Propolfenol® against LPO induced by tert-Butyl Hydroperoxide were evaluated using the C11-BODIPY581/591 probe. Chemical analysis of Propolfenol® revealed low quantities of galangin, pinocembrin and CAPE; cyclic voltammetry experiments showed that Propolfenol® may exert an antioxidant activity. A protective action of Propolfenol® (20 and 100 μg/ml) on induced LPO in human spermatozoa was detected. Propolfenol® may be proposed as the supplement in media for sperm preparation techniques or cryopreservation to counteract the increased presence of reactive oxygen species generated by these methods.
  • NOS3 gene variants and male infertility: Association of 4a/4b with oligoasthenozoospermia
    Results of recent studies confirmed that oxidative stress negatively affects sperm motility and causes sperm DNA damage. Produced by nitric oxide synthase 3 (NOS3), nitric oxide is considered to be one of the important mediators of oxidative stress in testis tissue. The aim of this study was to assess the possible association of three genetic variants (rs2070744, rs1799983 and intron variant 4a/4b) in NOS3 gene and infertility occurrence in two groups of infertile men (idiopathic azoospermia and oligoasthenozoospermia) and fertile controls. Genotypes for the single-nucleotide genetic variants rs1799983 and rs2070744 were determined by PCR-RFLP, while genotyping of intron 4 variant 4a/4b was performed by gel electrophoresis of PCR products. Statistical analysis was performed by SNPStats software. No significant association between the three genetic variants of the NOS3 gene and infertility risk was determined comparing allele and genotype frequencies among group of patients diagnosed with azoospermia and the control group. Nevertheless, there was a significant positive association between 4a/4b and infertility in the group of males diagnosed with oligoasthenozoospermia, under overdominant genetic model. Our findings suggest that tandem repeat variant within intron 4 of the NOS3 gene is associated with an increased risk of infertility in men diagnosed with idiopathic oligoasthenozoospermia.
  • Selection of viable human spermatozoa with low levels of DNA fragmentation from an immotile population using density gradient centrifugation and magnetic-activated cell sorting
    We aimed to determine whether density gradient centrifugation and magnetic-activated cell sorting (DGC-MACS) could select viable spermatozoa, with lower levels of DNA fragmentation, from an immotile population. Analysis involved sixteen patients, each with a sperm count ≥107/mL. All samples were immotile despite exhibiting a live population >40%. Spermatozoa were prepared using DGC-MACS and selected spermatozoa evaluated for membrane and DNA integrity using the hypo-osmotic swelling (HOS) test, vital staining and the TUNEL test. The mean proportion of spermatozoa with an intact membrane in control, DGC and DGC-MACS populations, was 52.5 ± 12.21%, 69.38 ± 7.87% and 81.81 ± 5.29%. The mean proportion of live spermatozoa in control, DGC and DGC-MACS populations, was 65.88 ± 12.77%, 77.25 ± 7.39% and 85.81 ± 5.2%. DGC-MACS reduced the within-sample discrepancy between HOS test and vital stain results from 13.18% to 4.12%. The mean proportion of spermatozoa exhibiting DNA damage in control, DGC and DGC-MACS populations, was 9.56 ± 3.39%, 5.25 ± 1.61% and 2.75 ± 1.13%. Finally, analysis showed that 71.23% of the DNA-fragmented spermatozoa in unprocessed samples were removed following DGC-MACS and that the addition of MACS to an existing DGC protocol reduced fragmented spermatozoa by a further 26.15% compared to DGC alone. Consequently, DGC-MACS is a clinically viable method able to select viable spermatozoa with lower levels of DNA fragmentation from an immotile population.
  • The influence of male age on treatment outcomes and neonatal birthweight following assisted reproduction technology involving intracytoplasmic sperm injection (ICSI) cycles
    The aim of this study was to investigate the effect of male age on treatment outcomes and neonatal birthweight following intracytoplasmic sperm injection (ICSI). This study included 2,474 ICSI cycles. Male partners were stratified into 5-year age categories (up to 25, 26–30, 31–35, 36–40 and 41 and up). Multilevel logistic regression was used to evaluate the relationship between male age and treatment outcomes. After adjusting for confounders, we found no difference in the clinical pregnancy rate. However, we observed that the 31- to 35-year group had a higher odds of live birth than that of the >41-year group (aOR 1.63, p = .03), and that the risk of abortion in the 31- to 35-year group was lower than that of the reference group (aOR 0.41, p = .02). A total of 754 single-foetus newborns and 556 twin newborns were analysed. Among the singletons, none of the variables differed among the five groups (p > .05). Among the twins, the infants in the 36- to 40-year group had a lower neonatal birthweight and a higher low-birthweight rate than those of the other groups (p < .05). Our study indicates that increased paternal age negatively affects the live birth and miscarriage rates. In addition, advanced paternal age may affect the birthweight of twins.
  • Protective effect of L-carnitine and L-arginine against busulfan-induced oligospermia in adult rat
    Busulfan is an anticancer drug caused variety of adverse effects for patients with cancer. But it could cause damage to the male reproductive system as one of its adverse effects. This study aimed to investigate the protective effect of L-carnitine and L-arginine on semen quality, oxidative stress parameters and testes cell energy after busulfan treatment. Adult male rats were divided into four groups: control (Con), busulfan (Bus), busulfan plus L-arginine (Bus + L-arg) and busulfan plus L-carnitine (Bus + L-car). After 28 days, the semen was collected from the epididymis and the testes were assessed. Sperm count, motility and velocity were measured by CASA, and smears were prepared for assessment of sperm morphology. Serum and testes supernatants were separated for DNA metabolites, oxidative stress and cell energy parameters. Testes tissues also subjected for caspase-3. The results showed significant improvement in sperm morphology, motility, velocity and count in the groups treated with L-arginine and L-carnitine and accompanied with an increase in MDA, GSSG and ATP, reduction in GSH, AMP, ADP, NO and 8-OHDG also recorded. These results are supported by caspase-3. Conclusions: Administration of L-arg and L-car attenuated the cytotoxic effects of busulfan by improving semen parameters, reducing oxidative stress and maintaining cell energy.
  • Possible role of serum testosterone, gonadotropins and prolactin in patients with premature ejaculation
    Premature ejaculation (PE) is the most common male sexual dysfunction. This study aimed to investigate the role of serum testosterone, gonadotropins and prolactin in patients with PE. In a prospective a case-controlled study, it was conducted on 90 male patients with PE and 90 male healthy participants as controls. Patients were evaluated by Premature Ejaculation Diagnostic Tool (PEDT) and intravaginal ejaculatory latency time (IELT). Patients with mean IELT values ≤60 s and PEDT total scores ≥11 were considered to have PE. Serum levels of total testosterone (TT), free testosterone (FT), follicle-stimulating hormone (FSH), luteinising hormone (LH) and prolactin (PL) were investigated in patients with PE and controls. There was no statistically significant difference between patients with PE and controls regarding the serum levels of TT, FT, FSH, LH and PL (p value ˃.05). There was no significant correlation between the sex hormones levels (TT, FT, FSH, LH and PL) and (age, body mass index (BMI), IELTS and total PEDT scores of the patients; p value ˃.05). This study concluded that there was no disturbance in serum levels of testosterone, gonadotropins and prolactin in patients with PE and controls. These hormones could not relate to pathogenesis of PE.
  • Molecular detection of Chlamydia trachomatis and semen quality of sexual partners of infertile women
    Chlamydia trachomatis is considered as the bacterium that is more sexually transmitted as cause of male urethritis, epididymitis, orchitis and infertility. A total of 116 semen samples of men whose couples are infertile women were analysed. The quality of the semen was measured by standard procedures recommended by WHO while C. trachomatis was detected by the PCR assay. Thirty-seven semen samples were positive for C. trachomatis (31.9%). Regarding semen analysis, no different values were observed between positive and negative samples to C. trachomatis. However, the presence of leucocytes and erythrocytes suggests an inflammatory process; however, these were high in negative samples to C. trachomatis. Furthermore, an association between low seminal volume at 1, 5 ml and the positivity to C. trachomatis was observed (OR=2, 1; CI95% 1,16-3,07). The total semen volume is a contribution by the various accessory glands (this reflects the secretory activity of the glands); a low semen volume could be due to an obstruction of the ejaculatory duct or infection of accessory glands by C. trachomatis. More studies are necessary to identify the causes of a reduced semen volume.
  • Efficacy of varicocelectomy in the treatment of hypogonadism in subfertile males with clinical varicocele: A meta-analysis
    To reassess the efficacy of varicocelectomy in the treatment of hypogonadism in subfertile males, we carried out a meta-analysis of clinical trials and retrospective studies that compared the pre-operative and postoperative serum testosterone. We searched Embase and PubMed (1980 to May 2016) for studies. Eight studies and 712 patients were included. The combined analysis of seven studies discovered that the mean serum testosterone of patients post-operation improved by 34.3 ng/dl (95% CI: 22.57–46.04, p < .00001, I² = 0.0%) compared with their pre-operative levels. In subgroup analysis, testosterone improvements in the hypogonadal treated subgroup were more significant (improved by 123 ng/dl, 95% CI: 114.61–131.35, p < .00001, I2 = 37%) than in the eugonadals, or the untreated controls. In an analysis of surgery versus untreated control (three studies included), results showed that mean testosterone among hypogonadals increased by 105.65 ng/dl (95% CI: 77.99–133.32), favouring varicocelectomy, as the differences were significant (p < .00001), However, there were insignificant differences in eugonadals (p = .36). In conclusion, varicocelectomy significantly improved testosterone in hypogonadal men with subfertility. Active surgical treatment of varicocele might have a benefit of maintaining healthy androgen levels in subfertile men.
  • Association of the c.-9C>T and c.368A>G transitions in H2BFWT gene with male infertility in an Iranian population
    The H2BFWT (H2B family, member W, testis specific) gene is a testis-specific histone which is involved in the spermatogenesis process. This study aimed to investigate the association of H2BFWT gene c.-9C>T and c.368A>G polymorphisms with male infertility in an Iranian population. The 232 independent individuals, including 109 infertile men and 123 healthy controls, were recruited from IVF centre (Kashan, Iran). The allele types of c.-9C>T and c.368A>G polymorphisms were detected by using PCR-RFLP method. In overall analysis, we found that the c.-9T (OR: 1.75, 95% CI: 1.04–2.95, p = .035) and c.368G (OR: 1.71, 95% CI: 1.02–2.89, p = .042) alleles are associated with male infertility. The c.-9T allele was also associated with nonobstructive azoospermia (OR: 2.08, 95% CI: 1.01–4.25, p = .046), while c.368G allele was associated with oligozoospermia (OR: 2.10, 95% CI: 1.15–3.85, p = .016). It is concluded that H2BFWT gene c.-9C>T and c.368A>G polymorphisms might be genetic risk factors for idiopathic male infertility.
  • Total flavonoids of Epimedium reduce ageing-related oxidative DNA damage in testis of rats via p53-dependent pathway
    Increased DNA damage in testis is considered as a major factor for ageing-related dysfunction. Total flavonoids of Epimedium (TFE), the main active compositions of Epimedium, have been used to treat sexual dysfunction and delay ageing. However, whether TFE could improve ageing-related testicular dysfunction remains unknown. Therefore, we investigated the protection effects of TFE and its mechanisms of action in a naturally ageing rat model. Eighteen-month-old SD rats were randomised to receive either vehicle or TFE (10 and 20 mg/kg). Nine-month-old SD rats were used as adult controls. Morphology, protein expression and immunohistochemistry were determined. Compared with adult control group, intragastric administration of TFE for 6 months significantly improved testicular morphology, increased the activities of SOD and decreased the levels of MDA of testis. In addition, TFE decreased γH2AX expression levels and γH2AX focal formation in spermatogonia and primary spermatocyte with concomitant downregulation of 8-OHdG levels. Furthermore, TFE inhibited p-P53/p21 and chk1/chk2 expression levels. Collectively, TFE effectively reduce oxidative DNA damage in testis of ageing rats via a p53-dependent pathway. Thus, inhibition of oxidative DNA damage is likely to represent a promising strategy for restoration of ageing-related testicular dysfunction.
  • Honey improves spermatogenesis and hormone secretion in testicular ischaemia–reperfusion-induced injury in rats
    This study was conducted to survey the protective effect of pre-treatment with Persian honey during post-ischaemia reperfusion on ischaemia–reperfusion (IR)-induced testis injury. Animals were divided into four groups of IR, honey + ischaemia- reperfusion (HIR), vitamin C + ischaemia- reperfusion (VIR) and carbohydrates + ischaemia- reperfusion (CIR). The testes were examined for spermatogenesis index. Detection of single- and double-stranded DNA breaks at the early stages of apoptosis was performed. Total serum concentration of FSH, LH and testosterone was measured using ELISA. All data were expressed as mean ± SD in each group, and significance was set at p ≤ .05. Spermatogenesis index was significant in the HIR group (p < .001). Serum levels of FSH and LH were significantly higher in the CIR and HIR groups. Serum levels of testosterone were significantly higher in VIR and HIR groups. Apoptotic cells in IR and CIR groups increased significantly statistically (p < .001), while in HIR and VIR groups, the number of apoptotic cells decreased and the positive cells of TUNEL staining were detected in spermatocytes and spermatid. The present study indicates that honey decreases the cellular damage and apoptosis during testicular I/R injury, with significant protective effects on reproductive hormone production.
  • Effects of taking tadalafil 5 mg once daily on erectile function and total testosterone levels in patients with metabolic syndrome
    We aimed to evaluate the efficacy of tadalafil 5 mg once-daily treatment on testosterone levels in patients with erectile dysfunction (ED) accompanied by the metabolic syndrome. A total of 40 men with metabolic syndrome were evaluated for ED in this study. All the patients received 5 mg tadalafil once a day for 3 months. Erectile function was assessed using the five-item version of the International Index of Erectile Function (IIEF) questionnaire. Serum testosterone, follicle-stimulating hormone and luteinising hormone levels were also evaluated, and blood samples were taken between 08.00 and 10.00 in the fasting state. All participants have three or more criteria of metabolic syndrome. At the end of 3 months, mean testosterone values and IIEF scores showed an improvement from baseline values (from 3.6 ± 0.5 to 5.2 ± 0.3, from 11.3 ± 1.9 to 19 ± 0.8 respectively). After the treatment, serum LH levels were decreased (from 5.6 ± 0.6 to 4.6 ± 0.5). There was significantly difference in terms of baseline testosterone and luteinising hormone values and IIEF scores (p < .05). Based on our findings, we recommend tadalafil 5 mg once daily in those men with erectile dysfunction especially low testosterone levels accompanied by metabolic syndrome.
  • Cumene hydroperoxide induced changes in oxidation–reduction potential in fresh and frozen seminal ejaculates
    Oxidation–reduction potential (ORP) is a newer integrated measure of the balance between total oxidants (reactive oxygen species—ROS) and reductants (antioxidants) that reflects oxidative stress in a biological system. This study measures ORP and evaluates the effect of exogenous induction of oxidative stress by cumene hydroperoxide (CH) on ORP in fresh and frozen semen using the MiOXSYS Analyzer. Semen samples from healthy donors (n = 20) were collected and evaluated for sperm parameters. All samples were then flash-frozen at −80°C. Oxidative stress was induced by CH (5 and 50 μmoles/ml). Static ORP (sORP—(mV/106 sperm/ml) and capacity ORP (cORP—μC/106 sperm/ml) were measured in all samples before and after freezing. All values are reported as mean ± SEM. Both 5 and 50 μmoles/ml of CH resulted in a significant decline in per cent motility compared to control in pre-freeze semen samples. The increase in both pre-freeze and post-thaw semen samples for sORP was higher in the controls than with 50 μmoles/ml of CH. The change from pre-freeze to post-thaw cORP was comparable. The system is a simple, sensitive and portable tool to measure the seminal ORP and its dynamic impact on sperm parameters in both fresh and frozen semen specimens.
  • Fertility enhancing activity and toxicity profile of aqueous extract of Chasmanthera dependens roots in male rats
    This study evaluated the fertility-enhancing activity and safety of aqueous extract of Chasmanthera dependens root (AECDR) in male rats. In the fertility study, twenty, sodium arsenite (10 mg/kg) body weight (BW)-treated male rats (171.02 ± 3.36 g), assigned into four groups (I–IV), received 1 ml of distilled water (DW), 25, 50 and 100 mg/kg BW of AECDR for 60 days, whereas the control received DW. After 7 days of pairing with female rats (153.67 ± 2.24 g), spermatogenic, fertility, testicular function indices and enzymatic antioxidant activities were evaluated. The animal groupings in the toxicity study were similar to the fertility study except no administration of sodium arsenite. Sodium arsenite treatment-related decreases (p < .05) in the semen and sperm parameters, testicular function indices, antioxidant activities and female rat fertility indices were reversed/ameliorated by AECDR. AECDR significantly altered the function indices of the liver and kidney and the lipid profile and selectively altered the haematological parameters. There was no treatment-related histoarchitectural changes in the organs. Overall, the aqueous extract of C. dependens roots exhibited pro-spermatogenic, fertility enhancing, antioxidant and androgenic activities in male rats. It also exhibited functional toxicity. Therefore, the chronic use of AECDR may not be completely safe as oral remedy.
  • Sperm DNA fragmentation as a result of ultra-endurance exercise training in male athletes
    Intensive sports practice seems to exert negative effects on semen parameters; in order to assess these effects, the objective of this study was to assess semen, including DNA fragmentation, and hormone parameters in elite triathletes. Twelve high-level triathletes preparing for a National Triathlon Championship participated in the study. The qualitative sperm parameters analysed were volume, sperm count, motility, morphology and DNA fragmentation; when needed, additional testing was performed. Assessed hormones were testosterone (T), cortisol (C) and testosterone–cortisol ratio (T/C). Maximum oxygen consumption and training characteristics were also assessed. Hormonal values and physical semen parameters were within normal ranges. DNA fragmentation showed high values (20.4 ± 6.1%). Round cells in semen were higher than normal (2.8 ± 1.5 million/ml), with the presence of macrophages. Correlations were found for several parameters: concentration of round cells positively correlated with progressive sperm motility (p = .01) and sperm morphology (p = .02); contrarily, the correlation found with DNA fragmentation was negative (p = .04). Sperm DNA fragmentation and the T/C ratio, however, were correlated in a positive manner (p = .03). As evidenced by the observed results, sperm DNA fragmentation is affected by high-level sports practice; therefore, high loads of endurance training could potentially interfere with the athlete's fertility potential.
  • Icariin improves the sexual function of male mice through the PI3K/AKT/eNOS/NO signalling pathway
    We aimed to investigate the effect and mechanism of icariin on male sexual function. Forty-eight Crl:CD1(ICR) male mice were randomly divided into control, low-, medium- and high-dose icariin group (intragastric administration of 50, 100 and 200 mg/kg/d for 21 days). Mating experiment was then performed at a ratio of 1: 3 (male: female). The mating behaviours of male mice were recorded. The genital indexes and serum testosterone, nitric oxide (NO), hypothalamic dopamine (DA) and 5- hydroxy tryptophan (5-HT) concentrations were measured. The expression of endothelial nitric oxide (eNOS), phosphatidylinositol tallow alcohol 3-kinase (PI3K) and phosphorylated protein kinase (p-AKT) in penile tissue was detected by Western blot. All icariin groups exhibited shorter capture latency and ejaculation latency, increased number of capture and ejaculation, higher capture and ejaculation rate, and higher testicular and prostate indexes compared with controls (p < .001). These groups had higher serum testosterone and NO concentrations (p < .001), hypothalamic DA and 5-HT levels, and eNOS, PI3K and phosphorylated AKT expressions in penile tissue (p < .05). The effect of icariin was dose-dependently increased. Our study suggests that icariin improves the sexual function of male mice, which might be associated with the hypothalamic–pituitary–gonadal axis and the PI3K/AKT/eNOS/NO signalling pathway.
  • Sperm DNA methylation of H19 imprinted gene and male infertility
    Infertility affects up to 15% of reproductive-aged couples worldwide, with male factor being detected in 40%–50% of the cases. Proper sperm production is associated with the establishment of appropriate epigenetic marks in developing germ cells. Several studies have demonstrated the association between abnormal spermatogenesis and epigenetic disturbances with the major focus on DNA methylation. Imprinted genes are expressed in a parent-of-origin-specific manner, and the role of their DNA methylation in proper spermatogenesis has been documented recently. The existing evidence along with the absence of relevant data in south of Iran prompted us to study the methylation of H19 imprinted gene in spermatozoa of idiopathic infertile patients (males with abnormalities in sperm parameters) and healthy controls by Combined Bisulfite Restriction Analysis. According to our results, the lowest methylation percentage of H19 imprinted gene belongs to three cases with sperm characteristics under normal range (two cases Oligoasthenoteratozoospermia and one case Oligoteratozoospermia). However, our results show that the median of methylation percentage for H19 is not statistically significant between case and control groups. Our results and those of others introduce DNA methylation as a potential marker of fertility and should be investigated with more patients in future studies.
  • Effects of various cryoprotectants on the quality of frozen–thawed immature bovine (Qinchuan cattle) calf testicular tissue
    To investigate the effects of different concentrations of various cryoprotectants (CPs) on the cell viability as well as expression of spermatogenesis-related genes, such as CREM, Stra8 and HSP70-2 in frozen–thawed bovine calf testicular tissue, immature bovine (Qinchuan cattle) calf testicular tissue was collected and cryopreserved in the cryomedia containing different concentrations (5%, 10%, 15% and 20%) of the following three CPs: glycerol, ethylene glycol (EG) and dimethyl sulphoxide (DMSO) respectively. After 1 month cryopreservation in liquid nitrogen, cell viability was evaluated using Trypan blue exclusion under a bright-field microscope. The mRNA expression of the three genes was also evaluated using qRT-PCR. The results indicated that different concentrations of glycerol, EG and DMSO in cryomedia during cryopreservation could protect bovine calf testicular tissue in various ways to avoid freezing or cryopreservation-induced expression changes in spermatogenesis-related genes. The highest cell viability and the three spermatogenesis-related genes (CREM, Stra8 and HSP70-2) expression level came from the cryomedia containing glycerol, EG and DMSO at 10% concentration respectively (p < .05). Meanwhile, compared with the other CPs, the frozen–thawed bovine calf testicular tissue treated with 10% DMSO exhibited the highest cell viability and mRNA expression level of the spermatogenesis-related genes (CREM, Stra8 and HSP70-2).
  • Androgen deficiency impairs erectile function in rats through promotion of corporal fibrosis
    The aim of this study was to investigate the underlying mechanism of androgen deficiency inducing corporal fibrosis, thereby causing erectile dysfunction (ED). Forty 12-week-old healthy male rats were divided randomly into four groups: normal control group (Control); castration group (Castration); the other 20 rats were castrated followed by testosterone (T) (orally) each day: castration + 10mg/kg T group (Castration + 10T) and castration + 20 mg/kg T group (Castration + 20T). After 8 weeks' treatment, the main outcome measures were the following: serum levels of T; the ratios of intracavernous pressure (ICP) to mean arterial pressure (MAP); histologic changes in penile smooth muscle cells; the Smad and non-Smad pathways; and extracellular matrix (ECM) protein deposition. Castration group showed lower level of T and ratio of ICP/MAP, reduced ratio of penile smooth muscle cells/collagen, increased extracellular matrix protein deposition, and a higher expression of the Smad and non-Smad pathways. Castration + 10T partially preserved erectile function and histology stabilisation. However, the Castration + 20T group showed significantly better erectile function and molecular changes. Better efficacy could be expected with ART of adequate dose. Androgen deficiency induces corporal fibrosis through activation of the Smad and non-Smad pathways, and accumulation of ECM proteins.
  • Single nucleotide polymorphisms associated with nonsyndromic cryptorchidism in Mexican patients
    Cryptorchidism is a frequent genitourinary malformation considered as an important risk factor for infertility and testicular malignancy. The aetiology of cryptorchidism is multifactorial in which certain SNPs, capable of inhibiting the development of the gubernaculum, are implicated. We analysed 16 SNPs by allelic discrimination and automated sequencing in 85 patients and 99 healthy people, with the objective to identify the association between these variants and isolated cryptorchidism. In two different patients with unilateral cryptorchidism, we found the variants rs121912556 and p.R105R of INSL3 gene in a heterozygous form associated with cryptorchidism, so we could considered them as risk factors for cryptorchidism. On the other hand, SNPs rs10421916 of INSL3 gene, as well as the variants rs1555633 and rs7325513 in the RXFP2 gene, and rs3779456 variant of the HOXA10 gene were statistically significant, when the patients and controls were compared and could be considered as protective factors since are predominantly present in controls. The genotype–phenotype correlation did not show statistical significance. With these results, we could conclude that these polymorphisms can be considered as important variants in our population and would contribute in the future knowledge of the aetiology and physiopathology of cryptorchidism.
  • Significant predictive factors for subfertility in patients with subclinical varicocele
    We made a retrospective study to determine useful parameters for predicting subfertility in patients with subclinical varicocele (SV). One hundred and fifty men with SV and 17 age-matched men without SV were divided into three groups: Group 1, subfertile patients (n = 15); Group 2, fertile patients (n = 135); and Group 3, control patients (n = 17). Their age, body mass index (BMI), semen analysis, scrotal temperature, testicular volume, resistive index (RI), pulsatility index (PI) and peak retrograde flow (PRF) were compared. Subfertile patients (Group 1) with SV had significantly lower testicular volume and higher scrotal temperature, RI, PI and PRF than fertile men with SV (Group 2) and the control group (Group 3). Elderly men (>50 years, n = 30) with SV had a significantly higher incidence of bilateral SV than young men with SV (10/30; 33.3% vs. 12/120; 10%). There was no difference in age and BMI among the three groups. Patients with SV and RI >0.55 ml/s, PI >0.99 ml/s, total testicular volume <27 cc, scrotal temperature >34.9°C and PRF >29 cm/s have higher incidence of subfertility. Patients with SV may suffer from subfertility regardless of age. Close follow-up with colour Doppler ultrasound may be beneficial.
  • Ferulic acid prevents lead-induced testicular oxidative stress and suppressed spermatogenesis in rats
    Lead affects multiple organ systems including testis. We investigated the effects of ferulic acid (FA) on lead-induced oxidative stress and spermatogenesis suppression in rats. Animals received lead acetate (500 mg/L in drinking water) and/or FA (50 mg/kg, i.g.) for eight weeks. Lead increased testicular malondialdehyde (MDA) and nitrite levels and decreased glutathione (GSH) content and catalase (CAT) activity. Lead decreased testis weight and testosterone level. Sperm parameters decreased in lead group. FA ameliorated the decreased testis weight, serum testosterone as well as sperm count, viability, motility and normal morphology in lead group. FA improved antioxidant capacity as well as sperm count, viability, motility and normal morphology. FA decreased Johnsen's mean testicular biopsy score (MTBS) criteria by restoring degeneration, atrophy and tubular disarrangement. FA also normalised spermatogonia, spermatocytes and spermatids numbers in lead group and led to increases in number of Leydig and Sertoli cells. FA showed beneficial effects in lead-induced testicular oxidative stress and spermatological disorders, through inhibiting lipid peroxidation and enhancing antioxidant defence systems. The positive effects of FA on Leydig cells may be involved in restoring testosterone levels in lead group. FA can be considered a potential candidate to protect testis against the deleterious effect of lead intoxication.
  • Testosterone therapy has positive effects on anthropometric measures, metabolic syndrome components (obesity, lipid profile, Diabetes Mellitus control), blood indices, liver enzymes, and prostate health indicators in elderly hypogonadal men
    To alleviate late-onset hypogonadism, testosterone treatment is offered to suitable patients. Although testosterone treatment is commonly given to late-onset hypogonadism patients, there remains uncertainty about the metabolic effects during follow-ups. We assessed the associations between testosterone treatment and wide range of characteristics that included hormonal, anthropometric, biochemical features. Patients received intramuscular 1,000 mg testosterone undecanoate for 1 year. Patient anthropometric measurements were undertaken at baseline and at each visit, and blood samples were drawn at each visit, prior to the next testosterone undecanoate. Eighty-eight patients (51.1 ± 13.0 years) completed the follow-up period. Testosterone treatment was associated with significant increase in serum testosterone levels and significant stepladder decrease in body mass index, total cholesterol, triglycerides and glycated haemoglobin from baseline values among all patients. There was no significant increase in liver enzymes. There was an increase in haemoglobin and haematocrit, as well as in prostate-specific antigen and prostate volume, but no prostate biopsy intervention was needed for study patients during 1-year testosterone treatment follow-up. Testosterone treatment with long-acting testosterone undecanoate improved the constituents of metabolic syndrome and improved glycated haemoglobin in a stepladder fashion, with no adverse effects.
  • Improvement in erectile function in a rat model of high cholesterol diet-induced atherosclerosis by atorvastatin in a manner that is independent of its lipid-lowering property
    The purpose of the present study is to explore the effects of a lipid-lowering drug atorvastatin, a three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in the treatment of erectile dysfunction (ED) in a rat model of atherosclerosis (AS) and the possible mechanisms underneath. A high-cholesterol diet was administrated to Sprague-Dawley rats in an attempt to induce an ASED model, which was later confirmed by abdominal aorta histopathology and erectile function evaluation. ASED rats were further assigned to non-treatment group, atorvastatin low-dose treatment group (5 mg kg−1 day−1), high-dose group (10 mg kg−1 day−1) and sildenafil (1.5 mg kg−1 day−1) treatment group. Lipid profile, erectile function, oxidative stress biochemical markers, endothelial nitric oxide synthase (eNOS) and extracellular superoxide dismutase (SODEX) mRNA expression were evaluated after 8-week treatment duration. Erectile function was impaired in AS rat model, which was preserved in atorvastatin and sildenafil intervention groups. The oxidative stress biochemical markers were attenuated, while eNOS and SODEX mRNA expression were restored in atorvastatin and sildenafil groups, which were found to be involved in ED pathogenesis. However, the lipid profile remained unaltered in the treatment group, and it was elevated in ASED rats. This kind of lipid-lowering agent, or atorvastatin, has the utilisation potential in ASED treatment, even before lipid profiles altered. This effect on erectile function preservation of atorvastatin was attributed to its preservation of endothelial function, possibly through amelioration of oxidative stress and improvement in eNOS expression.
  • Efficacy and safety of testosterone replacement gel for treating hypogonadism in men: Phase III open-label studies
    Efficacy and safety of testosterone gel 2% (TG) were evaluated in two phase 3, open-labelled, single-arm, multicentre studies (000023 and extension study 000077). Hypogonadal men having serum testosterone levels <300 ng/dl at two consecutive measurements were included. Study duration was 9 months (000023: 3 months; 000077: 6 months). Starting dose of TG (46 mg) was applied on upper arm/shoulder. The primary endpoint (000023) was responder rate (subjects with average 24-hour serum testosterone concentration 300-1050 ng/dl on Day 90). Study 000077 evaluated the safety of TG in patients rolling over from study 000023 over a period of 6 months. Of 180 subjects in 000023, 172 completed and 145 rolled over to 000077, with 127 completers. The responder rate was 85.5%. Fewer subjects in 000077 (12.7%) versus 000023 (31.8%) had maximum testosterone concentration (Cmax) >1500 ng/dl, with no significant safety concerns. Significant improvements in sexual function and quality of life were noted in both studies. Subjects experienced few skin reactions without notable increases in prostate-specific antigen and haematocrit levels. TG was efficacious with an acceptable safety profile. Cmax >1500 ng/dl did not exhibit distinct impact on safety parameters. However, further optimisation of titration schema to reduce Cmax is warranted while maintaining the average steady state total testosterone concentration.
  • Long-term feeding of hydroalcoholic extract powder of Lepidium meyenii (maca) enhances the steroidogenic ability of Leydig cells to alleviate its decline with ageing in male rats
    This study examined whether feeding hydroalcoholic extract of Lepidium meyenii (maca) to 8-week-old (sexually maturing) or 18-week-old (mature) male rats for more than a half year affects serum testosterone concentration and testosterone production by Leydig cells cultured with hCG, 22R-hydroxycholesterol or pregnenolone. Testosterone concentration was determined in the serum samples obtained before and 6, 12, 18 and 24 weeks after the feeding, and it was significantly increased only at the 6 weeks in the group fed with the maca extract to maturing rats when it was compared with controls. Testosterone production by Leydig cells significantly increased when cultured with hCG by feeding the maca extract to maturing rats for 27 weeks (35 weeks of age) and when cultured with 22R-hydroxycholesterol by feeding it to mature rats for 30 weeks (48 weeks of age). Overall testosterone production by cultured Leydig cells decreased to about a half from 35 to 48 weeks of age. These results suggest that feeding the maca extract for a long time to male rats may enhance the steroidogenic ability of Leydig cells to alleviate its decline with ageing, whereas it may cause only a transient increase in blood testosterone concentration in sexually maturing male rats.
  • Effect of Kaempferia parviflora on sexual performance in streptozotocin-induced diabetic male rats
    Kaempferia parviflora Wall. Ex.Baker or Krachidum (KP) has been used locally in medicine and food. It has been claimed that KP has aphrodisia properties; however, no scientific data in support of this function in diabetic model have been reported. This study aimed to investigate the efficacy of KP on sexual behaviour and sperm parameter in streptozotocin (STZ)-induced diabetic male rats. Diabetes was induced in twenty male rats by STZ and divided into four groups: diabetic control group, and 3 treatment groups where KP was dose at 140, 280 and 420 mg/kg orally once a day for 6 weeks. Five normal control rats were treated with vehicle. The body weight, blood glucose, food intake, epididymal sperm parameter, sexual behaviour and serum testosterone level were evaluated. The results showed that KP treatment has no effect on the body weight, blood glucose and food intake in diabetic rats. A significant increase in sperm density in diabetic rats was observed (p < .05) at highest dose of KP. Furthermore, KP treatment demonstrated a significant recovery of sexual behaviour and serum testosterone levels in diabetic rats. These results confirm that KP exhibits aphrodisiac properties that improve the sperm density, testosterone level and sexual performance of STZ-induced diabetic rats.
  • Mean platelet volume, platelet distribution width and platelet count in erectile dysfunction: A systematic review and meta-analysis
    The aim of this study was to investigate the relationship between mean platelet volume (MPV), platelet distribution width (PDW), platelet count (PC) and erectile dysfunction (ED). We searched for observational studies from PubMed, EMBASE, Web of Science and CNKI up to 31 March 2016. Two reviewers independently selected the studies and extracted the data. MPV, PDW, and PC and mean differences in these platelet indices between healthy subjects and ED patients were explored using the Comprehensive Meta-Analysis software package. Seven studies including 795 patients and 524 healthy subjects met the inclusion criteria. The MPV was significantly larger in patients with ED than controls with the standardised mean difference of 0.596 fL (95% CI: 0.378, 0.815, p < 0.001). In ED patients, the pooled mean difference in MPV between vasculogenic ED patients and nonvasculogenic ED patients was 0.706 fL in case–control studies (95% CI: 0.410, 1.002, p < 0.001). There was no significant difference in PDW and PC between healthy subjects and ED patients. The available data suggest that larger MPV was associated with ED. Patients with vasculogenic ED tend to have higher MPV than nonvasculogenic ED patients. Further studies are needed to assess whether increased MPV in ED patients is associated with increased cardiovascular disease.
  • Testosterone in human studies: Modest associations between plasma and salivary measurements
    Testosterone is involved in many processes like aggression and mood disorders. As it may easily diffuse from blood into saliva, salivary testosterone is thought to reflect plasma free testosterone level. If so, it would provide a welcome noninvasive and less stressful alternative to blood sampling. Past research did not reveal consensus regarding the strength of the association, but sample sizes were small. This study aimed to analyse the association in a large cohort. In total, 2,048 participants (age range 18–65 years; 696 males and 1,352 females) were included and saliva (using cotton Salivettes) and plasma were collected for testosterone measurements. Levels were determined by enzyme-linked immunosorbent assay and radioimmunoassay respectively. Free testosterone was calculated by the Vermeulen algorithm. Associations were determined using linear regression analyses. Plasma total and free testosterone showed a significant association with salivary testosterone in men (adjusted β = .09, p = .01; and β = .15, p < .001, respectively) and in women (adjusted β = .08, p = .004; and crude β = .09, p = .002 respectively). The modest associations indicate that there are many influencing factors of both technical and biological origin.
  • Transformation, migration and outcome of residual bodies in the seminiferous tubules of the rat testis
    Experiments were performed to study the transformation, migration and outcome of residual bodies (RBs) in the seminiferous tubules of the rat testes. One part of the testes from adult Sprague–Dawley rats was used to generate paraffin sections to observe RBs and RB precursors through specific staining, and the other part of the testes was used to generate ultrathin sections to observe RBs under a transmission electron microscope. Deep blue particles of different sizes were observed in some seminiferous tubules through specific staining for RBs and RB precursors. These particles first appeared in the seminiferous tubules at stage I of the spermatogenic cycle, and after spermiation, the particles travelled rapidly towards the deeper region of the seminiferous epithelium and soon appeared close to the basement membrane of the seminiferous tubule. All of the particles in the tubules disappeared at stage IX. Using transmission electron microscopy, components of different electron densities were observed in the RBs on the surface of the seminiferous epithelium, all of which gradually formed in the cytoplasm of spermatozoon in later stages of spermiogenesis. After the spermatozoa were released, the RBs in the epithelium travelled quickly to the edge of the tube and were gradually transformed into lipid inclusions. These lipid inclusions ultimately became lipidlike particles. The lipidlike particles were discharged into the interstitial tissue. RBs initiate their own digestive process before their formation during spermiation in the rat testes. After spermiation, the RBs transform into lipid inclusions and finally into lipidlike particles. These lipidlike particles can be eliminated from the seminiferous tubules.
  • A literature review of antithrombotic and anticoagulating agents on sexual function
    Although millions of people receive antithrombotic agents (ATAs) or anticoagulating agents (ACAs) for vascular prophylaxis daily, the negative impact of these agents on sexual function has not been systematically studied. Therefore, a literature search was conducted to determine the effects of the marketed ATAs and ACAs on sexual function. In regard to men, the results show that thienopyridine derivatives increase the risk of erectile dysfunction (ED) and decrease libido and sexual function. The relationship between aspirin use and ED is inconsistent, ranging from a moderate risk to beneficial effects. Nonetheless, aspirin appears to result in a lower risk for ED than does clopidogrel, and seems to benefit patients with lithium-induced ED. Coumarin can cause vasculogenic priapism. In regard to women, only a single report of genital haemorrhage was found. Available data exclusively focus on male subjects. Taken together, ATAs and ACAs can disturb sexual function in different aspects in men. Newer thienopyridine derivatives, such as prasugrel or ticagrelor, may be used as a substitute for clopidogrel when sexual dysfunction occurs. Priapism and genital haemorrhage were found to be uncommon but serious complications of ACA treatment. Additional studies examining the effects of ATAs and ACAs on sexual function are needed, especially in woman and elderly.
  • Clinical and molecular characteristics in 15 patients with androgen receptor gene mutations from South China
    A variety of mutations in the androgen receptor (AR) gene are linked to androgen insensitivity syndrome (AIS) or sexual development disorder. Here, we studied 15 patients with various degrees of disorders of genital hypoplasia from South China. Clinical data including basal hormone level, phenotype, karyotyping and SRY gene identification were documented. Exons with flanking intronic region of the AR gene were sequenced and analysed for mutations, and a total of eight mutations were identified in the AR gene. Of eight mutations, two novel mutations c.2518G>T (p.Asp840Tyr) and c.1186G>C (p.Gly396Arg) were predicted to be damaging by SIFT and Polyphen2 online software. Previously reported mutations: c.528C>A (p.Ser176Arg), c.1789G>A (p.Ala597Thr), c.2612C>T (p.Ala871Val), c.1752C>A (p.Phe584Leu), c.171_172insCTG (p.57_58insLeu) and c.2659A>G (p.Met887Val) were also detected in our subjects. Most of them are involved in hypospadias, penis dysplasia or other disorders of sexual development. A complete AIS case (p.Phe584Leu) with female phenotype and high serum concentrations of dihydrotestosterone (DHT) was also found. This study presented a wide range of spectrum of AIS (from partial AIS to complete AIS) caused by AR mutations in South China population. It suggests that further study with larger data set need to be performed to elucidate the differences of the phenotypes in our study.
  • Oxidative stress and motility impairment in the semen of fertile males
    The aim of the study was to determine the total oxidant status (TOS) and evaluate the influence of oxidative stress on sperm quality in fertile males. The study population consisted of 55 fertile males. Based on the seminal plasma TOS value, the study subjects were divided into the two subgroups: a group with a low (TOS-L) and a high (TOS-H) value. Comparing the TOS-H group with the TOS-L group, we found poorer sperm motility in the TOS-H group. We found lower total antioxidant capacity values and lower activity levels in the majority of the determined superoxide dismutase, glutathione peroxidase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase. Further, we found higher levels of copper and iron as well as lower levels of zinc in the TOS-H group. We observed lower medians of IL-2, 4, 6, 8 and INF-γ in the TOS-H group compared with the TOS-L group, whereas the medians of IL-1β, IL-10 and IL-12 were significantly higher. In fertile males, higher oxidative stress intensity was associated with poorer semen quality and decreased antioxidant capacity in semen. These negative effects might be a result of decreased activities of antioxidant enzymes and altered levels of trace metals and cytokines.
  • Genetic analysis of HOXA11 gene in Chinese patients with cryptorchidism
    Cryptorchidism is the most common congenital anomaly in male children. Its aetiology remains unknown in the majority of cases. Because HOXA11 plays a vital role in regulating testicular descent, genetic variants in HOXA11 genes may contribute to the risk of cryptorchidism. In this study, mutation analysis was performed on the HOXA11 gene in a cohort of 89 patients with cryptorchidism. Furthermore, an association analysis of the HOXA11 tag single nucleotide polymorphism rs6461992 was performed in 168 patients with unilateral cryptorchidism and 193 controls. No pathogenic mutations were found. A significant difference in genotype and allele distribution was detected between cases and controls (p = .029 and .022 respectively). These results suggest that mutations in the coding sequence of HOXA11 might not be a common cause of cryptorchidism, while common polymorphisms in the HOXA11 gene might contribute to the risk of developing unilateral cryptorchidism.
  • Stenotrophomonas maltophilia isolated from prostatic fluid as an infertility factor in a male dog
    The aim of this case was to describe very rare infection of canine prostate gland with Stenotrophomonas maltophilia which had influence on male fertility. The bacterium was cultured from third fraction of the ejaculate collected by manual manipulation. The sperm concentration and motility parameters were evaluated by Hamilton-Thorne Sperm Analyser, version IVOS 12.3, sperm morphology by Diff-Quick staining and live/dead spermatozoa by eosin/nigrosin staining. After 3 weeks of treatment with targeted antibiotic, ciprofloxacin, there was no bacterial growth in prostate fluid. Semen parameters were improved after 60 days from the end of treatment, and females were successfully mated.
  • Impact of physical activity on patient self-reported outcomes of lifelong premature ejaculation patients: Results of a prospective, randomised, sham-controlled trial
    Previous studies have investigated whether physical activity increases serotonin hormone levels. Serotonin receptor dysfunction is one of the frequently accused factors of premature ejaculation (PE). Nevertheless, no studies up to date have demonstrated that the association between physical activity and premature ejaculation. We aimed to investigate the relationship between physical activity and PE and determine whether moderate physical activity might delay ejaculation time or be an alternative treatment for PE. A total of 105 patients diagnosed with PE were enrolled in this study. Of the patients, 35 were treated with dapoxetine, (30 mg) on demand (Group 1), 35 performed moderate physical activities (Group 2), and 35 performed minimal physical activity (Group 3-sham). Demographic characteristics, metabolic equivalents (MET), premature ejaculation diagnostic tool (PEDT) and intravaginal ejaculatory latency time (IELT) were recorded. There were no significant differences among three groups in terms of age, BMI, MET, PEDT or IELT before treatment. At the end of the study, there was significant decrease in PEDT scores, and increase in IELT in groups 1 and 2 as compared to Group 3. In conclusion, a moderate physical activity longer than 30 min at least 5 times a week leads to ejaculation delay, and appears as an alternative to dapoxetine on demand for the treatment of PE.
  • Trimetazidine has protective effects on spermatogenesis in a streptozotocin-induced diabetic rat model
    The aim of this study was to investigate the protective effects of trimetazidine (TMZ), as an antioxidant agent, on streptozotocin (STZ)-induced diabetic rats. A total of 50 male Sprague Dawley rats were randomly classified into five groups as follows: Group 1 (control), Group 2 (STZ-induced diabetic rats), Group 3 (STZ-induced diabetic rats treated orally with 1 cc/day isotonic saline), Group 4 (diabetic rats treated orally with 10 mg/kg/day TMZ) and Group 5 (diabetic rats treated orally with 20 mg/kg/day TMZ). After 8 weeks, orchiectomy was carried out. Histopathological and electron microscopic examinations were performed in all groups. In groups 1 and 5, the structural and ultra-structural findings of the testicular tissue and spermatogenesis were found normal. In groups 2, 3 and 4, similar results were obtained in terms of the impaired testicular architecture and degeneration of spermatogenesis. The administration of an optimal dose of TMZ protects against the harmful effects of diabetes mellitus on spermatogenesis in rats. TMZ therapy can be used to maintain normal spermatogenesis in diabetic rats.
  • The presence of human papillomavirus in semen does not affect the integrity of sperm DNA
    It remains unknown whether human papillomaviruses (HPVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen samples of 22 normozoospermic patients undergoing infertility treatment, nine fertile donors and seven fertile men with a risk of HPV infection (genital warts or condylomas) were included in the study. The samples were examined by an INNO-LiPA test PCR-based reverse hybridisation array that identifies 28 types of HPVs as simple or multiple infections. Sperm DNA integrity was determined by sperm chromatin dispersion assay (SCD). Our preliminary findings demonstrate an increase in HPV infection in infertile men with respect to fertile men. However, the sperm DNA fragmentation index was not increased in semen containing these viruses.
  • Sperm RNA preparation for transcriptomic analysis: Review of the techniques and personal experience
    In the last 10 years, several approaches, including microarrays, have been applied to investigate sperm transcript levels. However, success using microarray profiling is highly dependent of the quality of the RNA obtained. Therefore, the development of methods that deliver highly purified and intact RNA is of utmost importance. The three steps used to achieve this goal, purification of spermatozoa, RNA extraction and evaluation of RNA quality, are reviewed. Following that review and preliminary experiments, we processed sperm samples from seven normozoospermic men with a combination of gradient centrifugation and somatic cell lysis. RNA was extracted using the NucleoSpin RNA XS kit (Macherey-Nagel) and its purity checked using the BioAnalyzer. Hybridisation was done on Agilent SurePrint G3 Human GE 8 × 60K V2 microarrays. We identified 900 transcripts among the 1000 high abundance sperm transcripts reported in the literature. These genes are known to be involved in several biological processes, notably spermatogenesis, transcription regulation, cell growth and differentiation, sperm motility and capacitation, fertilisation, and embryogenesis. Therefore, our methodology is highly suitable for sperm transcriptomic analyses and can be used, notably, to compare mRNA profiles between fertile and infertile males.
  • Feeding hydroalcoholic extract powder of Lepidium meyenii (maca) enhances testicular gene expression of 3β-hydroxysteroid dehydrogenase in rats
    Although feeding diets containing the extract powder of Lepidium meyenii (maca), a plant growing in Peru's Central Andes, increases serum testosterone concentration associated with enhanced ability of testosterone production by Leydig cells in male rats, changes in testicular steroidogenesis-related factors by the maca treatment are not known. This study examined the effects of maca on testicular gene expressions for luteinizing hormone receptor, steroidogenic acute regulatory protein and steroidogenic enzymes. Eight-week-old male rats were given the diets with or without (control) the maca extract powder (2%) for 6 weeks, and mRNA levels were determined by reverse transcription quantitative real-time PCR. The results showed that the testicular mRNA level of HSD3B1 (3β-hydroxysteroid dehydrogenase; 3β-HSD) increased by the treatment, whereas the levels of the other factors examined did not change. These results suggest that increased expression of 3β-HSD gene may be involved in the enhanced steroidogenic ability by the maca treatment in rat testes.
  • Radiofrequency electromagnetic radiation from cell phone causes defective testicular function in male Wistar rats
    Cell phones have become an integral part of everyday life. As cell phone usage has become more widespread, concerns have increased regarding the harmful effects of radiofrequency electromagnetic radiation from these devices. The current study was undertaken to investigate the effects of the emitted radiation by cell phones on testicular histomorphometry and biochemical analyses. Adult male Wistar rats weighing 180–200 g were randomly allotted to control, group A (switched off mode exposure), group B (1-hr exposure), group C (2-hr exposure) and group D (3-hr exposure). The animals were exposed to radiofrequency electromagnetic radiation of cell phone for a period of 28 days. Histomorphometry, biochemical and histological investigations were carried out. The histomorphometric parameters showed no significant change (p < .05) in the levels of germinal epithelial diameter in all the experimental groups compared with the control group. There was no significant change (p < .05) in cross-sectional diameter of all the experimental groups compared with the control group. Group D rats showed a significant decrease (p ˂ .05) in lumen diameter compared with group B rats. There was an uneven distribution of germinal epithelial cells in groups B, C and D. However, there was degeneration of the epithelia cells in group D when compared to the control and group B rats. Sera levels of malondialdehyde (MDA) and superoxide dismutase (SOD), which are markers of reactive oxygen species, significantly increased (MDA) and decreased (SOD), respectively, in all the experimental groups compared with the control group. Also sera levels of gonadotropic hormones (FSH, LH and testosterone) significantly decreased (p < .05) in groups C and D compared with the control group. The study demonstrates that chronic exposure to radiofrequency electromagnetic radiation of cell phone leads to defective testicular function that is associated with increased oxidative stress and decreased gonadotropic hormonal profile.
  • Sperm DNA fragmentation affects epigenetic feature in human male pronucleus
    To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation.
  • Ascorbic acid reduces redox potential in human spermatozoa subjected to heat-induced oxidative stress
    Oxidation–reduction potential (ORP) is a new measure of oxidative stress. It is a balance between the total available oxidants and reductants. This study measures the efficiency of ascorbic acid (AA) against oxidative stress induced by either heat alone or heat and hydrogen peroxide in sperm suspensions using the MiOXSYS System. Two concentrations of ascorbic acid (400 and 600 μmol/L) were tested against heat- and heat plus hydrogen peroxide-induced oxidative stress in sperm suspensions after 2 and 4 hr of incubation. Sperm motility and static oxidation reduction potential (sORP) were measured at 2 and 4 hr of incubation at three different temperatures. A significant decrease in sORP was observed as a function of AA concentration. The 600 μmol/L AA had more pronounced reduction in sORP compared to 400 μmol/L AA (p = .001). Significant decreases in sperm motility ranging from 4.89% to 14.02% were observed both as a function of incubation time and addition of H2O2 (p < .001). Ascorbic acid is efficacious to reduce heat-induced oxidative stress in sperm preparations in vitro. The supplementation of ascorbic acid may be advantageous for semen preparations in IUI, IVF and ICSI.
  • Response to cooling of pony stallion semen selected by glass wool filtration
    The aim of this study was to compare the sperm separation technique using filtration through glass wool compared with just diluted cooled semen. Eighteen ejaculates were collected from 6 pony stallions of the Brazilian pony breed. Evaluations were done on pH, osmolarity, total motility, membrane functionality (HOST), membrane integrity (CFDA/PI), morphology and mitochondrial viability (MTT) in fresh, 24 and 48 h of cooled semen at 5°C. After dilution, the half of the extended semen was cooled (control group). The other half was cooled after filtration trough glass wool (filtered group). Retained semen was considered the portion of cells that did not transpose glass wool barrier. Total motility from the control, filtered and retained groups after 24 h of cooling was 35.5%, 43.3% and 10% (p < .0001) respectively. Sperm membrane integrity percentage at the CFDA/PI test was 37.9%, 44.8% and 14.8% (p < .0001), on the control, filtered and retained groups respectively. The results confirmed that the passage of spermatozoa through glass wool increased the selection of spermatozoa from pony stallions with higher motility, mitochondrial viability and membrane integrity for cooling in milk extender up to 24 h. Moreover, it was not obtained higher sperm parameters to control after cooling 48 h under the conditions that the study was conducted.
  • Issue Information
  • Melatonin abrogates nonylphenol-induced testicular dysfunction in Wistar rats
    Endocrine-disrupting chemicals present in the environment can bring about hormonal imbalance and be potentially harmful to the human health. Alkylphenols are omnipresent in the environment as they are constituents of several products. The aim of this study was to evaluate the effect of exogenous melatonin treatment on nonylphenol (NP)-induced oxidative stress and testicular toxicity in Wistar rats using biochemical and histopathological parameters. The oxidative stress biomarkers, activities of enzymatic and non-enzymatic antioxidants and histopathological evaluation were performed in testicular tissues. NP caused elevated TBARS levels and marked alteration of both nonenzymatic and enzymatic biomarkers. Furthermore, severe histopathological alterations were observed in the testis of NP-exposed animals as compared with that of the control rats. Melatonin supplementation ameliorated the alterations in these biochemical and histopathological variables in rats. In conclusion, our results demonstrated that melatonin through its antioxidant activity effectively protected against the NP-induced testicular toxicity.
  • Stemness of spermatogonial stem cells encapsulated in alginate hydrogel during cryopreservation
    This study investigated the effect of spermatogonial stem cell encapsulated in alginate hydrogel during cryopreservation, as cells were protected against damage during cryopreservation within the hydrogel. Spermatogonial stem cells were isolated from the testes of Balb/c mice pups (6 days old), purified in laminin-coated dishes and CD90.1 microbeads, encapsulated in alginate hydrogel and then cryopreserved. After thawing, cell viability and Spermatogonial stem cell (SSC) colony diameter were evaluated. After RNA was isolated and cDNA was synthesised, the expression of stemness genes was considered using RT real-time PCR. Finally, spermatogonial stem cells labelled with BrdU were transplanted to busulfan azoospermic mouse models. Lin28a and Sall4 genes were significantly upregulated after cryopreservation in alginate hydrogel. However, cell viability was significantly decreased. The diameter of colonies consisting of spermatogonial stem cells freeze-thawed in alginate microbeads showed no significant difference with fresh spermatogonial stem cells and the control group. The injection of freeze-thawed spermatogonial stem cells encapsulated in alginate hydrogel resulted in spermatogenesis recovery. Alginate mimics the extracellular matrices (ECM) for spermatogonial stem cells; therefore, it can support stemness potential during the cell cryopreservation process and restart spermatogenesis after transplantation.
  • Impact of titanium dioxide on androgen receptors, seminal vesicles and thyroid hormones of male rats: possible protective trial with aged garlic extract
    The aim of this study was to evaluate the effect of titanium dioxide (TiO2), a widely produced and consumed pigment in various food products, on the post-natal development of male albino rat seminal vesicle and thyroid hormones, as well as to evaluate the ameliorative effect of aged garlic extract (AGE) on TiO2-induced alterations. Forty male rat pups (3 weeks old) were divided into four equal groups. The 1st group received distilled water orally (control group), 2nd group was given 2 ml kg−1 AGE, 3rd group was administered TiO2 (5 g kg−1 BW) day after day for 65 days, and the 4th group administered AGE 6 hr prior to TiO2 gavage. TiO2-exposed rats showed nonsignificant changes in the serum testosterone, TSH, T3 and T4, while serum glucose showed a significant decrease. Androgen receptor (AR) mRNA expression was significantly down-regulated and weak signal of AR immune labelling. Histopathologically, the epithelium cell lining of seminal vesicles showed focal areas of necrosis and fibrous tissue with the prominent fibrous stroma of the atrophied glands. Meanwhile, AGE supplementation ameliorated the deleterious effects of TiO2 intoxication through protecting the tissues from oxidative stress caused by TiO2. In summary, oral administration of TiO2 resulted in abnormal developmental events in male rat seminal vesicle and AGE able to reduce TiO2 toxicity.
  • An experimental study: quantitatively evaluating the change of the content of collagen fibres in penis with two-dimensional ShearWave™ Elastography
    The purpose of this study was to explore the value of two-dimensional ShearWave™ Elastography (2D-SWE) on quantitatively evaluating the change of the content of collagen fibres in penis. Twenty male Sprague Dawley rats were divided into the pre-sexual maturity group (Group 1) and the sexual decline group (Group 2) according to age. The ultrafast ultrasound device Aixplorer® (SuperSonic Imagine, Aix-en-Provence, France) was used for 2D-SWE imaging of penis, and the measurement index was shear wave stiffness (SWS). The immunohistochemistry was used to analyse the content of collagen fibres in penis, and the measurement index was positive area percentage (PAP). The differences of SWS between the two groups and PAP between the two groups were analysed. SWS of Group 1 and Group 2 was 10.18 ± 1.09 and 8.02 ± 1.34 kPa, and SWS of Group 2 was significantly lower than Group 1 (p < .01). PAP of Group 1 and Group 2 was 4.83 ± 3.61% and 16.41 ± 10.02%, and PAP of Group 2 was significantly higher than Group 1 (p < .01). Our results indicate that when the content of collagen fibres changes, SWS of penis measured with 2D-SWE would change significantly as well. Two-dimensional SWE can be used to quantitatively evaluate the change of the content of collagen fibres in penis.
  • MMP2, MMP9 and TIMP2 polymorphisms affect sperm parameters but not fertility in Polish males
    Proper function of the blood–testis barrier is pivotal to spermatogenesis. Synchronised action of matrix metalloproteinases (MMP) and their inhibitors (TIMP) is mandatory to maintain dynamic balance of the barrier. Therefore, the association of functional genetic variants of MMP-2, MMP-9 and TIMP-2 and male infertility was studied. A total of 416 infertile males and 421 healthy subjects were genotyped for 7 SNPs within MMP2, MMP9 and TIMP2 genes, along with the assessment of semen parameters (concentration, motility and morphology of spermatozoa). No association was observed between the studied genotypes and male infertility. However, higher sperm concentration was associated with TIMP2 rs8080623 C and rs2277698 T variants among infertile men, and with MMP9 rs17576 A minor allele in controls (p < .05). TIMP2 rs9900972 T and rs2277698 T allele were associated with higher percentage of morphologically normal spermatozoa among controls. MMP2 rs2285053 TT homozygous infertile patients presented higher percentage of spermatozoa displaying nonprogressive motility. Haplotype analysis revealed strong linkage disequilibrium between the studied loci (5 of 8 possible TIMP2 haplotypes, and 3 of 4 possible MMP2 and MMP9 were found). None of the haplotypes showed association with infertility. This study results suggest an association between MMP9 and TIMP2 SNPs with sperm parameters, but not infertility.
  • Effect of oral administration of Tribulus terrestris extract on semen quality and body fat index of infertile men
    Male fertility can be evaluated through complete semen analysis. Plants belonging to the Tribulus genus are known for their role in enhancing sex hormone levels and semen quality. The aim of this study was to evaluate the effects of T. terrestris on semen quality and physiological parameters. Sixty-five men with abnormal semen evaluation were included in this study, in which they were prescribed with oral administration of Androsten® (250 mg of Tribulus terrestris dried extract per capsule). Body fat percentage, lean muscle mass gain, fluctuation in steroid hormone levels and all semen parameters were analysed during the period of treatment. The results demonstrated that decrease in the percentage of body fat and increase in lean mass were significant, as well as increase in dihydrotestosterone levels. Complete semen analysis evaluated at the end of treatment showed significant enhancement in sperm concentration, motility and liquefaction time. Protodioscin, the main phytochemical agent of the Tribulus genus, acts on sertoli cells, germ cell proliferation and growth of seminiferous tubules. This component is known to convert testosterone into dihydrotestosterone, which plays important roles in male attributes. Our results indicate the therapeutic use of Tribulus terrestris by men presenting altered semen parameters, and/or undergoing infertility treatment.
  • Capsaicin attenuates spermatogenic cell death induced by scrotal hyperthermia through its antioxidative and anti-apoptotic activities
    This study was performed to examine whether capsaicin, the main pungent ingredient of red peppers, exerts protective effects against testicular injuries induced by transient scrotal hyperthermia. Capsaicin (0.33 mg kg−1) was administered subcutaneously to mice one hour before heat stress (HS) in a 43°C water bath for 20 min. After 7 days, mice exposed to HS showed low testicular weight, severe vacuolisation of seminiferous tubules followed by loss of spermatogenic cells, and appearance of multinucleated giant cells and remarkable TUNEL-positive apoptotic cells, as well as weak immunoreactivity of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in spermatogenic cells. Levels of lipid peroxidation and heat shock 70-kDa protein 1 (Hsp72) and BCL2-associated X protein (Bax) mRNA were greatly increased, but PHGPx, manganese superoxide dismutase (MnSOD), and B-cell lymphoma-extra large (Bcl-xL) mRNAs were significantly diminished in the testes by HS. However, capsaicin pre-treatment significantly suppressed the spermatogenic cell death, oxidative stress (levels of MDA, PHGPx immunoreactivity, and Hsp72, PHGPx, and MnSOD mRNA) and apoptosis (levels of TUNEL-positive cells, and Bcl-xL and Bax mRNA) in testes by HS. These suggest that capsaicin has a protective effect against spermatogenic cell death induced by scrotal hyperthermia through its antioxidative and anti-apoptotic activities.
  • Relation between blood vitamin B12 levels with premature ejaculation: case–control study
    The aim of this study was to investigate whether vitamin B12 levels are associated with premature ejaculation (PE). A total of 109 subjects (56 PE and 53 controls) were included in this study. PE was defined as self-reported intravaginal ejaculatory latency time (IELT) based on the Diagnostic and Statistical Manual of Mental Disorders IV criteria and those who had had an IELT of <2 min was considered as PE. All participants were evaluated using premature ejaculation diagnostic tool (PEDT), International Index of Erectile Function (IIEF) and Beck Depression Inventory (BDI). The vitamin 12 levels were measured in all subjects. The mean age between the PE and controls was comparable (p = .084). Mean IIEF and BDI scores between the two groups did not statistically differ. The mean IELT values in the PE group were significantly lower than in the control group (p < .0001). PE patients reported significantly lower vitamin B12 levels compared with the controls (213.14 vs. 265.89 ng ml−1; p < .001). The ROC analysis showed a significant correlation between the diagnosis of PE and lower vitamin B12 levels. This study has demonstrated that lower vitamin B12 levels are associated with the presence of PE. This work also shows a strong correlation between vitamin B12 levels and the PEDT scores as well as the IELT values.
  • 6-Gingerol-rich fraction from Zingiber officinale ameliorates carbendazim-induced endocrine disruption and toxicity in testes and epididymis of rats
    This study evaluated the protective effects of 6-gingerol-rich fraction (6-GRF) from Zingiber officinale on carbendazim (CBZ)-induced reproductive toxicity in rats. Adult male rats were treated with either CBZ (50 mg/kg) alone or in combination with 6-GRF (50, 100 and 200 mg/kg) for 14 consecutive days. Gas chromatography–mass spectrometry (GCMS) analysis revealed that 6-GRF consists of ten bioactive chemical components with 6-gingerol being the most abundant (30.76%). Administration of 6-GRF significantly (p < .05) prevented CBZ-mediated increase in absolute and relative testes weights as well as restored the sperm quantity and quality in the treated rats to near control. In testes and epididymis, 6-GRF significantly abolished CBZ-mediated increase in oxidative damage as well as augmented antioxidant enzymes activities and glutathione level in the treated rats. Moreover, CBZ administration alone significantly decreased plasma levels of testosterone, thyrotropin, triiodothyronine and tetraiodothyronine, whereas follicle-stimulating hormone was significantly elevated without affecting luteinising hormone and prolactin levels when compared with the control. Conversely, 6-GRF ameliorated the disruption in the hormonal levels and restored their levels to near normalcy in CBZ-treated rats. Collectively, 6-GRF inhibited the adverse effects of CBZ on the antioxidant defence systems, hormonal balance and histology of the testes and epididymis in rats.
  • Are oxidative stress markers associated with unexplained male infertility?
    Male infertility can be responsible for up to 20% of the cases attending fertility consultation facilities; nonetheless, the underlying molecular mechanisms that could explain it are still elusive. Therefore, we aimed to evaluate conventional and functional parameters of semen samples from patients who presented with male infertility of unknown origin. Conventional semen parameters and functional parameters (i.e. intracellular reactive oxygen species production, mitochondrial membrane potential, sperm chromatin structure assay, sperm membrane lipid peroxidation and antioxidant capacity of seminal plasma) were evaluated on semen samples from 54 healthy donors, 23 patients with idiopathic infertility and 34 fertile controls. No significant differences were observed in the conventional seminal parameters between the fertile and infertile men. However, increased intracellular reactive oxygen species (ROS) production and DNA fragmentation were observed in the infertile patients compared to the fertile group. Alterations in intracellular ROS production and DNA fragmentation could be associated with male idiopathic infertility. These parameters could eventually distinguish both groups more accurately than the conventional parameters. Our current results are encouraging, and the efficacy of these parameters in the clinical settings needs to be further assessed to establish their predictive potential as a marker of unexplained male infertility.
  • Cross-sex hormone therapy in transgender persons affects total body weight, body fat and lean body mass: a meta-analysis
    Weight gain and body fat increase the risk of cardiometabolic disease. Cross-sex hormone therapy in transgender persons leads to changes in body weight and body composition, but it is unclear to what extent. We performed a meta-analysis to investigate the changes in body weight, body fat and lean body mass during cross-sex hormone therapy in transgender persons. We searched the PubMed database for eligible studies until November 2015. Ten studies reporting changes in body weight, body fat or lean mass in hormone naive transgender persons were included, examining 171 male-to-female and 354 female-to-male transgender people. Pooled effect estimates in the male-to-female group were +1.8 kg (95% CI: 0.2;3.4) for body weight, +3.0 kg (2.0;3.9) for body fat and −2.4 kg (−2.8; −2.1) for lean body mass. In the female-to-male group, body weight changed with +1.7 kg (0.7;2.7), body fat with −2.6 kg (−3.9; −1.4) and lean body mass with +3.9 kg (3.2;4.5). Cross-sex hormone therapy increases body weight in both sexes. In the male-to-female group, a gain in body fat and a decline in lean body mass are observed, while the opposite effects are seen in the female-to-male group. Possibly, these changes increase the risk of cardiometabolic disease in the male-to-female group.
  • Short-term storage of salmonids semen in a sodium alginate-based extender
    Short-term storage of semen is a useful strategy for preservation of fish spermatozoa. However, there is a significantly decrease on sperm function mainly due to oxidative stress. In this way, sodium alginate plays an important role as free radical scavenger compound. Accordingly, the aim of our study was to analyse the effect of a sodium alginate-based extender on sperm function in the short-term storage of salmonids semen. Samples of Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss were stored in Storfish® (Ext-C) and Storfish® supplemented with sodium alginate (Ext-A) during 10 days at 4°C. After storage, motility, viability, mitochondrial membrane potential (ΔΨmit), superoxide anion (O2−) level and DNA fragmentation (DNA Frag) were assessed. Ext-A had positive effect in preservation of sperm motility, viability, ΔΨmit, O2− level and DNA integrity in the three species analysed compared to control samples. In Ext-A, the spermatozoa of S. salar and O. mykiss showed significantly higher motility, viability and ΔΨmit than O. kisutch. However, O. kisutch and O. mykiss had significantly lower O2− level than S. salar, and DNA fragmentation in O. kisutch and S. salar was significantly lower than in samples of O. mykiss (p < 0.05). Dilution of salmonids semen in a sodium alginate-based extender is effective for protecting sperm quality during 10 days of short-term storage.
  • Multicentre study of Y chromosome microdeletions in 1,808 Chinese infertile males using multiplex and real-time polymerase chain reaction
    Azoospermia factor (AZF) genes on the long arm of the human Y chromosome are involved in spermatogenesis, and microdeletions in the AZF region have been recognised to be the second major genetic cause of spermatogenetic failure resulting in male infertility. While screening for these microdeletions can avoid unnecessary medical and surgical treatments, current methods are generally time-consuming. Therefore, we established a new method to detect and analyse microdeletions in the AZF region quickly, safely and efficiently. In total, 1,808 patients with spermatogenetic failure were recruited from three hospitals in southern China, of which 600 patients were randomly selected for screening for Y chromosome microdeletions in AZF regions employing real-time polymerase chain reaction with a TaqMan probe. In our study, of 1,808 infertile patients, 150 (8.3%) were found to bear microdeletions in the Y chromosome using multiplex PCR, while no deletions were found in the controls. Among the AZF deletions detected, two were in AZFa, three in AZFb, 35 in AZFc, three in AZFb+c and two in AZFa+b+c. Our method is fast—it permits the scanning of DNA from a patient in one and a half hours—and reliable, minimising the risk of cross-contamination and false-positive and false-negative results.
  • Assessment of antisperm antibodies in a sample of Egyptian patients with hepatitis C virus infection
    Association of hepatitis C virus (HCV) with autoimmune phenomena and impaired semen parameters has been previously reported. The aim of this study was to investigate the influence of HCV infection on the development of antisperm antibodies (ASAs) in HCV-positive males. The study was conducted on 30 HCV-infected individuals and 30 healthy control subjects. In both patients and control groups, liver enzymes and reproductive hormones were measured; computer-assisted semen analysis (CASA) was performed; HCV-RNA in serum was measured and IgG and IgA ASAs in semen were determined. Free testosterone, sperm concentration, progressive and total motility were significantly lower in HCV patients than in the control group, whereas ASAs of the IgG and IgA classes were significantly higher in HCV patients. However, correlations between viral load and the examined semen parameters and ASAs were nonsignificant. In conclusion, HCV may be responsible for the increased ASAs detected in HCV patients in the present study, possibly providing another plausible explanation for the decreased sperm motility reported in HCV patients. These findings could be of value in fertility management of HCV patients.
  • Pregnancy after intracytoplasmic sperm injection following extended sperm preparation and hormone therapy in an azoospermic man with maturation arrest and microlithiasis: a case report and literature review
    In the management of azoospermia, a combination of testicular sperm extraction and intracytoplasmic sperm injection (ICSI) is usually the most successful option for fatherhood. However, an outstanding question remains: How can at least a few spermatozoa be obtained from the ejaculate, thus avoiding the need for a surgical procedure? A 36-year-old man presented to Assisted Reproduction Unit with his 26-year-old wife. The ultrasound assessment revealed bilateral microlithiasis. Two spermograms revealed absolute azoospermia. Levels of follicle-stimulating hormone (FSH) and luteinising hormone were normal–low. The patient underwent 10 months of treatment with clomiphene citrate. A bilateral testicular sperm extraction failed to retrieve spermatozoa and revealed a maturation arrest at spermatocyte/spermatid stages depending on the tubules. Clomiphene citrate was replaced with recombinant FSH (rFSH). After 9-month treatment with rFSH, motile spermatozoa from droplets of ejaculate pellet were cryopreserved as a single straw. Ovarian stimulation was provided using classic antagonist protocol, and five mature oocytes were collected. Two consecutive fresh semen samples on the day of ICSI yielded seven motile spermatozoa, and fertilisation was achieved in all five oocytes. On day 3, two embryos were transferred, yielding positive beta-human chorionic gonadotropin and a healthy delivery of a boy and a girl.


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Carlos Tello Royloa


Actualizada el: 08-Abr-2013