Overlapping sperm damages from vitamin B or D deficiency in mice: Insights into the role of clinical supplementations.

In this study, the effect of 14 weeks of standard diet (controls) or folate and vitamin B12-free diet (VBD group) or vitamin D-free diet (VDD group) were assessed on mice testicular function, and sperm function. Vitamin D deprivation caused increased body weight with no effect from VBD confirming the calcium-independent role of vitamin D on body weight homeostasis. The two deprivations caused convergent damages including decreased testosterone, worsened Johnson scores, tubular differentiation index and spermatogenesis index, and serious worsening of sperm parameters and of sperm functional tests (DNA methylation, protamination, DNA damage and lipid peroxidation). From a metabolic point of view, the damage from both models converged on the one carbon cycle (methylations) and the transsulfuration pathway (GSH and antioxidant defences) and increased circulating homocysteine, although with different mechanisms: VBD appeared to hamper methylations due to lower ability to regenerate homocysteine to methionine whereas VDD appeared to interfere with homocysteine transsulfuration to cysteine and, thereafter, GSH. VDD also caused a huge paradox increase of vitamin B12, which was likely in a non-functional form and warrants further investigation. These findings strongly endorse the potential benefit of combined folate/B12 and vitamin D supplementation in infertile patients.

Effect of curcumin, and nano-curcumin on sperm function in varicocele rat model.

Varicocele is one of the most important causes of infertility in men which gradually leads to testicular dysfunction. Testicular heat stress-induced oxidative stress is considered the main cause of pathology in these individuals. In this study, the effects of curcumin and nano-curcumin, as natural antioxidants, were investigated on spermatogenesis and sperm function in varicocele-induced rats. Seventy Wistar rats were randomly divided into seven groups; sham, control, varicocele, varicocele + curcumin 50 mg, varicocele + curcumin 100 mg, varicocele + nano-curcumin 4 mg and varicocele + nano-curcumin 8 mg. After 2 months of antioxidant therapy, all the rats were sacrificed. The results demonstrated that the mean sperm concentration and motility were significantly lower while the mean of abnormal morphology, lipid peroxidation, intracytoplasmic ROS and DNA damage was significantly higher in varicocelised rats compared to control and sham groups (p < .05). Both doses of curcumin and also nano-curcumin were significantly effective in improving the aforementioned parameters except for abnormal sperm morphology, and motility where nano-curcumin (4 mg) was significantly more effective than other groups (p < .05). The results of the current study suggest the application of nano-curcumin is more preferable to curcumin in infertile individuals with varicocele.

The effect of green coffee and/or endurance exercise on sperm function in pre-diabetic mice.

This study aimed to investigate the effect of green coffee (GC), chlorogenic acid (CA) as an active ingredient of GC and exercise, alone or in a combination with exercise, on spermatogenesis and sperm function in pre-diabetic mice. Results revealed that pre-diabetic status can have a significant adverse effect on spermatogenesis (Johnson score), and sperm concentration, motility, DNA damage and persistent histone in compared to the control group. Although lipid peroxidation, intracellular ROS production, and persistent histones in sperm were high in pre-diabetic mice, exercise only can improve sperm motility. GC alone only improved sperm motility in pre-diabetic mice while CA alone, even did not have this beneficial effect. However, GC along with exercise, did not improve motility but reduce DNA damage, while CA with exercise, significantly improved motility compared to pre-diabetic stage and to the level comparable to control. Therefore, based on this result in individuals with high DNA damage, GC supplementation and exercise could be useful approach while in asthenozoospermia, CA supplementation and exercise should be considered as an alternative approach. However, such an interpretation awaits validation.

A report of pregnancy following ICSI in one of two sisters with familiar primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a disorder of structure and function of motor ciliary and dyskinetic activity of ciliary in the fallopian tubes of affected women and could lead to infertility in some cases. In vitro fertilisation (IVF) is a choice of treatment in infertile women with PCD, which could conquer the tubal dysfunction. In this case study, we report a PCD affected woman with infertility who was treated by IVF and pregnancy was achieved but it failed due to the spontaneous abortion. We also performed whole-exome sequencing for this case and her PCD-affected sister, which did not reveal any genetic abnormality related to the PCD or infertility.

Analysis of leukocyte and sperm telomere length in oligozoospermic men.

Telomere length is considered one of the most relevant biological markers of genomic stability since it protects DNA from impairment and also ensures chromosome alignment during DNA replication. The negative impact of telomere shortening on sperm quality has been suggested as an important indicator of male infertility. Therefore, we aimed to assess leucocyte and sperm telomere length (LTL&STL), as well as sperm parameters, DNA damage and protamine deficiency in men with oligozoospermia as compared to fertile men. Our results demonstrated a significant reduction in sperm parameters (concentration, motility, morphology), LTL & STL and a significant increase in sperm DNA damage and protamine deficiency in oligozoospermic men compared with fertile individuals. These outcomes revealed that low sperm concentration in men is possibly a sign of impaired meiotic and/or meiotic division during the spermatogenesis process. It is not only associated with proper chromatin packaging but also with telomere length as a key player in the process of mitosis and meiosis, assisting in chromosomal alignment, pairing, synapsis and crossing over during spermatogenesis.

Sperm chromatin integrity in a man with macrocephaly syndrome.

This study reports chromatin status and ICSI outcomes in a case of sperm macrocephaly syndrome(SMS), showing 100% of spermatozoa with abnormal morphology. Percentages of sperm DNA fragmentation for TUNEL (31.7% versus 6.5%), SCSA (33% versus 25%) assays, chromatin maturity tests, CMA3 (58% versus 29%) and aniline blue (63% versus 35%) staining were higher in case sample compared to the fertile sample. Artificial oocyte activation resulted in a similar fertilisation rate between case and control samples (71% versus 66.7%), but the case showed delayed embryo development on day 3 post-insemination. Unlike fertile case, no embryos reached the blastocyst stage. The result of this case study shows that macrocephaly is associated with reduced chromatin maturity and DNA integrity. Although both cases showed a similar chance for fertilisation through artificial chemical activation for only macrocephalic man, the developmental competency is jeopardised in such cases.

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.

Alpha-lipoic acid improves sperm motility in infertile men after varicocelectomy: a triple-blind randomized controlled trial.

RESEARCH QUESTION: Does supplementation with alpha-lipoic acid (ALA) enhance sperm parameters and/or the status of sperm lipid peroxidation and DNA fragmentation in men who have undergone microsurgical repair of a varicocele? DESIGN: Individuals with a varicocele who had undergone varicocelectomy were divided into two groups receiving either 600 mg of ALA or an identical placebo for 80 days. Semen samples obtained from the participants before surgery and after completion of the course of medication were analysed and compared. Participants, clinicians and data analysts were blinded to the randomization sequence. RESULTS: In the ALA group, total motility (P = 0.01) and progressive motility (P = 0.002) of the spermatozoa were significantly higher compared with the placebo group after surgery. Sperm lipid peroxidation and DNA damage (assessed by sperm chromatin structure assay) showed significant decreases in both the ALA and placebo groups (P ≤ 0.02) after treatment. CONCLUSIONS: An 80-day course of ALA medication after surgical repair improves total motility and progressive motility of the spermatozoa in individuals with a varicocele.

Effect of recombinant ß-defensin 1 protein on human sperm motility and viability.

The reduction of sperm motility and subsequently reduced ability to undergo capacitation and acrosome reaction are considered as common causes of male infertility. The ß-defensin family is a group of well-known secretory proteins with antimicrobial activity that contribute to the process of "sperm maturation" during the passage of spermatozoa in the epididymis when spermatozoa attain its motility. One member of this family is "ß-defensin 1" which is present in seminal plasma and spermatozoa. The aim of this study was the incubation of human processed spermatozoa with recombinant ß-defensin 1 (500 ng/ml) for 1, 2 and 3 hr at 37°C under 5% CO2 atmosphere and assessment of sperm viability and motility in 59 semen samples. The analysis of semen samples such as sperm concentration, motility, viability, morphology and semen volume was performed according to the World Health Organization (2010; World health organization laboratory manual for the examination and processing of human semen (p. 287). Geneva, Switzerland: World Health Organization) criteria. The result of the current study shows that the incubation of spermatozoa with recombinant ß-defensin significantly maintained percentage of sperm viability and motility compared to processed spermatozoa incubate in the absence of ß-defensin in the studied time intervals (p < .05). Therefore, we concluded that recombinant ß-defensin 1 protein as an agent with antimicrobial activity can maintain sperm viability and motility in in vitro condition.

Expression of RXFP2 receptor on human spermatozoa and the anti-apoptotic and antioxidant effects of insulin-like factor 3.

Insulin-like factor 3 (INSL3) has an important role in the human reproductive system; however, its detailed function is still mysterious. We aimed to investigate the possibility of expression of RXFP2 receptor on human spermatozoa and to determine the anti-apoptotic and antioxidant mechanism derived the binding of INSL3 and RXFP2. In this experimental study, the expression/location of the RXFP2 receptor was determined on the spermatozoa of fertile and infertile men. Twenty samples from 20 fertile men were collected and divided into 6 parts (control group, and five groups treated with INSL3 10, 100, 250, 500, 1,000 ng/ml). DNA damage, active caspase, reactive oxygen species (ROS) and sperm parameters were evaluated by TUNEL, flow cytometry, optical microscope and computer-assisted sperm analysis. The expression of RXFP2 was confirmed by Western blot. Immunocytochemistry illustrated that this receptor is expressed in the posterior half of the spermatozoa's head. The INSL3 at concentrations of 500 and 1,000 ng/ml reduced the active caspase and mitochondrial ROS, and also reduced DNA fragmentation at 1,000 ng/ml. Besides, INSL3 500 and 1,000 ng/ml significantly increased the sperm motility. This study confirmed the presence of RXFP2 receptor in fertile and infertile men's spermatozoa, indicating the highly dose-dependent efficacy of the INSL3, which may have promising impacts on the in-vitro fertilisation outcomes.

Relationship between sperm telomere length and sperm quality in infertile men.

Telomeres, noncoding and repetitive DNA sequences play a significant function in chromatin integrity. Telomere length is age-dependent in somatic cells, while it increases in sperm cell with age. Therefore, we aimed to assess sperm chromatin, leucocyte and sperm telomere length (LTL, STL) in spermatozoon of 38 infertile and 19 fertile men aged between 20 and 50 years. Protamine deficiency (chromomycin A3 test), DNA fragmentation (TUNEL assay), lipid peroxidation (Bodipy probe) and telomere length (quantitative real-time PCR) were assessed. A significant decrease in mean of sperm concentration and motility and a significant increase in means of sperm abnormal morphology, DNA fragmentation, lipid peroxidation and protamine deficiency were observed in infertile compared with fertile men. In addition, the mean of LTL and STL were significantly shorter in infertile men compared with fertile individuals. We observed significant associations between telomere length with sperm concentration, DNA fragmentation and lipid peroxidation. We hypothesised that increased oxidative stress in spermatozoa of infertile men can result in abnormal packaging of chromatin, damage of DNA and shorter sperm telomere length. Together, these anomalies may account for fertility failure in these individuals.

Capsaicin improves sperm quality in rats with experimental varicocele.

Capsaicin is the main capsaicinoid in chilli peppers that have numerous biological and pharmaceutical roles in the body such as antioxidant, anti-inflammatory, anticarcinogenic, analgesic, counterirritant and antiarthritic properties. Numerous studies have shown increased oxidative stress in men with varicocele that is caused by dilation of the spermatic vein and increase of testicular temperature. Therefore, we aimed to assess the effect of Capsaicin on sperm parameters in rats with experimental varicocele. At first, we induced varicocele in 30 Wistar rats and, verify varicocele model only in 10 rats by assessment of sperm parameters, oxidative stress, DNA damage and persistent histone after 2 months. Of the remaining 20 varicocelised rats, half of them were treated with 2.5 mg/kg Capsaicin for two months and the other half served as control. Then, sperm tests were assessed, and the results showed that Capsaicin can restore the mean of sperm oxidative stress (38.78 ± 3.75 versus 58.37 ± 4.34; p < .05), sperm concentration (60.14 ± 7.66 versus 34.87 ± 5.78; p < .05) and motility (62.43 ± 3.10 versus 41.22 ± 5.11; p < .05) in varicocelised rats treated with Capsaicin compared to varicocelised rats that were not treat. Therefore, Capsaicin possibly with reduction of oxidative stress level could improve mean of sperm concentration and motility in varicocele condition.

Evaluation of sperm telomere length in infertile men with failed/low fertilization after intracytoplasmic sperm injection.

RESEARCH QUESTION: Telomeres are non-coding, repetitive DNA sequences (TTAGGG repeats) that play an important role in maintaining genome integrity. Unlike in somatic cells, telomere length in spermatozoa increases with male age and is considered as a molecular marker of sperm quality. The aetiology of failed fertilization following intracytoplasmic sperm injection (ICSI) is multifactorial; perhaps one of the reasons for this failure in these individuals is shortened sperm telomere length. This study therefore aimed to assess sperm telomere length in addition to DNA damage, lipid peroxidation and protamine deficiency in infertile men with previously failed/low fertilization post-ICSI. DESIGN: Semen samples were obtained from infertile men with previous failed/low fertilization rates (n = 10). Chromatin integrity (chromomycin A3 staining and TUNEL assay), lipid peroxidation (BODIPY probe) and telomere length (real-time PCR) for semen samples from these men were compared with samples obtained from fertile individuals (n = 10). RESULTS: The results showed significantly higher mean values for sperm DNA damage, lipid peroxidation and reduced telomere length in spermatozoa of infertile men with previous failed/low fertilization compared with fertile individuals (P < 0.05). CONCLUSIONS: Failed/low fertilization rates could be related to oxidative stress resulting in short telomere length, and also increased sperm chromatin damage and lipid peroxidation. From literature sources, shortened telomere length may lead to detachment of chromosomes from the nuclear membrane, the consequences of which are defects in the process of spermatogenesis, pronuclei formation, and delayed or arrested cell cycle post-ICSI.

Alpha-lipoic acid minimises reactive oxygen species-induced damages during sperm processing.

Shearing forces during sperm preparation for assisted reproduction techniques may lead to excessive production of reactive oxygen species (ROS) which may have an unpleasant effect on embryonic development. In the current study, we assessed the effect of alpha-lipoic acid (ALA) on ROS-induced damages during sperm preparation process. Semen samples were collected from 15 normozoospermic men. Each semen sample was divided into two parts; one part was washed and centrifuged with sperm washing medium plus 0.02 mM ALA. Then, sperm pellet was diluted and incubated for 1 hr at 37°C in sperm washing media in the absence (ALA-) or presence of 0.02 mM ALA (ALA+). The second part was washed and centrifuged with sperm washing media in the absence of ALA, and then, sperm pellet was incubated for 1 hr at 37°C in sperm washing media in the absence (ALA-) or presence of 0.02 mM ALA (ALA+). Sperm viability, motility, intracellular oxidative stress and DNA fragmentation were assessed by eosin-nigrosin, computer-assisted sperm analysis system, H2 DCFDA staining and acridine orange staining respectively. Our results showed that addition of ALA as a fat- and water-soluble antioxidant to sperm washing media maintains sperm viability and motility by reduction in ROS production and can also protect sperm DNA integrity.

Zeta method: A noninvasive method based on membrane charge for selecting spermatozoa expressing high level of phospholipaseCζ.

Intracytoplasmic sperm injection (ICSI) is a technique developed to help attain successful fertilisation for couples with severe male factor. However, a small percentage of couples confront low or failed fertilisation, mainly due to failed oocyte activation. Several studies have introduced phospholipase Cζ (PLCζ) as the main sperm factor inducing oocyte activation and thereby has the potential to act as a biomarker for the prediction of ICSI fertilisation outcome. On the other hand, researchers have focused on novel sperm selection procedures based on cellular characteristics of spermatozoa such as surface electrical charge (Zeta potential) to isolate normal sperm subpopulation with intact chromatin. Therefore, we aimed to compare PLCζ between Zeta method and routine sperm preparation procedure: density gradient centrifugation (DGC). Our results showed that number of PLCζ-positive spermatozoa was significantly low in the Zeta method, but the intensity of PLCζ protein in such spermatozoa was significantly higher than DGC procedure. Therefore, the combination of DGC with Zeta procedure may allow selecting the population of spermatozoa with a high percentage of PLCζ which may also contain a high amount of PLCζ and with intact chromatin. This sperm selection procedure can open a new approach for infertile men with previously failed fertilisation.

Effects of branched-chain amino acid supplementation and/or aerobic exercise on mouse sperm quality and testosterone production.

Individuals who regularly exercise utilise dietary supplements to enhance their exercise routine and to increase lean mass. Branched-chain amino acids (BCAAs) are a popular supplement and have been shown to produce a number of beneficial effects in rodent and human models. Therefore, in the present study, the effect of exercise and/or BCAA on sperm parameters and testes tissue was assessed. C57BL6 male mice were divided to six groups; Control, Exercise (Exc), BCAA (consumes 20 mg BCAAs), BCAA+ (consumes 60 mg BCAAs), BCAA/Exc (consumes 20 mg BCAAs during aerobic training) and BCAA+/Exc (consumes 60 mg BCAAs during aerobic training). After 8 weeks of exercise and oral treatment with BCAA; testes and epididymides were dissected, and sperm function and plasma testosterone were assessed. Exercise significantly improved sperm motility and plasma testosterone in Exercise groups with or without BCAA. Percentage of sperm lipid peroxidation was significantly decreased in Exercise group, while intensity of lipid peroxidation at the same group has significantly increased. Epithelium diameters, meiotic index and Johnson' grade did not show any changes between groups. Unlike intensive exercise, endurance exercise along with modest supplementation of BCAAs, but not an overdose, may have some synergic effect on sperm function and testosterone production.

The effect of vitamin E & vitamin B on sperm function in rat varicocele model.

We assessed the effect of vitamin E and vitamin B (Vit E & Vit B) and their combination on sperm functional parameters in the rat varicocele model. Male rats (n = 120) were divided into control (n = 30), sham (n = 30) and varicocele induction (n = 60) groups. After 2 months, 10 rats from each group were sacrificed to verify varicocele model. This part of results showed that sperm parameters, DNA damage, lipid peroxidation and residual histone were adversely effected in the varicocele group. From the 50 remaining rats in varicocele group, 10 rats received Vit B complex (6, 9.6, 30.4, 9.6 and 0.006 mg/kg for B6, B2, B9, B1 and B12 respectively), 10 rats received Vit E (40 mg/kg), 10 rats received Vit B & E, 10 rats only received water and 10 rats were only received sesame oil as a solvent for Vit E, for 2 months. From 40 remaining rats in control and sham groups, 20 rats only received water and other 20 rats only received sesame oil for 2 months. Then, all the aforementioned parameters were assessed. These results showed that Vit B antioxidant was more efficient in improvement of sperm parameters, chromatin integrity and lipid peroxidation in varicocelized rats compared with Vit E.

Effect of aerobic exercise, low-fat and high-fat diet on the testis tissue and sperm parameters in obese and nonobese mice model.

Semen quality and male fertility depend on numerous factors such as age, environment, lifestyle, physical activity, genetic background and occupation. We aimed to access the effect of aerobic exercise, low- and high-fat diet on mice testis tissue, and sperm function. Obese and nonobese male mice C57BL/6 were exposed to high fat (Hf) or low fat (Lf) and/or activity (Exe: exercise or Sed: sedentary). Finally, testicular morphometric characteristics, sperm concentration and motility (light microscopy), sperm morphology (eosin/nigrosin dye), lipid peroxidation (BODIPY C11 Probe), chromatin (acridine orange and chromomycin A3 staining) were compared within obese groups (Hf/Exe, Lf/Exe, Lf/Sed, Hf/Sed) and nonobese groups (Hf/Exe, Lf/Exe, Lf/Sed, Hf/Sed). Both exercise and diet interventions did not show any alteration in testicular morphological characteristics, sperm morphology and DNA fragmentation within both obese and nonobese groups (p > 0.05). Exercise and/or diet resulted in a significant increase in sperm concentration and motility within both groups (p < 0.05). Exercise in both groups leads to high percentage of lipid peroxidation (p < 0.05). Exercise intervention significantly improved sperm protamine deficiency within obese group (p < 0.05). We concluded that exercise intervention was more effective than diet in improvement of sperm function within obese groups.

Comparison of main molecular markers involved in autophagy and apoptosis pathways between spermatozoa of infertile men with varicocele and fertile individuals.

Abnormal dilatation and tortuosity of the pampiniform plexus within the spermatic cord are termed varicocele which leads to impaired spermatogenesis due to heat-related oxidative stress and cell death. Previously, it was shown that both apoptosis and autophagy pathways were activated by heat in germ cells of mouse in vivo and in vitro. But, status of these pathways is not clear in chronic state of heat stress such as varicocele. Therefore, we aimed to access sperm apoptotic markers (active caspases 3/7 and DNA fragmentation), and autophagic markers (Atg7 and LC3 proteins) as primary outcomes, and also sperm parameters and protamine deficiency as secondary outcomes between 23 infertile men with varicocele and 16 fertile individuals. Sperm parameters were assessed according to World Health Organization 2010 protocol. Apoptotic markers (active caspases 3/7 and DNA fragmentation), autophagic markers (Atg7 and LC3 proteins), and protamine deficiency were evaluated by flow cytometry, fluorescence microscope, and western blotting techniques. Mean of autophagy and apoptosis markers, and also protamine deficiency have significantly increased in infertile men with varicocele compared to fertile individuals, but autophagy and apoptosis markers did not significantly correlate with each other. In conclusion, it seems that both apoptosis and autophagy pathways are independently active in spermatozoa of infertile men with varicocele.

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.