Evaluation of the NOX5 protein expression and oxidative stress in sperm from asthenozoospermic men compared to normozoospermic men.

PURPOSE: NADPH oxidase 5 (NOX5), the main isoform of NOX in spermatozoa, has been recognized as the main active generators of reactive oxygen species (ROS), including superoxide anion (O 2 -. ) and hydrogen peroxide (H2O2). ROS have been shown to play important roles in many physiological and pathological conditions in spermatozoa. The present study aims to investigate the alterations of NOX5 protein expression and oxidative stress (OS) status in asthenozoospermic men compared to normozoospermic men. METHODS: Semen samples were collected from 25 asthenozoospermic men and 28 normozoospermic men. In this study, NOX5 protein expression was evaluated by Western blotting. An OS status was evaluated by measuring of ROS (O 2 -. and H2O2), DNA damage and plasma membrane integrity in spermatozoa. RESULTS: The protein expression of NOX5 (p < 0.0001) was remarkably higher in asthenozoospermic men in comparison to normozoospermic men. In addition, the percentages of intracellular O 2 -. (p < 0.0001), H2O2 (p < 0.0001) in viable spermatozoa, apoptotic sperm cells with altered plasma membrane (p < 0.001) and DNA damage (p = 0.001) were significantly increased in asthenozoospermic men compared to normozoospermic men. CONCLUSIONS: The present study provides evidence that the overexpression of NOX5 protein may induce excessive ROS production and oxidative stress damages to DNA and plasma membrane integrity in asthenozoospermic men.

Nutrient patterns and asthenozoospermia: a case-control study.

The association of dietary nutrient patterns and sperm motility is not yet well elucidated, and previous studies have just focused on the isolated nutrients. This case-control study examined the association of nutrient patterns with asthenozoospermia among Iranian men. In total, 107 incident asthenozoospermic men and 235 age-matched controls were interviewed through the infertility clinics in Tehran, Iran, from January 2012 to November 2013. Semen quality data were analysed according to the fifth edition of WHO guideline. Nutrient patterns were identified using principal component analysis based on semiquantitative 168-item food frequency questionnaires. All nutrient intakes were energy-adjusted by the residual method. In principal component analysis, three dietary patterns emerged. The first pattern, which was high in vitamin E, vitamin D, vitamin C, zinc, folate, total fibre, selenium and polyunsaturated fatty acids, was significantly associated with lower risk of asthenozoospermia. After adjustment for potential confounders, participants in the highest tertile of the first pattern scores had 51% lower risk of asthenozoospermia compared with those in the lowest (p-trend: .004). Our findings suggest that adherence to the pattern comprising mainly of antioxidant nutrients may be inversely associated with asthenozoospermia.

Effect of vitamins A, E, C and omega-3 fatty acids supplementation on the level of catalase and superoxide dismutase activities in streptozotocin-induced diabetic rats.

BACKGROUND: Since free radicals and antioxidant enzymes may play an important role in the development of diabetes, the present study was designed to assess the effect of supplementation with vitamins A, E and C and ω-3 fatty acids on catalase and superoxide dismutase activity in streptozotocin (STZ)-induced diabetic rats. METHODS: A total of 64 male Wistar rats weighing 250 g were divided into four groups as normal control, diabetic control, diabetic supplemented with vitamin A, E and C and diabetic supplemented with ω-3 fatty acids. After four weeks the rats were anesthetized and catalase (CAT) and superoxide dismutase (SOD) activities were investigated in blood samples, liver and heart homogenates. RESULTS: In diabetic rats, the activity levels of heart SOD (p < 0.001) and heart and liver CAT (p < 0.001) were significantly lower than in normal control rats. Supplementation with vitamins A, E and C significantly increased heart CAT (p = 0.05). No significant change was observed in diabetic rats supplemented with ω-3 fatty acids. CONCLUSION: Supplementation with vitamins A, E and C and ω-3 fatty acids was found to increase heart CAT activity in diabetic rats and they can be valuable candidates in the treatment of the complications of diabetes (Tab. 6, Ref. 26).

Immobilising effect of Ruta graveolens L. on human spermatozoa: coumarin compounds are involved.

This study was designed to find out Ruta graveolens L. functional components, which have immobilisation effect on human spermatozoa for contraceptive use. A five-step fractionation method was used to derive different components from rue aqueous extract by using hexane, chloroform, ethanol, acetone and ultrapure water. Gas chromatography-mass spectrophotometery (GC-MS) of all fractions and the aqueous extract were performed to determine the chemical components. The immobilisation assay and membrane integrity test were also performed with four different coumarins, which were found in GC-MS in a concentration of 10 µm. Hexane, chloroform, acetone and ethanol fractions could significantly decrease motility of sperms within the first and the second hours. Hexane fraction had also significant immediate effect. The aqueous fraction had no effect on sperm motility. Meanwhile, GC-MS revealed that aqueous extract and effective fractions had similar coumarin compounds. We performed the immobilisation assay on four different coumarins, which were found in GC-MS in a concentration of 10 µm. Reduction of sperm motility was only significant for xanthotoxin. In the sperm viability and membrane integrity tests, hexane and ethanolic fractions could impair sperm vitality significantly, in contrast to coumarins. These results indicated that a part of immobilising effect of rue could be due to its coumarins. The possible mechanism could be blocking of spermatozoa potassium channels.

Regeneration of spermatogenesis in a mouse model of azoospermia by follicle-stimulating hormone and oestradiol.

Busulfan is a chemotherapeutic drug that induces sterility, azoospermia and testicular atrophy. To induce degeneration of spermatogenesis, we used different amounts of busulfan. Adult male C57Bl/6 mice were treated with 15, 30 and 45 mg kg(-1) of busulfan. After 5 weeks, animals had daily injections of 7.5 IU human follicle-stimulating hormone (hFSH) and 12.5 µg kg(-1) oestradiol benzoate (EB), separately or simultaneously. After this time, the animals were killed and blood samples were taken through cardiac puncture. Testes were used for histopathology experiments, DNA flow cytometry and RNA extraction for expression of c-kit and cyclin B1 genes. EB unlike FSH has induced stimulatory effects on spermatogenesis, increased the level of serum testosterone 2-fold and caused a 2-fold increase in the number of haploid cells. The result showed that hFSH with EB multiplied EB stimulatory effects on spermatogenesis up to four times. Expression of c-kit and cyclin B1 genes increased in EB and hFSH+EB groups. These findings suggest that EB regulates spermatogonial stem cells via hFSH. hFSH with EB had synergistic effect on regeneration of spermatogenesis.

The micronutrient supplements, zinc sulphate and folic acid, did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men.

We investigated the effects of folic acid and zinc sulphate supplementation on the improvement of sperm function in subfertile oligoasthenoteratozoospermic (OAT) men. Eighty-three OAT men participated in a 16-week intervention randomised, double-blind clinical trial with daily treatment of folic acid (5 mg day(-1) ) and zinc sulphate (220 mg day(-1) ), or placebo. Before and after treatment, semen and blood samples were obtained for determining sperm concentration, motility, and morphology, sperm viability, sperm mitochondrial function, sperm chromatin status using toluidine blue, aniline blue, acridine orange and chromomycin A3 staining; and semen and blood folate, zinc, B12 , total antioxidant capacity (TAC) and malondialdehyde (MDA) concentrations. Sperm concentration (×10(6)  ml(-1) ) increased in subfertile men receiving the combined treatment of folic acid and zinc sulphate and also in the group receiving only folic acid treatment; however, it was not statistically significant (P = 0.056 and P = 0.05, respectively). Sperm chromatin integrity (%) increased significantly in subfertile men receiving only zinc sulphate treatment (P = 0.048). However, this improvement in sperm quality was not significant after adjusting placebo effect. This study showed that zinc sulphate and folic acid supplementation did not ameliorate sperm quality in infertile men with severely compromised sperm parameters, OAT. Male infertility is a multifactorial disorder, and also nutritional factors play an important role in results of administration of supplementation on sperm parameters. However, these results should be confirmed by multiple studies in larger populations of OAT men.

Effect of Coenzyme Q10 supplementation on antioxidant enzymes activity and oxidative stress of seminal plasma: a double-blind randomised clinical trial.

Low seminal plasma concentrations of coenzyme Q10 (CoQ10) have been correlated with impaired sperm parameters, but the exact mechanism remains of dominating interest. This randomised, placebo-controlled study examined the effect of CoQ10 on catalase, superoxide dismutase (SOD) and F2 -isoprostanes in seminal plasma in infertile men and their relation with CoQ10 concentration. Sixty infertile men with idiopathic oligoasthenoteratozoospermia (OAT) were randomised to receive 200 mg d(-1) of CoQ10 or placebo for 3 months. 47 persons of them completed the study. Semen analysis, anthropometric measurements, diet and physical activity assessment were performed for subjects before and after treatment. Independent and paired t-test, chi-square test and ancova were compared outcomes of supplementation between two groups. CoQ10 levels increased from 44.74 ± 36.47 to 68.17 ± 42.41 ng ml(-1) following supplementation in CoQ10 (P < 0.001). CoQ10 group had higher catalase and SOD activity than the placebo group. There was a significant positive correlation between CoQ10 concentration and normal sperm morphology (P = 0.037), catalase (P = 0.041) and SOD (P < 0.001). Significant difference was shown between the mean of changes in seminal plasma 8-isoprostane in two groups (P = 0.003) after supplementation. Three-month supplementation with CoQ10 in OAT infertile men can attenuate oxidative stress in seminal plasma and improve semen parameters and antioxidant enzymes activity.

Regulation of luteinizing hormone receptor in hippocampal neurons following different long-lasting treatments of castrated adult rats.

The aim of this study was to investigate the effects of different Luteinizing hormone (LH) and steroid hormones levels on LH receptor (LHR) expression in the hippocampal cells. Rats (24 males and 24 females) were assigned to four groups: one control and three experimental [gonadectomy (GDX), gonadectomy + gonadotropin releasing hormone analogue (GDX+GnRHa) and GDX+GnRHa+estradiol (E2) or testosterone (T)] independently for each gender. All experimental rats were gonadectomized; then GnRHa was administrated to GDX+GnRHa group, and GnRHa plus steroid hormone to GDX+GnRHa+E2 or T group in both genders for four-month. LHR mRNA expression and its protein level in hippocampal cells were measured using QRT-PCR and Western blotting. Quantification of mRNA revealed a decrease in LHR transcripts level in GDX+GnRHa group of females. A significant change was observed between GDX groups and GDX+GnRHa+E2 or T versus GDX+GnRHa group in females. High levels of LH decreased significantly the immature isoform of LHR in GDX group compared to control group in both genders, but low LH concentrations in GDX+GnRHa group induced immature LHR isoform production only in females. Therefore increased LH concentration induces production of incomplete LHR transcripts in hippocampal cells and decreases immature LHR at the protein level. This implies that LH decreases the efficiency of translation through either producing non-functional LHR molecules or preventing their translation.

The Association between Y Chromosome Microdeletion and Recurrent Pregnancy Loss.

BACKGROUND: To date, the role of male factor contributing in evaluation of spontaneous recurrent pregnancy loss (RPL) has been less investigated and there is discrepancy in the role of Y chromosome microdeltions in RPL. Therefore, the current study was designed to examine whether Y chromosome microdeletions were associated with RPL in an Iranian population. METHODS: One hundred men from couples, experiencing three or more RPLs, and one hundred normal men from couples with at least one child and no history of miscarriages as control group were included. Genomic DNA was extracted from peripheral blood and tested for Y chromosome microdeletions in AZFa, AZFb and AZFc regions using two multiplex PCR. RESULTS: None of the men in the case and control groups had any microdeletions in the AZFa, AZFb and AZFc regions. CONCLUSION: It seems that Y chromosome microdeletion is not associated with recurrent pregnancy loss, therefore performing this test in Iranian couples with RPL is not recommended.

Comparison of intracytoplasmic sperm injection outcomes between spermatozoa retrieved from testicular biopsy and from ejaculation in cryptozoospermic men.

The infrequent presence of spermatozoa in cryptozoospermic men ejaculate is a limiting factor in the treatment of them. Sometimes, this consideration impels us to apply meticulous microscopic search in ejaculate or testicular sperm extraction (TESE) method. The aim of this study was to assess putative effectiveness of sperm origin, ejaculated or testicular, in cryptozoospermia treatment. In this context, were evaluated intracytoplasmic sperm injection (ICSI) outcomes in two parameters including fertilisation rate (2PN) and embryo quality, independently. We compared the outcome in two groups: patients who underwent ejaculate/ICSI and ones who underwent TESE/ICSI process. Nineteen ICSI cycles performed with testicular spermatozoa and the rest of cycles (n = 208) carried out with ejaculated spermatozoa. Result analysis showed similar fertilisation rate between testicular and ejaculated spermatozoa (respectively, 60% versus 68%, P ≥ 0.05). Also, on the other hand, embryo quality did not show significant differences between two groups, except grade A with low significance. With regard to almost equal performance of both methods in results and being invasive of TESE as surgical sperm retrieval method, the use of ejaculated sperm more than testicular sperm should be recommended in patients with cryptozoospermia whenever possible.

The effects of stenosis severity on the hemodynamic parameters-assessment of the correlation between stress phase angle and wall shear stress.

To study the effects of increase in the degree of stenosis severity and subsequent complexity of hemodynamic patterns on hemodynamic parameters, experimental investigations and numerical simulations were performed. The correlations between the large negative Stress Phase Angle (SPA), the low mean Wall Shear Stress (WSS) and high Oscillatory Shear Index (OSI) were investigated at the distal shoulder and post-stenotic regions as the outcomes of elevated stenosis severity. Models included non-Newtonian fluid flow in stenotic arteries with 30-80% symmetrical stenoses. To study the interactions between pulsatile WSS and pulsatile wall circumferential stress (WCS) acting on endothelial cells, SPA as the phase difference between WSS and WCS waves was used. Moreover, the distribution of SPA on the lumen axis was compared to the distributions of the mean WSS and OSI that have been regarded until now as the determinants of atherosclerosis-prone regions. Results indicate that an increase in stenosis severity, not only affects the mean WSS, mean WCS and pulse amplitudes, but also influences the phase difference between them. The SPA is large negative on the distal shoulder and post-stenotic areas where atherosclerotic plaque develops. The increasing stenosis severity and the subsequent increasing complexity of hemodynamic patterns affect the correlation between any of the low mean WSS and high OSI with large negative SPA, such that it not only leads to create and develop some regions where the correlation between any of the low mean WSS and high OSI with large negative SPA is well but also leads to create and develop other regions where such correlations fail.

Expression profiling of vitamin D receptor in placenta, decidua and ovary of pregnant mice.

OBJECTIVES: The presence of vitamin D receptor (VDR) and the identification of localized vitamin D3 synthesis in placenta and decidua implicate the importance of vitamin D3 in reproductive function. There is, however, no data on the expression profile of VDR in the mouse placenta and endometrium throughout the pregnancy period. STUDY DESIGN: In the present work expression of VDR in reproductive tissues of pregnant mice at different gestational phases has been addressed. Expression of VDR was determined by semi-quantitative RT-PCR, Western blotting and immunohistochemistry. RESULTS: The results showed that VDR mRNA and protein were expressed in decidua, placenta and ovary throughout the pregnancy. VDR gene expression in placenta was significantly elevated in late pregnancy when compared to that of mid pregnancy. Additionally, VDR expression level in decidua rose significantly as pregnancy progressed from early to mid stages. VDR expression in decidua of pregnant mice was higher in comparison to endometrium of non-pregnant mice. Immunohistochemical analysis revealed that VDR protein is consistently expressed by luminal and glandular epithelial cells of decidua, giant cells, glycogen rich cells and labyrinth cells of placenta and by almost all follicular cell types of ovary. Surveying the expression of VDR at the protein level by Western blotting confirmed PCR results. CONCLUSION: It seems that expression of VDR in reproductive organs is finely tuned during pregnancy indicating its eminent role in reproductive biology.

Relationship between sperm chromatin status and ICSI outcome in men with obstructive azoospermia and unexplained infertile normozoospermia.

This study was done to evaluate the effect of sperm source on chromatin integrity and ICSI outcomes. One hundred and thirteen samples containing epididymal aspirates of 57 obstructive azoospermic men and 56-ejaculated semen of normozoospermic men were included in this study. Sperm chromatin status was evaluated by Chromomycin A3 (CMA3), Aniline Blue (AB) and Toluidine Blue (TB). Fertilization rate and embryo quality were recorded. In epididymal group the percentage of sperms stained with AB, CMA3 and TB were significantly higher compared to ejaculate group while fertilization rate (60.6% vs. 74.04%) was significantly lower. However, embryo quality was not significantly different between two groups. In addition, abnormal sperm chromatin condensation and DNA fragmentation were not correlated with fertilization rate and embryo quality. Our results highlight the role of epididymis in sperm maturation and confirm that ICSI using ejaculated sperm is the gold standard for treatment of infertile men.

Numerical simulation of blood pulsatile flow in a stenosed carotid artery using different rheological models.

Symmetrical 30-60% stenosis in a common carotid artery under unsteady flow condition for Newtonian and six non-Newtonian viscosity models are investigated numerically. Results show power-law model produces higher deviations, in terms of velocity and wall shear stress in comparison with other models while generalized power-law and modified-Casson models are more prone to Newtonian state. Comparing separation length of recirculation region at different critical points of cardiac cycle confirms the necessity of considering blood flow in unsteady mode. Increasing stenosis intensity causes flow patterns more disturbed downstream of the stenosis and WSS appear to develop remarkably at the stenosis throat.

Coenzyme Q10 improves seminal oxidative defense but does not affect on semen parameters in idiopathic oligoasthenoteratozoospermia: a randomized double-blind, placebo controlled trial.

BACKGROUND: Several lines of evidence show the implication of oxidative stress in the etiology of male infertility. Recently, the role of coenzyme Q10 (CoQ10) in the prevention and treatment of disease has been intensively probed. However, definitive efficacy studies in oligoasthenoteratozoospermia (OAT) have not been completed yet. AIM: To evaluate the effect of CoQ10 supplementation on semen parameters in idiopathic OAT (iOAT). MATERIAL/SUBJECTS AND METHODS: A double-blind placebo controlled clinical trial was carried out. A total of 47 infertile men with iOAT were randomly assigned to receive 200 mg CoQ10 daily or placebo during a 12- week period. Semen parameters were determined using microscopic evaluation according to World Health Organization guidelines. Lipid peroxidation was assessed by measuring the concentration of plasma malondialdehyde. We evaluated the total antioxidant capacity of seminal plasma. To compare variables between and within the 2 groups we used independent t-test and Paired t-test. RESULTS: The trial showed non-significant changes in semen parameters of CoQ10 group. However, concentrations of thiobarbituric acid-reactive substances were significantly (p<0.05) reduced in serum of treated groups compared with the control. Furthermore, total antioxidant capacity of seminal plasma significantly increased in the CoQ10 group (p<0.05). CONCLUSION: Our results provide further evidence suggesting that CoQ10 supplementation is associated with alleviating oxidative stress, although it does not show any significant effects on sperm concentration, motility and morphology. It may be suggested that CoQ10 could be taken as an adjunct therapy in cases of OAT. Further studies are needed to draw a final conclusion.

Numerical simulation of LDL mass transfer in a common carotid artery under pulsatile flows.

Symmetrical 30-60% stenosis in carotid artery with a semi-permeable wall under steady/unsteady flows for Newtonian/non-Newtonian fluids is investigated numerically. The results show that the unsteadiness of blood flow, blood pressure rise and LDL component size increase the luminal concentration, LC, of the surface. The maximum LC occurring immediately after the separation point and the non-Newtonian fluid predicts higher LDL accumulation. LC decreased as the recirculation length is increased and reaches maximum at 40% stenosis. This process is used to estimate the time-dependent growth of the arterial wall.

Increased sperm ubiquitination correlates with abnormal chromatin integrity.

Ubiquitin, a 8.5 kDa peptide that marks other proteins for proteasomal degradation, tags defective spermatozoa during epididymal passage and is proposed as a biomarker for sperm quality. The present study was designed to evaluate the relationships between sperm ubiquitination, sperm chromatin integrity and semen parameters. Semen samples from 63 couples were collected and analysed according to World Health Organization criteria. Each sample was evaluated for sperm ubiquitination by the direct immunofluorescence method, using anti-ubiquitin antibodies. Chromatin integrity of the same samples was analysed using acridine orange (AO) and toluidine blue (TB) tests. A positive correlation was found between ubiquitinated spermatozoa and the percentage of spermatozoa with abnormal chromatin (AO: r = 0.58, P < 0.001 and TB: r = 0.48, P < 0.001). Negative correlations were obtained between sperm ubiquitination and: sperm count (r = -0.2, P = 0.048), sperm morphology (r = -0.36, P = 0.003), rapidly progressive motility (r = -0.25, P = 0.044) and slow progressive motility (r = -0.28, P = 0.022). Sperm ubiquitination was positively correlated with the percentage of immotile spermatozoa. These results show that among semen parameters, chromatin abnormality is more closely associated with sperm ubiquitination and further validate sperm ubiquitination as a suitable marker for sperm quality.

Physical sequelae and self-perceived attachment in adult survivors of childhood cancer.

The association between the physical sequelae of childhood cancer and self-reported attachment behavior was investigated in 97 adult survivors. Attachment was assessed using standardized self-report questionnaires. Functional disability and cosmetic sequelae of the cancer and its treatment were evaluated by the attending oncologist using a standardized rating scale. Results suggest that attachment is not significantly associated with physician-rated physical sequelae, but may be related to the time of onset of the functional deficits, independent of the current age or age at diagnosis. Further, survivors with functional sequelae in adulthood describe themselves as more insecure in their relationships in general and more ambivalent in their relationship with their parents. These findings, if replicated, suggest that the duration of the functional deficits as well as the developmental stage of the individual when they began may be important variables which affect the survivor's capacity to develop and maintain intimate relationships that are secure and satisfying.