Assessment of MUSASHI 1 and MUSASHI 2 expression in spermatozoa and testicular tissue

MUSASHI (MSI) family plays the main role in the spermatogenesis process. The purpose of this study was the assessment of sperm MSI1 and MSI2, and sperm functional tests in infertile men (n = 30) with varicocele and fertile men (n = 30). Furthermore, MSI1 and MSI2 proteins were assessed in testicular tissue of azoospermic men (n = 9) as well as epididymal spermatozoa and testis of mice. Expression of MSI1 and MSI2 was assessed at RNA and protein levels in human spermatozoa. Sperm concentration and motility were significantly lower, while abnormal sperm morphology, lipid peroxidation, DNA fragmentation and protamine deficiency were significantly higher in men with varicocele compared to fertile individuals. Any significant difference was not observed in the expression of MSI1 and MSI2 mRNA between the two groups. Unlike MSI1 protein that was not detectable in humans, the relative expression of MSI2 protein was similar in varicocele and fertile individuals. The expression level of both Msi1 and Msi2 proteins was also observable in mouse spermatozoa. No significant relationship was observed between sperm functional parameters with expression of these genes. The data of this study demonstrated that although MSI1 and MSI2 play important roles during spermatogenesis, their relative expression in spermatozoa was not affected by varicocele.     

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.     

A simple sperm nuclear vacuole assay with propidium iodide

Our aim was to develop a new simple sperm nuclear vacuole assay (SNVA) with propidium iodide (PI) to determine the status of nuclear vacuole (NV) of individual spermatozoa. After PI staining, sperm nuclei were classified into the 14 categories according to both nuclear morphology and the status of NV. The incidence was 57.8% (range 28–84%) in fertile controls (n = 40), and 85.1% (range 67–99%) in men with varicocele (n = 40). In the fertile group, normal nuclear‐shaped spermatozoa without NV or with one small NV located in the ante‐nuclear region were significantly more in comparison with the varicocele group. In the varicocele group, abnormal nuclear‐shaped spermatozoa with one large NV and with multiple NVs located in the ante‐nuclear region were most frequent findings. Besides, spermatozoa with NVs in both ante‐ and post‐nuclear regions in the varicocele group were significantly more than those in the fertile group. In both fertile and varicocele groups, normal or abnormal nuclear‐shaped spermatozoa with one or more vacuoles only located in the post‐nuclear region occurred sparingly. The SNVA provides a useful additional approach to identify the status of NV in human spermatozoa for diagnostic purposes. A good sperm sample would have more spermatozoa without NV or with one small NV located in the ante‐nuclear region.     

Fumaria parviflora regulates oxidative stress and apoptosis gene expression in the rat model of varicocele induction

Varicocele is one of the leading causes of male infertility in which oxidative stress induces DNA damages in spermatozoa of patients with varicocele. Recent studies indicated that the treatment with antioxidant agents has protective effects against the formation of reactive oxygen species (ROS). Our research aimed to evaluate the impact of Fumaria Parviflora (FP) on the varicocele-induced testicular injury. For this purpose, 32 adult male Wistar rats (n = 8 per group) were randomly assigned to four groups as follows: sham group, varicocele group, varicocele treatment group and the control treatment group. The experimental groups daily received FP (250 mg/kg) for 8 weeks. The induction of varicocele was conducted by partial occlusion on the left renal vein. The diameter of seminiferous tubules, Johnsen's score and the epithelium thickness improved in the treated-varicocele group as compared to the varicocele group. FP extract could increase the biochemical parameters including superoxide dismutase and glutathione peroxidase, and also decrease malondialdehyde level in the varicocele group. Furthermore, varicocele markedly increased both mRNA and intensity of Bax, while treatment with FP could alleviate them. We concluded that FP could alleviate varicocele, possibly by lowering oxidative stress and testicular damage.     

4977‐bp mitochondrial DNA deletion in infertile patients with varicocele

Varicocele is the abnormal inflexion and distension of veins of the pampiniform plexus within spermatic cord and is one of the amendable causes of male infertility. It can increase reactive oxygen species (ROS) production in semen and cause oxidative stress. The purpose of this study was to analyse spermatozoa mtDNA 4977‐bp deletion in infertile men with varicocele. To detect 4977‐bp deletion in spermatozoa mtDNA, semen samples of 60 infertile patients with clinical varicocele and 90 normal men from northern Iran were prepared. After extraction of spermatozoa total DNA, Gap polymerase chain reaction (Gap PCR) was performed. 4977‐bp deletion was observed in 81.66% of patients with varicocele, while approximately 15.55% of controls had this deletion. As spermatozoa from patients with varicocele had a high frequency of occurrence of 4977‐bp deletion in mtDNA [OR = 24.18, 95% confidence interval (CI) = 10.15–57.57, P < 0.0001], varicocele may induce mtDNA deletion in spermatozoa and cause infertility in north Iranian men. However, to determine the relation between sperm mtDNA 4977‐bp deletion and varicocele‐induced infertility, larger population‐based studies are needed. It is concluded that there is an association between sperm mtDNA 4977‐bp deletion and varicocele‐induced infertility in the population studied.     

Seminal reactive oxygen species‐antioxidant relationship in fertile males with and without varicocele

The aim of this study was to assess seminal reactive oxygen species (ROS)‐antioxidants relationship in fertile and infertile men with and without varicocele. One hundred and seventy six males were studied; fertile healthy volunteers (n = 45), fertile men with varicocele (n = 45), infertile oligoasthenozoospermia (OA, n = 44) without varicocele and infertile OA with varicocele (n = 42). In their seminal plasma, two ROS parameters (malondialdehyde, hydrogen peroxide) and five antioxidants (superoxide dismutase, catalase, glutathione peroxidase, vitaminE, vitaminC) were estimated. Compared with fertile healthy men, in all other studied groups, estimated seminal ROS were significantly higher and estimated antioxidants were significantly lower. Infertile men with varicocele showed the same relationship as infertile men without varicocele. Sperm concentration, total sperm motility as well as sperm normal forms were negatively correlated with seminal malondialdehyde and were positively correlated with vitaminC. It is concluded that varicocele has an oxidative stress (OS) in fertile normozoospermic bearing conditions. This may allow understanding that, within men with varicocele, there is a threshold value of OS over which male fertility may be impaired.     

The correlation between mammalian target of rapamycin (mTOR) gene expression and sperm DNA damage among infertile patients with and without varicocele

This study aimed to assess the possible correlation between mammalian target of rapamycin (mTOR) gene expression and sperm DNA damage among infertile patients with and without varicocele. The study included sixty infertile males and fifty fertile males as controls. The infertile group was subdivided into the following subgroups: thirty males with varicocele and thirty males without varicocele. All subjects underwent medical history collection, clinical examination, semen analysis, sperm DNA integrity assessment, mTOR gene expression assessment and scrotal colour Doppler ultrasound. The mean mTOR gene expression in infertile patients with varicocele (23.52 ± 14.65) was significantly higher than that in infertile patients without varicocele (12.24 ± 12.44) and fertile control subjects (3.92 ± 3.26; p = 0.003 and p < 0.001 respectively). In the infertile varicocele‐positive group, mTOR gene expression showed a significant negative correlation with sperm count (p = 0.028, r = ?0.400) and progressive sperm motility (p = 0.038, r = ?0.381), as well as a significant positive correlation with the sperm DNA fragmentation index (DFI; p = 0.001, r = 0.578). In the infertile varicocele‐negative group, mTOR gene expression showed a significant negative correlation with progressive sperm motility (p = 0.018, r = ?0.429) and a significant positive correlation with sperm DFI (p < 0.001, r = 0.673). In conclusion, according to these results, there is a significant positive correlation between mTOR gene expression and sperm DFI among infertile patients with and without varicocele.     

Oxidative stress‐induced alterations in seminal plasma antioxidants: Is there any association with keap1 gene methylation in human spermatozoa?

Kelch‐like ECH‐associated protein 1 (keap1)‐nuclear factor‐erythroid 2‐related factor 2 (Nrf2) pathway is one of the master regulators of cellular defence against oxidative stress. Epigenetic alterations like hypermethylation of keap1 gene impair keap1‐Nrf2 system in several oxidative stress–associated diseases. The objective of this study was to evaluate the epigenetic status of keap1 in sperm DNA of normozoospermic subjects, having different levels of reactive oxygen species (ROS) in seminal plasma. Semen samples were obtained from 151 apparently healthy male partners of couples who attended the Avicenna infertility clinic. Samples were categorised into four groups according to their ROS levels: group A (n = 39, ROS < 20 RLU/s per 10⁶ spermatozoa), group B (n = 38, 20 ≤ ROS < 40 RLU/s per 10⁶ spermatozoa), group C (n = 31, 40 ≤ ROS < 60 RLU/s per 10⁶ spermatozoa) and group D; (n = 43, ROS ≥ 60 RLU/s per 10⁶ spermatozoa). Keap1 methylation status was assessed using methylation‐specific PCR along with seminal total antioxidant capacity. The results showed no significant alterations in keap1 methylation in any groups, whereas the total antioxidant capacity enhanced with increasing levels of ROS exposure. These results indicate that keap1 was not methylated during ROS elevation and oxidative stress, suggesting that the cells have adopted other mechanisms to elevate antioxidant level.     

Seminal plasma oxytocin and oxidative stress levels in infertile men with varicocele

This study aimed to assess seminal plasma oxytocin (OT) and oxidative stress (OS) levels in infertile men with varicocele (Vx). A total of 131 men were divided into fertile men (n = 20), fertile men with Vx (n = 17), infertile men without Vx (n = 40) and infertile men with Vx (n = 54). OT, malondialdehyde (MDA) and glutathione peroxidase (GPx) were estimated in seminal plasma. Mean levels of seminal OT, MDA were significantly decreased, and the mean level of GPx was significantly increased in fertile men with/without Vx compared with infertile men with/without Vx. Mean levels of OT, MDA were increased, and mean level of GPx was significantly decreased in Vx grade III cases compared with Vx grades I, II cases and in bilateral Vx cases compared with unilateral Vx. There was significant negative correlation between seminal OT with sperm count, sperm motility, seminal GPx and significant positive correlation with sperm abnormal forms, seminal MDA. It is concluded that seminal OT is significantly decreased in fertile men with/without Vx compared with infertile men with/without Vx. Seminal OT demonstrated significant negative correlation with sperm count, sperm motility, seminal GPx and significant positive correlation with sperm abnormal forms, seminal MDA. Seminal OT is associated with Vx grade and its bilaterality.     

Hypoxia pathway has more impact than inflammation pathway on etiology of infertile men with varicocele

This study aimed to compare main molecular markers of hypoxia (HIF1‐α and P53) and inflammation (TLR‐2, TLR‐4 and TNF‐α) pathways between infertile men with varicocele and fertile individuals. Sperm parameters such as sperm concentration, motility and morphology were assessed according to World Health Organization (Laboratory manual for the examination and processing of human semen. Geneva, Switzerland, 2010) guideline in 20 infertile men with grade II or III varicocele, and 20 fertile men candidate of family balancing. In addition, sperm DNA fragmentation and molecular markers involved in hypoxia and inflammation pathways were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and real‐time PCR respectively. Mean of sperm parameters (concentration, motility and morphology) and DNA integrity were significantly lower in infertile men with varicocele compared to fertile individuals. Unlike markers involved in inflammation pathway, mean expression of markers of hypoxia pathway (HIF1‐α and P53) was significantly higher in infertile men with varicocele compared to fertile individuals (p < 0.05), and also a significant correlation was observed between expression of HIF1‐α and P53 (r = 0.461; p = 0.003). Overall, the result of this study suggests higher likelihood of involvement of hypoxia pathway, in comparison with inflammation pathway, in pathogenesis varicocele associated with male infertility.     

Assessment of seminal granulysin in infertile men with varicocele

Varicocele has a common association with male infertility, but its exact role is still debated. Apoptosis has been suggested as one of the mechanisms of varicocele‐associated infertility. Granulysin is a molecule that plays a role in apoptosis with no previous study about its role in male infertility. This case‐controlled study aimed to assess seminal plasma granulysin level in infertile patients with varicocele. This study involved 90 men that were allocated into fertile normozoospermic men (n = 20), infertile men without varicocele (n = 30) and infertile men with varicocele (n = 40). These men were subjected to history taking, clinical examination, semen analysis and estimation of seminal granulysin. In general, seminal granulysin level was significantly elevated in infertile men compared with fertile men. Infertile men with varicocele showed significantly higher seminal granulysin compared with infertile men without varicocele, in bilateral varicocele cases and in grade III varicocele. Seminal granulysin level was negatively correlated with sperm concentration, sperm motility, sperm normal forms percentage and testicular volumes. It is concluded that increased seminal granulysin has a negative impact on spermatogenesis in infertile men in general and in infertile men associated with varicocele in particular.     

Sperm DNA damage and seminal antioxidant activity in subfertile men

Supraphysiological ROS levels can lead to apoptosis, lipid peroxidation, and DNA and protein damage. This pilot study aimed to investigate the sperm oxidative damage in subfertile men, to describe the relationship between the antioxidant system and ROS. Sixty-four semen samples were categorised according to the evaluated routine parameters (WHO, WHO laboratory manual for the examination and processing of human semen, 2010). Results were cross-referenced with the DNA damage [Comet (n = 53) and TUNEL (n = 49) assays], antioxidant enzyme activity [SOD (n = 51), CAT (n = 48) and GST (n = 48)], and content of total thiols (n = 36), lipid hydroperoxides (n = 35) and MDA (n = 31). Compared to pathospermic samples, normozoospermic presented 40%–45% fewer spermatozoa with fragmented DNA, 19% fewer hydroperoxides, and slightly higher total thiols and MDA levels. Asthenozoospermic/asthenoteratozoospermic samples had the lowest GST activity. SOD and CAT showed a similar trend. Our results evidenced significant positive correlations between DNA damage and immotile spermatozoa; SOD and CAT, GST and total thiols; CAT and GST; total thiols and sperm concentration; and MDA levels and head/midpiece abnormalities and hydroperoxides. This work contributes to the existing body of knowledge by showing that the oxidative status correlates with the classic sperm analysis parameters. Oxidative stress and DNA damage evaluation might be a valuable diagnostic and prognostic tool in cases of idiopathic male subfertility.     

Oxidative stress status and sperm DNA fragmentation in fertile and infertile men

Evidence suggests that disturbing the balance between reactive oxygen species levels and antioxidant contents in seminal plasma leads to oxidative stress resulting in male infertility. This study was carried out to identifying clinical significance of seminal oxidative stress and sperm DNA fragmentation in treatment strategies of male infertility in southwest Iran. Sperm parameters, lipid peroxidation and activity of antioxidant enzymes were assessed in fertile (n = 105) and infertile (n = 112) men. Malondialdehyde (MDA) levels in seminal plasma were found to be higher significantly (p < .001) in patients. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in seminal plasma were significantly (p < .001) lower in infertile men. Significant negative correlations were observed between MDA levels and sperm motility and normal morphology. Spermatozoa with fragmented DNA were higher (p < .001) in infertile men and significantly correlated with MDA levels and SOD and GPx activities. MDA of 4.2 nmol/ml, SOD of 4.89 U/ml and GPx of 329.6 mU/ml were optimum cut‐off limits to discriminate infertile patients from fertile men. The results show the leading role of oxidative stress in aetiology of male infertility in southwest Iran and indicate that evaluation of seminal antioxidant status and DNA integrity can be helpful in men attending infertility clinics during fertility assessment.     

Seminal Tumour necrosis factor‐related apoptosis‐inducing ligand and its relationship to infertility in Egyptian patients with varicocele

Germ cell apoptosis has been proposed as one of the mechanisms by which varicocele can influence fertility. The aim of this study was to investigate the relationship between seminal tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) levels and male infertility in patients with varicocele. This study included 112 males: 30 fertile males with varicocele, 44 infertile males with varicocele and 38 healthy fertile control subjects without varicocele. Semen analysis was performed, and serum levels of reproductive hormones were measured. Seminal TRAIL levels in the infertile varicocele group were significantly higher than in the fertile varicocele and the control groups (P = 0.014). A significant negative correlation was found between seminal TRAIL and progressive (P < 0.001) and total motility scores (P < 0.001) in the infertile varicocele group. A significant negative correlation was also detected between seminal TRAIL levels and normal sperm morphology in the fertile varicocele (P = 0.007) and infertile varicocele patients (P = 0.047). Seminal TRAIL was significantly correlated with varicocele grade whether the patients were fertile (P = 0.001) or infertile (P = 0.035). Seminal TRAIL may thus have a potential role in varicocele‐associated male infertility through its negative effect on sperm motility and morphology.     

Cost‐effective diagnosis of male oxidative stress using the nitroblue tetrazolium test: useful application for the developing world

Seminal oxidative stress plays an important role in male factor infertility (MFI), worldwide. A study was thus undertaken for the first time to establish seminal reactive oxygen species (ROS) as a clinical marker of MFI in a cohort of Sri Lankan males. The nitro blue tetrazolium (NBT) assay for ROS estimation and modified Endtz test for detecting leucocytes were carried out on semen samples (N = 102) of subfertile males. Age‐matched individuals (N = 30) with proven past paternity served as controls. Significantly higher ROS production was evident in individuals with asthenozoospermia and unexplained infertility (Mann–Whitney U‐test, P = 0.000), than in the fertile and the other subfertile groups tested. Receiver operating characteristic plot analysis established cut‐off points of 40.57 and 42.02 μg formazan/10⁷ spermatozoa for ROS to distinguish fertile males from asthenozoospermics (71.4% sensitivity: 70% specificity; AUC = 0.82), and from unexplained infertile males (74.1 % sensitivity: 73.3% specificity; AUC = 0.85) respectively. As ROS appear to be a potential marker of male infertility, it is imperative to validate this test as a simple, cost‐effective hence a widely accessible diagnostic tool to be included in MFI investigations in the developing world.     

MicroRNA-based regulatory circuit involved in sperm infertility

miRNAs (MicroRNAs), known as noncoding and important endogenous factors regulating the expression protein-coding genes, are vital regulators in each biological process. Thus, this study aims to explore the key role of four microRNAs in regulating the spermatogenesis. To conduct this experiment, 55 infertile and fertile men provided the study with the sperm and testicular tissue samples. To study the spermatozoa in terms of the morphology, Diff-Quick was applied. Then, quantitative real-time polymerase chain reaction (RT-PCR) was conducted on samples. Our data indicated that in contrast to the miR-15b, significant increasing of miR-383 and miR-122 occurred in both severe oligoasthenoteratozoospermia (SOAT) and moderate oligoasthenoteratozoospermia (MOAT) compared to normal sperm group (N). In addition, it was observed that miR-15b and miR-122 increased in patients with nonobstructive azoospermia (NOA) compared with obstructive azoospermia (OA) group. Expression levels of target genes including P53, CASPASE-9 and CYCLIN D1 underwent principle changes according to miRNAs expression level. Our finding indicated that miRNAs had essential role in the regulation of spermatogenesis, and their expression altering was associated with sperm abnormalities. Thus, microRNAs can be introduced as useful biomarkers to determine male infertility reasons to choose the effective treatment.     

Supplementing post-wash asthenozoospermic human spermatozoa with coenzyme Q10 for 1 hr in vitro improves sperm motility,but not oxidative stress

We investigated the effect of supplementing post-wash asthenozoospermic spermatozoa with coenzyme Q10 (CoQ10) in vitro, which may reduce oxidative stress and improve sperm motility. Semen samples were collected from 39 men with asthenozoospermia, and their spermatozoa were isolated by two-layer Percoll density-gradient centrifugation. Kinetic parameters of the isolated spermatozoa (baseline before intervention) were determined immediately by computer-aided semen analysis. Total anti-oxidant capacity and protein carbonyl levels, as markers of oxidative stress, were also measured in the baseline spermatozoa. The baseline spermatozoa suspension was divided equally into two portions, one for CoQ10 supplementation (50 µg/ml for 1 hr) and the other as an un-supplemented vehicle control. The total motility of the CoQ10-supplemented spermatozoa was significantly higher than in the control (p = .009) and progressive motility tended to be higher (p = .053). Immotile sperm concentration in the CoQ10-supplemented spermatozoa was significantly lower than in both the baseline (p = .026) and control (p = .009). Total anti-oxidant capacity and protein carbonyl levels between the baseline, CoQ10-supplemented and control spermatozoa were not significantly different. Our data suggest that CoQ10 treatment reactivated sperm motility. We propose short-term supplementation of post-wash asthenozoospermic spermatozoa with CoQ10 before intrauterine insemination.     

An additional marker for sperm DNA quality evaluation in spermatozoa of male partners of couples undergoing assisted reproduction technique (IVF/ICSI): Protamine ratio

The aim of this study was to evaluate the relationship between the protamine ratio (P1/P2), DNA fragmentation of spermatozoa and protamine deficiency. Patients were grouped into fertile (G1; n = 151) and sub‐fertile (G2; n = 121). DNA fragmentation in spermatozoa was analysed by a TUNEL assay (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labelling), and the protamination was determined by CMA3 staining, while Western blot was used to measure protamine P1 and P2. While sperm DNA fragmentation (SDF) and protamine ratio were significantly elevated in G2 compared with G1 (12.31 ± 7.01% vs. 17.5 ± 9.5%; p = .001) and (0.91 ± 0.43 vs. 0.75 ± 0.42; p = .003); respectively, the CMA3 positive showed no difference at all between G1 and G2. In G1, the CMA3 positive correlated negatively with the P1/P2 ratio and SDF (r = ?.586, r = ?.297; p = .001 respectively). In contrast, the protamine ratio correlated positively with SDF (r = .356; p = .001). In G2, no correlation was observed between CMA3 positive, SDF and the P1/P2 ratio but the P1/P2 ratio showed a positive correlation with SDF (r = .479; p = .001). In conclusion, the spermatozoa DNA deterioration was closely associated with abnormal protamination but showed an association with the protamine ratio, more than with CMA3 positive. Therefore, for the evaluation of DNA damage in spermatozoa, the P1/P2 ratio might act as an additional biomarker.