Evaluation of cannabinoid receptors Type 1 and Type 2 mRNA expression in mature versus immature spermatozoa from fertile and infertile males.

The expression of the main cannabinoid receptors (CBR1 and CBR2) was investigated to evaluate the possible association with the sperm maturation from fertile and infertile individuals. One hundred subjects were classified into fertile (n = 50) and infertile groups (n = 50). Fresh semen samples were collected. Computer-assisted semen analysis and acrosin activity test were done. RNA was extracted from mature and immature sperm pellets. Reverse transcriptase reaction and real-time PCR were done to assess the levels of both CBR1 and CBR2 genes expression in all samples. Mature spermatozoa from both groups showed significantly higher levels of both CBR1 and CBR2 compared with the immature spermatozoa (p < .05). This increment was significantly more important in the fertile group (p < .05). In mature spermatozoa, CBR1 expression was significantly related to variation in sperm morphology, and CBR2 was significantly related to both sperm morphology and linearity index. In conclusion, CBR1 and CBR2 mRNA expression may closely direct the sperm maturation at different steps of the reproductive process.

The possible role of NF-κB1 Rs28362491 polymorphism in male fertility of Egyptian population.

Male reproductive impairment is responsible for at least 50% of cases of couple infertility. Nuclear factor-kappa B (NF-κB) has been functionally linked to germ cell apoptosis, which may affect human fertility. The aim of this study was to determine the association between the rs28362491 SNP of the NF-κB1 gene and infertility in Egyptian men. In this case-control study, semen and blood samples of 247 infertile men, constituting the case group, and of 113 fertile healthy men as the control group were analysed. All study participants were genotyped for polymorphism of the NF-κB1 gene (rs28362491) by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Heterozygous I/D genotype of the NF-κB1 rs28362491 polymorphism was associated with a significantly lower risk of poor semen quality, including asthenozoospermia, astheno-teratozoospermia, and oligo-astheno-teratozoospermia, when compared to I/I genotype (odds ratio = 0.25, 0.26, 0.18, p < .0005, <.0005, <.0005) respectively. Overall, the presence of the D allele was associated with a significantly decreased risk of poor sperm quality as compared to the I allele (odds ratio = 0.56, 0.64, 0.49, p = .050, .038, .001). In conclusion, these results suggest that heterozygosity of the NF-κB1 gene may play a protecting role against male infertility in Egyptians.

Relationship between DYNLT1 and Beclin1 expression and the fertilising potential of human spermatozoa.

This study aimed to evaluate dynein light chain type 1 (DYNLT1) mRNA expression in mature spermatozoa and to investigate its association with Beclin1 expression to help in understanding of pathogenesis of male infertility. It included 60 infertile men divided into idiopathic (n = 20), accessory gland inflammation (n = 20), and varicocele (n = 20) groups, and 20 healthy fertile men as a control group. Semen parameters were evaluated according to the 2010 World Health Organization criteria. Mature spermatozoa were isolated by Sil-select gradient. Relative quantification of DYNLT1 and Beclin1 mRNA expression in whole sperm pellet and mature spermatozoa was done using real-time PCR. Beclin1 protein was assessed in whole sperm pellet and mature spermatozoa by ELISA. Beclin1 mRNA and protein were significantly increased in spermatozoa from infertile patients of different aetiologies in comparison to healthy controls (p < .05). However, DYNLT1 mRNA expression was significantly decreased in infertile groups than controls (p < .05). Mature spermatozoa extracted from all studied subjects showed increased DYNLT1 mRNA and decreased Beclin1 mRNA and protein expression compared with the whole sample. It is concluded that decreased Beclin1 and increased DYNLT1 mRNA expression in mature spermatozoa may provide an insight into the biological processes that are activated or suppressed during sperm maturation.

The Role of Protamine 2 Gene Expression and Caspase 9 Activity in Male Infertility.

PURPOSE: Approximately 15% of couples are affected by infertility with the man responsible in almost half of the cases. PRMs (protamines) confer a higher order of DNA packaging in sperm than that in somatic cells. Because of the critical roles of PRMs in spermatid differentiation, aberrations in PRM expression or changes in protein structure could be causes of certain types of idiopathic human male infertility. The aim of this study was to give insight into the role of PRM2 gene expression and caspase 9 activity in the pathogenesis of male infertility. MATERIALS AND METHODS: The current study included 70 men with idiopathic infertility and 64 fertile men who attended the andrology outpatient clinic at Mansoura University Hospital. Semen sample analyses were done according to WHO recommendations. The acrosome reaction of spermatozoa recovered from each sample was assessed. Samples were separated using discontinuous gradient separation. From each semen sample mature sperm were separated from immature sperm. The resulting samples were divided into 2 parts, including one to determine caspase 9 activity and the other for RNA extraction and reverse transcriptase-polymerase chain reaction of PRM2 gene expression. The polymerase chain reaction product was electrophoresed on 2% agarose gel. RESULTS: PRM2 gene expression was significantly decreased in immature sperm extracted from the fertile and infertile groups. Caspase 9 activity was significantly increased in immature sperm extracted from both groups. CONCLUSIONS: Low levels of PRM2 may be associated with morphological abnormalities, initiation of the apoptotic pathway and decreasing sperm motility. PRM2 may be an important marker to better understand the key regulatory pathway of spermatogenesis and it may act as a crucial part of fertilization.

Androgen receptor expression relationship with semen variables in infertile men with varicocele.

PURPOSE: Androgen receptor, a member of the nuclear receptor superfamily, has important roles in male reproductive function. It is required for sexual differentiation, pubertal development, spermatogenesis regulation, meiosis completion and spermatocyte transition to haploid round spermatids. We assessed the association of androgen receptor expression and semen variables in infertile men with varicocele. MATERIALS AND METHODS: A total of 299 men were grouped into healthy, fertile controls, infertile men without varicocele and men with infertility associated with varicocele. A history was obtained, clinical examination and semen analysis were done and reproductive hormones were estimated. Androgen receptor expression and the acrosome reaction were determined in recovered spermatozoa. RESULTS: Androgen receptor expression was significantly decreased in infertile men with varicocele more than in infertile men without varicocele compared to fertile controls. Androgen receptor correlated positively with sperm count, motility, normal forms, velocity, linear velocity, acrosome reaction and α-glucosidase. It correlated negatively with serum follicle-stimulating hormone and estradiol. Multiple stepwise regression analysis of androgen receptor expression revealed that the sperm acrosome reaction and linearity index were the most affected independent variables. CONCLUSIONS: Androgen receptor expression was significantly decreased in infertile men with varicocele more than in infertile men without varicocele compared to fertile men. Androgen receptor expression correlated positively with sperm count, motility, normal forms, velocity, linear velocity and acrosome reaction.

Seminal clusterin gene expression associated with seminal variables in fertile and infertile men.

PURPOSE: CLU is a disulfide linked, heterodimeric protein associated with the clearance of cellular debris and apoptosis. We assessed the association of seminal CLU gene expression with seminal variables in fertile and infertile men. MATERIALS AND METHODS: A total of 124 men were divided into healthy, fertile men with normozoospermia, and men with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia. History was obtained, and clinical examination and semen analysis were done. In semen we assessed sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression. RESULTS: CLU RNA and CLU protein gene expression were significantly increased in semen samples of infertile men with oligoasthenoteratozoospermia > asthenoteratozoospermia > asthenozoospermia compared with healthy, fertile controls. CLU gene expression significantly correlated negatively with sperm count, motility, acrosin activity index, linearity index and linear velocity, and significantly correlated positively with the percent of sperm abnormal forms and DNA fragmentation. CONCLUSION: CLU gene expression was significantly increased in the semen samples of infertile men. It correlated negatively with sperm count, motility, acrosin activity, linearity index and linear velocity, and positively with the percent of sperm abnormal forms and DNA fragmentation.

ACE gene insertion/deletion polymorphism seminal associations in infertile men.

PURPOSE: We assessed seminal associations of the ACE gene insertion/deletion polymorphism in infertile men. MATERIALS AND METHODS: A total of 405 men were investigated, divided into healthy fertile men, and those with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia, respectively. They underwent semen analysis, and assessment of sperm acrosin activity, hypo-osmotic swelling, seminal 8-iso-prostaglandin-F(2α), total antioxidant capacity, α-glucosidase and ACE gene polymorphisms. RESULT: The ACE insertion/insertion genotype was noted in 182 men, including 76.5% of healthy fertile men, and 47.4%, 39.8% and 17.6% of those with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia, respectively. The ACE insertion/deletion genotype was noted in 133 men, including 13.7% of healthy fertile men, and 42.3%, 27.5% and 47.2% of those with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia, respectively. The ACE deletion/deletion genotype was identified in 90 men, including 9.8% of healthy fertile men, 10.3%, 32.70% and 35.2% of those with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia, respectively. Men with the ACE deletion/deletion and insertion/deletion genotypes showed a significant decrease in sperm count, motility, linear velocity and normal forms, acrosin activity index, hypo-osmotic swelling test and seminal α-glucosidase, and significantly increased seminal 8-iso-prostaglandin-F(2α) than those with the ACE insertion/insertion genotype. CONCLUSIONS: ACE gene deletion polymorphism is associated with abnormal seminal variables, such that carriers of the ACE deletion/deletion genotype have higher seminal oxidative stress.

Increased DNA damage in children caused by passive smoking as assessed by comet assay and oxidative stress.

The present study aimed to evaluate the association between the environmental tobacco smoke (ETS) and DNA damage in relation to oxidative stress (OS) in children. Sixty-four children of age 1-8 years, selected from the outpatient clinic of Mansoura University Children Hospital were divided into two groups (23 children/group) based on high (>20 cigarettes/day) or low (<20 cigarettes/day) exposure to ETS at home. Twenty symptom-free children with normal cotinine level and with no exposure to ETS were recruited as controls. The comet assay was used to quantify the level of DNA damage in lymphocytes isolated from all children. Spectrophotometric methods were used to assess the serum level of malondialdehyde (MDA) and activity of glutathione peroxidase (GSH-Px) in erythrocytes. Also, serum level of tocopherol fractions (alpha, gamma, delta) was assessed by high performance liquid chromatography (HPLC). Children exposed to ETS exhibited retarded growth, more chest problems, and gastroenteritis than the control. A significant increase in mean comet tail length indicating DNA damage was observed in ETS-exposed children (P<0.001) compared to controls. ETS-exposed children had significantly (P<0.001) higher MDA level paralleled with significant (P<0.001) decrease in the level of GSH-Px and tocopherol fractions compared with controls. The GSH-Px activity and tocopherol levels were inversely correlated with the increase of ETS exposure. These results show that inhalation of ETS is associated with an increase in the level of oxidants and a simultaneous decrease in the level of antioxidants in the children's blood. This status of oxidant-antioxidant imbalance (OS) may be one of the mechanisms leading to DNA damage detected in lymphocytes of ETS-exposed children. In conclusion, the present study gives an indication of an association between DNA damage and ETS exposure in children.

In vitro effect of cell phone radiation on motility, DNA fragmentation and clusterin gene expression in human sperm.

BACKGROUND: Use of cellular phones emitting radiofrequency electromagnetic field (RF-EMF) has been increased exponentially and become a part of everyday life. This study aimed to investigate the effects of in vitro RF-EMF exposure emitted from cellular phones on sperm motility index, sperm DNA fragmentation and seminal clusterin (CLU) gene expression. MATERIALS AND METHODS: In this prospective study, a total of 124 semen samples were grouped into the following main categories: i. normozoospermia (N, n=26), ii. asthenozoospermia (A, n=32), iii. asthenoteratozoospermia (AT, n=31) and iv. oligoasthenoteratozoospermia (OAT, n=35). The same semen samples were then divided into two portions non-exposed and exposed samples to cell phone radiation for 1 hour. Before and immediately after exposure, both aliquots were subjected to different assessments for sperm motility, acrosin activity, sperm DNA fragmentation and CLU gene expression. Statistical differences were analyzed using paired t student test for comparisons between two sub-groups where p<0.05 was set as significant. RESULTS: There was a significant decrease in sperm motility, sperm linear velocity, sperm linearity index, and sperm acrosin activity, whereas there was a significant increase in sperm DNA fragmentation percent, CLU gene expression and CLU protein levels in the exposed semen samples to RF-EMF compared with non-exposed samples in OAT>AT>A>N groups, respectively (p<0.05). CONCLUSION: Cell phone emissions have a negative impact on exposed sperm motility index, sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression, especially in OAT cases.

Relationship between acrosin activity of human spermatozoa and oxidative stress.

AIM: To study the association between seminal oxidative stress and human sperm acrosin activity. METHODS: It is a prospective study consisting of 30 infertile men and 12 fertile normozoospermic volunteers. A full history, clinical examination and scrotal ultrasound were done to exclude other related factors such as smoking and varicocele. Presence of white blood cells (WBCs) in semen samples was evaluated by peroxidase staining. Lipid peroxidation in spermatozoa was induced after incubating with ferrous sulphate (4 mmol/L) and sodium ascorbate (20 mmol/L). Induced peroxidation of spermatozoa was assessed by determining the production of thiobarbituric acid reactive substances (TBARS). Acrosin activity was measured using the gelatinolysis technique. The halo diameters around the sperm heads and the percentages of spermatozoa showing halo formation were evaluated. An acrosin activity index was calculated by multiplying the halo diameter by the halo formation rate. RESULTS: A significant difference was observed in acrosin activity parameters and TBARS levels between samples with WBCs (1 multiply 10(6)/mL of ejaculate) and those without. This difference was also noted between the normozoospermic and the oligoasthenoteratozoospermic semen samples. The TBARS production by spermatozoa had a significant negative correlation with the acrosin activity index (r = -0.89, P 0.001). CONCLUSION: The presence of oxidative stress in an individual with leukocytospermia and/or abnormal semen parameters is associated with impaired sperm function as measured by its acrosin activity.

Tumor necrosis factor-α gene polymorphism relationship to seminal variables in infertile men.

OBJECTIVE: To assess the tumor necrosis factor (TNF)-α gene polymorphism relationship with seminal variables in fertile men (N) and those with asthenozoospermia (A), asthenoteratozoospermia (AT), and oligoasthenoteratozoospermia (OAT). MATERIALS AND METHODS: A total of 50 infertile men without a female factor who were attending a fertility clinic and 48 fertile men were randomly screened for semen analysis, analysis of the TNF-α promoter region for polymorphism, seminal caspase-9, acrosin activity, α-glucosidase, and reproductive hormones. RESULTS: The TNF-α GG genotype was present in 83.9%, 72.7%, 66.7%, and 59.5%, the TNF-α AA genotype in 3.2%, 6.8%, 10.4%, and 11.9%, and TNF-α AG genotype in 12.9%, 20.5%, 22.9%, and 28.6% in the N, A, AT, OAT groups, respectively. The occurrence of A allele was significantly greater among infertile patients than among fertile controls (21.6% vs 9.7%; odds ratio 0.388, 95% confidence interval 0.2 to 0.75, P = .005). Men with the TNF-α AA genotype demonstrated a significant decrease in the sperm count, sperm motility, normal sperm morphology, acrosin activity, and seminal α-glucosidase and a significant increase in seminal caspase-9 compared with those with the TNF-α GG genotype. CONCLUSION: This single nucleotide polymorphism in the TNF-α(-308) gene was associated with significantly increased seminal caspase-9 and a significantly decreased sperm count, sperm motility, normal sperm morphology, acrosin activity, and seminal α-glucosidase.

Sperm caspase-9 in oligoasthenoteratozoospermic men with and without varicocele.

OBJECTIVE: To assess sperm caspase-9 activity in infertile oligoasthenoteratozoospermic (OAT) men with and without varicocele. DESIGN: Prospective. SETTING: Academic setting. PATIENT(S): Eighty men: healthy fertile control subjects (n = 20), OAT (n = 25), and OAT associated with left-side varicocele (n = 35). INTERVENTION(S): History taking, clinical examination, semen analysis,assessment of seminal caspase-9. MAIN OUTCOME MEASURE(S): Semen analysis, sperm caspase-9. RESULT(S): Sperm caspase-9 was significantly increased in infertile OAT men associated with varicocele compared with OAT men without varicocele and healthy fertile control subjects. Sperm casapse-9 activity demonstrated significant negative correlation with sperm count, sperm motility, sperm velocity, sperm linear velocity, sperm linearity index, and sperm normal morphology. CONCLUSION(S): Sperm caspase-9 is exaggerated in infertile OAT cases with varicocele compared with infertile OAT cases without varicocele or healthy fertile men. Sperm caspase-9 demonstrated significant negative correlation with semen variables.