Increased Sperm DNA Damage Research Articles

Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: An approach to non-surgical sterilization.

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades. However, this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection, would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in sperm DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability, we discuss how these reagents might be modified in order to increase target cell ablation and improve their efficacy as sterilization agents.

Increased Sperm DNA Damage in Sprague-Dawley Rats Exposed to Dextromethorphan as an Antitussive

Introduction: Male reproductive functions have been observed to be negatively impacted by the antitussive dextromethorphan (DM). It was also discovered that DM has an impact on micronutrients (such as zinc, calcium, and selenium) which are crucial for both capacitation and the acrosome reaction, which can result in infertility. Using Sprague-Dawley rats as models, we investigated the effects of quercetin and rutin on DM-induced toxicity in males in relation to apoptotic protein markers. Methodology: For a period of 16 weeks, 80 male rats, weighing 150 ± 30 g, were utilized and split into four groups. Twenty animals were employed in each group. For a period of 16 weeks, Group B received 20 mg/kg, Group C received 40 mg/kg, and Group D received 80 mg/kg of DM. Group A acted as the control group and received 1 ml of distilled water (DW). Five randomly chosen animals from each group were put to sleep at the end of the DM treatment period, and their testes were taken out to measure DNA fragmentation using comet assay. To ascertain the pace of recovery, the animals were then split into three groups, E–G, and kept for a total of 16 weeks. Group G received 1 ml of DW, Group F was given rutin (25 mg/kg), and Group E was given quercetin (50 mg/kg). Results: Comparing DM-treated groups to control revealed a significant dose-dependent decrease in DNA fragmentation. When DM-treated and recovery-alone groups were contrasted with rutin and quercetin groups, an improvement in DNA was seen. Conclusion: Significant improvements in the parameters were observed when rutin and quercetin supplements were used, which may lessen the harmful effects of DM and enhance male fertility.

Male reproductive ageing: a radical road to ruin.

In modern post-transition societies, we are reproducing later and living longer. While the impact of age on female reproductive function has been well studied, much less is known about the intersection of age and male reproduction. Our current understanding is that advancing age brings forth a progressive decline in male fertility accompanied by a reduction in circulating testosterone levels and the appearance of age-dependent reproductive pathologies including benign prostatic hypertrophy and erectile dysfunction. Paternal ageing is also associated with a profound increase in sperm DNA damage, the appearance of multiple epigenetic changes in the germ line and an elevated mutational load in the offspring. The net result of such changes is an increase in the disease burden carried by the progeny of ageing males, including dominant genetic diseases such as Apert syndrome and achondroplasia, as well as neuropsychiatric conditions including autism and spontaneous schizophrenia. The genetic basis of these age-related effects appears to involve two fundamental mechanisms. The first is a positive selection mechanism whereby stem cells containing mutations in a mitogen-activated protein kinase pathway gain a selective advantage over their non-mutant counterparts and exhibit significant clonal expansion with the passage of time. The second is dependent on an age-dependent increase in oxidative stress which impairs the steroidogenic capacity of the Leydig cells, disrupts the ability of Sertoli cells to support the normal differentiation of germ cells, and disrupts the functional and genetic integrity of spermatozoa. Given the central importance of oxidative stress in defining the impact of chronological age on male reproduction, there may be a role for antioxidants in the clinical management of this process. While animal studies are supportive of this strategy, carefully designed clinical trials are now needed if we are to realize the therapeutic potential of this approach in a clinical context.

P-717 Segmental aneuploidies are not related to paternal age in young women

Abstract Study question Is there an impact of paternal age on embryo segmental aneuploidy rate? Summary answer Even when segmental aneuploidies are most linked to paternal origin, results do not show a relationship between paternal age and embryo segmental aneuploidies. What is known already Most studies consider maternal age as a risk factor for spontaneous abortion, infertility and genetic defects in the offspring. However, attention has only recently turned to the impact of paternal age on reproductive outcome. In Assisted Reproduction Technology male age has been related to a decrease in sperm quality and clinical outcomes and an increase in sperm DNA damage has been identified among elderly males. Studies assessing the type of chromosomal aneuploidy associated with paternal aging have reported mixed relative risk results for chromosomes 13, 18, 21, and X. Segmental aneuploidies are most linked to paternal origin. Study design, size, duration Observational, retrospective and multicentric study. The study was approved by the local institutional review board (IRB). All the patients underwent an IVF cycle with oocytes from egg donors, followed by Preimplantation Genetic Testing for Aneuploidy (PGT-A) at IVI-RMA Madrid, IVI-RMA Valencia and IVI-RMA Barcelona from January 2018 to August 2022. A total of 3412 biopsied embryos obtained from 660 cycles were included in the study. Participants/materials, setting, methods Exclusion criteria were the following: altered karyotype, single gene disorder and male factor (sperm concentration lower than 5 million/ ml). Embryo biopsies were performed at blastocyst stage (day 5/ 6). After the trophoectoderm biopsy, all embryos were vitrified. Main outcome was segmental aneuploidy rate among different male age groups (<41 y.o./ ≥40 y.o.). Lineal regression analysis was used for continuous parameters and Chi square was used for categorical variables. Main results and the role of chance Descriptive parameters showed no differences, particularly in sperm concentration (43.2±15.6 vs. 40.1±14.3) and sperm motility (27.3±13.7 vs. 29.7±14.8). Lineal regression analysis showed no significant differences in segmental aneuploidy rate (p = 0.542). Statistical model showed no significant differences among male age groups (1.28% (0.76-2.01) vs 1.10% (0.69-1.66) p = 0.542). Limitations, reasons for caution The study has been conducted in three different clinics, there could be some unknown cofounding factors. Wider implications of the findings Male age don’t have an impact on the embryo segmental aneuploidy rate in young/donors’ oocyte Trial registration number Not applicable

Age-related DNA damage in stallions: an ongoing investigation

Foals sired by younger stallions record significantly higher racing speeds (Sharman.et.al. Royal Society Open Science. 2022;9:220691), and more race wins than foals sired by older stallions (Brazil.et.al. Honours Dissertation. University of Limerick. 2016). We hypothesize that this may be due to an age-associated increase in sperm DNA damage, as has been demonstrated in other species. Therefore, this study aimed to investigate the relationship between stallion age and sperm DNA damage. Post-coital dismount semen samples (n=75) were collected weekly from 46 commercial Thoroughbred stallions in the Hunter Valley (Australia). Samples were immediately diluted (2:1, EquiPlus:semen), and the high-density spermatozoa were isolated using EquiPure centrifugation to reduce contaminants (somatic cells and other debris). Any samples that were contaminated with urine were excluded from the study. Sperm count and motilities were recorded onsite (iSperm™), and samples were snap frozen for DNA damage analyses. Management information, including pregnancy scan results, were also collected. For this study, stallions aged ≤9 years were categorized as “young” while those aged ≥12 were categorized as “old”. Stallions between 9 and 12 years were excluded to improve the likelihood of observing an age-related difference. Mare age and fertility status were similar across both groups. All data were checked for normality (Shapiro-Wilk) and analyzed using unpaired t-tests. Sperm count and motility did not differsignificantly between age cohorts. DNA strand breaks were significantly higher in spermatozoa from older stallions compared to younger stallions (alkaline comet assay: 19.7±0.55% vs 15.4±0.57% mean tail intensity; P≤0.05, and sperm chromatin dispersion or ‘Halo’ assay; 0.48±0.045in vs 0.70±0.074in ‘halo’ area; P≤0.05, respectively). These breaks may be attributed to poor chromatin packaging, as older stallions had a concomitant deficiency of sperm protamines compared to young stallions (11.6±1.53 vs 6.9±1.07 chromomycin A3 positive cells; P≤0.05, respectively). Although younger stallions had lower levels of DNA strand breakage, they had more oxidized DNA adducts (8-hydroxy-2′-deoxyguanosine (8-OHdG) assay; P≤0.05). Interestingly, regardless of age, positive correlations between the proportion of oxidized DNA adducts and fertility were observed (R2=0.269 for younger, R2=0.483 for older stallions; P≤0.05). This is likely a reflection of the increased use of oxidative phosphorylation, and subsequent ROS production, in higher fertility ejaculates (Gibb.et.al. Biology of Reproduction. 2014;91(3):77). In conclusion, while stallion sperm DNA damage rises with age, it does not appear to influence fertility outcomes. However, further research is needed to determine if age-related sperm DNA damage compromises the next generation's genome. In addition, we must identify the most vulnerable genomic loci for sperm DNA damage and determine how these DNA lesions may contribute to reduced racing performance and health in offspring.

Sperm DNA damage: The possible link between obesity and male infertility, an update of the current literature.

Obesity prevalence worldwide is increasing significantly. Whilst maternal obesity has clear detrimental impacts on fertility, pregnancy and foetal outcomes, more recently there has been an increasing focus on the role of paternal obesity in human fertility. Recent meta-analyses have indicated that obesity in men negatively affects basic sperm parameters such as sperm count, concentration and motility, increases the incidence of infertility and reduces the chances of conception. Sperm DNA damage, typically characterised by DNA strand breaks and oxidation of DNA nucleotides, is a specialised marker of sperm quality that has been independently associated with recurrent miscarriage, reduced assisted reproduction success and increased mutational loads in subsequent offspring. Whilst, there are still conflicting data in humans as to the association of obesity in men with sperm DNA damage, evidence from rodent models is clear, indicating that male obesity increases sperm DNA damage. Human data are often conflicting because of the large heterogeneity amongst studies, the use of body mass index as the indicator of obesity and the methods used for detection of sperm DNA damage. Furthermore, comorbidities of obesity (i.e., heat stress, adipokines, insulin resistance, changes in lipids, hypogonadism and obstructive sleep apnoea) are also independently associated with increased sperm DNA damage that is not always modified in men with obesity, and as such may provide a causative link to the discrepancies amongst human studies. In this review, we provide an update on the literature regarding the associations between obesity in men and fertility, basic sperm parameters and sperm DNA damage. We further discuss potential reasons for the discrepancies in the literature and outline possible direct and indirect mechanisms of increased sperm DNA damage resulting from obesity. Finally, we summarise intergenerational obesity through the paternal linage and how sperm DNA damage may contribute to the transmission.

Alpha-Lipoic Acid Supplementation for Male Partner of Couples with Recurrent Pregnancy Loss: A Post hoc analysis in Clinical Trial.

Increased sperm DNA damage is known as one of the causes of recurrent pregnancy loss (RPL) which can be due to increased levels of oxidative stress. Therefore, the aim of this study was to assess the effect of alpha-lipoic acid (ALA) on sperm parameters and sperm functions in couples with a history of RPL. In this post hoc analysis in clinical trial study, a total of 37 couples with RPL (n=12 and n=25 for placebo and ALA groups, respectively) were considered. Men were treated with ALA (600 mg/day) or placebo for 80 days. Semen samples were acquired from the participants before initiation and after completion of the medication course and assessed regarding conventional sperm parameters, chromatin damage/integrity, intracellular oxidative stress, lipid peroxidation, and seminal antioxidant characteristics. Individuals were further followed up for twelve months for pregnancy occurrence and outcomes. Finally, after excluding patients with no history of RPL, the data was analyzed. No significant differences were observed between the baseline measures of the aforementioned parameters except for seminal volume. After the intervention, the mean sperm DNA damage, protamine deficiency, and persisted histones were significantly lower in the ALA group than in placebo receivers (P<0.05). A decrease in the mean of seminal total antioxidant capacity (P=0.03), malondialdehyde (P=0.02), and sperm DNA damage (P=0.004) as well as an increase in sperm total motility (P=0.04) after treatment with ALA was noticed. In addition, the mean of protamine deficiency and persisted histones were declined post-ALA therapy (P=0.003 and 0.002, respectively). The percentage of spontaneous pregnancy in the ALA group (4 of 25 cases; 16%) was higher than in the placebo group (1 of 12, 8.3%). ALA-therapy attenuates sperm DNA damage and lipid peroxidation while enhancing sperm total motility and chromatin compaction in the male partner of couples with PRL (registration number: IRCT20190406043177N1).

PMON259 Weight Loss using a Formula Low Energy Diet Reduces Seminal Oxidative Stress in Men with Obesity and Oligospermia: a Randomised Controlled Study

Abstract Background Poor sperm quality (male infertility) is present in 50% of couples requiring assisted reproductive technologies (ART). Few couples worldwide have access to ART, and there are currently no available drugs to improve sperm function. Obesity is a major cause of male infertility. Semen reactive oxygen species (ROS), is a recently identified mechanism for sperm DNA damage resulting in reduced fertilisation and pregnancy outcomes. Semen levels of ROS are positively correlated with body mass index. However, the acute energy restriction following bariatric surgery may paradoxically reduce sperm function, making it an unsuitable treatment for male infertility. No previous randomised controlled study has been performed investigating the effects of weight loss on sperm function and semen ROS in men with obesity and infertility. We hypothesized that milder weight loss through energy restriction could be a novel treatment to reduce semen ROS and improve sperm function in men with obesity and oligospermia. Objective Determine effects of a formula low energy diet (LED) on semen parameters and ROS in men with obesity and oligospermia. Methods Thirty-six men with obesity and oligospermia participated in a 16-week randomised, controlled study. After 2-weeks of baseline observation, all men were randomised to either LED (∼800kcal/day) or a control intervention of generalised healthy eating advice lasting 16-weeks; WHO semen analysis, semen ROS, metabolic parameters and reproductive hormones were monitored throughout the study. Semen ROS was measured in relative light years/sec/million sperm (RLU/s/mill) using an in-house, validated chemiluminescent luminol-based assay. Results At baseline, men with obesity and oligospermia had significantly elevated semen ROS levels when compared with the reference range generated from healthy men with normal BMI (median (inter-quartile range) ROS in RLU/s/mill: &amp;lt;3.8, healthy men; 60.3(113.1), control at baseline; 82.1(100.6) LED). Participants on the LED lost 9-fold more weight compared with control intervention (weight ± SD in kg: -1.5±4.3, control; -14.3±4.5, LED, p&amp;lt;0.0001). After 16 weeks, median semen ROS was reduced 12-fold greater in the LED versus control group (change in ROS vs. baseline in RLU/s/mill: -0.2(61.9), control; -6.0(137.5), LED; p&amp;lt;0.05). Total and progressive motility, and total motile count increased in the LED group versus the control intervention, but these changes were non-significant. Conclusions This is the first randomised controlled study investigating effects of dietary weight loss on spermatogenesis in men with obesity and oligospermia. Our study provides proof-of-concept, and that weight loss could be used therapeutically in men with obesity and infertility, to reduce seminal ROS which is associated with increased sperm DNA damage. These data have important potential implications for management of couples with male infertility. Larger studies are needed to investigate whether weight loss is sufficient to improve pregnancy outcomes in couples affected by male infertility due to obesity. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Relationship between serum vitamin D levels semen parameters and sperm DNA damage in men with unexplained infertility.

The aim of the study was to investigate the relationship between serum level of vitamin D, semen analysis parameters and sperm DNA damage in men with unexplained subfertility. Fifty-eight men diagnosed with unexplained infertility and 50 age and BMI matched fertile men were included in the study. A participant whose semen parameter is normal but pregnancy is not achieved was accepted as unexplained male infertility. Blood samples were taken from all participants following three-day abstinence for measurement of vitamin D. Sperm DNA damage was assessed by Aniline Blue staining of the collected samples. Compared with the fertile men, male patients with unexplained infertility had significantly lower vit D levels (27.00 ng/mL (12.63-39.30) vs. 23.66 ng/mL (7.50-55.00), p<0.004). While the number of patients with vitamin D levels lower than 20 ng/mL was 26 (44.8%) in the infertile group, it was recorded as 5 (10.0%) in the fertile group (p<0.001). DNA damage was found in 31.50% (9.0-71.0) of the infertile men and 26.00% (11.0-54.0) of the fertile men. DNA damage was found to be significantly higher in the unexplained infertile group (p<0.002). In men with unexplained male infertility, serum vit D levels were positively correlated with total sperm count (r = 0.527, p<0.001), total motility (r = 0.527, p<0.001) and sperm morphology (r = 0.416, p = 0.001). There was a negative and significant correlation between vit D levels and sperm DNA damage (r = -0.605, p<0.001). In the logistic regression analysis, serum vit D > 20 ng/mL led to an improvement in fertility outcome. Men with unexplained infertility exhibit decreased serum vit D levels and increased sperm DNA damage.

Genotoxicity of Polystyrene (PS) Microspheres in Short-Term Exposure to Gametes of the Sand Dollar Scaphechinus mirabilis (Agassiz, 1864) (Echinodermata, Echinoidea)

Microplastic pollution appears to be one of the major environmental problems in the world today, and researchers have been paying special attention to the study of the impact of microplastics on biota. In this article, we studied the short-term effects of polystyrene micro-spheres on genome integrity using the gametes of the Scaphechinus mirabilis sand dollar with the comet assay method. This highly sensitive method allowed us to identify the level of genome damage in both gametes before and after short-term exposure to PS microparticles. It was shown that primary polystyrene microspheres at concentrations of 104, 105, and 106 particles/L had a genotoxic effect during short-term exposure to the sperm of the sand dollar S. mirabilis, which was expressed as a significant increase in sperm DNA damage. The highest percentage of DNA damage (more than 20%) was detected in spermatozoa exposed for 1 h in water containing 105 microspheres of plastic per 1 L. Additionally, at all concentrations of microplastic studied in the experiment, the genetic damage index (GDI) values in spermatozoa exceeded the control level. However, regardless of the level of DNA damage, spermatozoa retained the ability to fertilise eggs with up to 97% efficiency. We must acknowledge that the genotoxic property of microplastic against sperm to some extent predicts the development of long-term adverse effects of environmental significance.

Adverse effects of oxytetracycline and enrofloxacin on the fertility of Saanen bucks.

This study aimed to determine the adverse effects of oxytetracycline and enrofloxacin application on the fertility of Saanen bucks. For this purpose, twenty-four bucks were divided into three groups. Group I (control group) received only 5ml of 0.9% NaCl for 7days, group II was given a single dose of 20mg/kg oxytetracycline and group III was given at a dose of 2.5mg/kg per day for 7days intramuscularly. Serum and semen samples were collected from the bucks at post-treatment 1, 3, 5, 7, and 9days and examined spermatological parameters (quantity, motility, density, abnormal sperm ratio, and live-dead sperm ratio), serum testosterone levels (with ELISA) and sperm DNA parameters (with Comet assay). The results showed no change in sperm volume, abnormal sperm rate, and dead-live sperm ratio in group II and III following oxytetracycline and enrofloxacin administration. However, a decrease in sperm density, sperm motility, mass activity, and testosterone levels, and an increase in sperm DNA damage were detected. These spermatological parameters (density, motility, mass activity) and testosterone levels were less decreased and sperm DNA damage was less increased in group II than group III. The greater damage in group III may be attributed to the longer duration of enrofloxacin administration compared to oxytetracycline and the effect of enrofloxacin on DNA. The results obtained from this study suggest that usage of oxytetracycline and especially enrofloxacin should be restricted and antibiotics with fewer side effects on sperm should be preferred in Saanen bucks during the reproduction period.

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.

Chronic Prostatitis/Chronic Pelvic Pain Syndrome Leads to Impaired Semen Parameters, Increased Sperm DNA Fragmentation and Unfavorable Changes of Sperm Protamine mRNA Ratio.

Background: Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a frequent disease affecting men of every age and accounting for a great number of consultations at urology departments. Previous studies suggested a negative impact of CP/CPPS on fertility. As increasing attention has been attributed to additional aspects, such as sperm DNA integrity and sperm protein alterations, besides the WHO standard semen analysis when assessing male fertility, in this prospective study, we aimed to further characterize the fertility status in CP/CPPS patients with a focus on these parameters. Methods: Sperm DNA fragmentation measured by sperm chromatin structure assay (SCSA) and protamine 1 to protamine 2 mRNA ratio assessed by RT-qPCR were analyzed along with conventional ejaculate parameters and inflammatory markers in 41 CP/CPPS patients and 22 healthy volunteers. Results: We found significant differences between the groups concerning multiple conventional ejaculate parameters. A significant increase in sperm DNA fragmentation was shown in CP/CPPS patients with association to other sperm parameters. The majority of CP/CPPS patients exhibited protamine mRNA ratios out of the range of regular fertility. Conclusions: This is a pioneering study with a strong practical orientation revealing that CP/CPPS leads to increased sperm DNA damage and changes in sperm protamine levels, emphasizing an unfavorable impact of CP/CPPS on fertility.

Risk of embryo aneuploidy is affected by the increase in sperm DNA damage in recurrent implantation failure patients under ICSI-CGH array cycles

This study investigates the relationship between sperm DNA damage in recurrent implantation failure (RIF) patients treated with comparative genomic hybridisation array-intracytoplasmic sperm injection (CGH array-ICSI) cycles and embryo aneuploidy screening. Forty-two RIF couples were selected. Sperm DFI was measured using TUNEL by flow cytometry. Two groups were defined as follows: (i) sperm with high DFI (> 20%); and (ii) low DFI (< 20%). Semen parameters, total antioxidant capacity (TAC), and malondialdehyde formation (MDA) were also measured in both groups. Following oocyte retrieval and ICSI procedure, blastomere biopsy was performed at the 4th day of development and evaluated with CGH-array. The high DFI group had a significant (p = 0.04) increase in the number of aneuploid embryos compared to the low one. According to Poisson regression results, the risk of aneuploidy embryos in the high DFI group was 55% higher than the low DFI group (RR = 1.55; 95% CI = 1.358–1.772). Moreover, chromosomal analysis showed an elevation of aneuploidy in chromosomes number 16 and 20 in the high DFI group compared to the low DFI group (p < 0.05). The high DFI in RIF patients may significantly affect the risk of aneuploidy embryos. Therefore, embryo selection by CGH-array should be considered for couples with high levels of sperm DNA fragmentation.

The Prevalence and Components of Metabolic Syndrome in Men from Infertile Couples and Its Relation on Semen Analysis.

PurposeMetabolic syndrome (MetS) has been reported as a deleterious factor in male fertility potential, associated with hypogonadism, impaired spermatogenesis, decreased sperm concentration and motility, and increased sperm DNA damage. This study aimed to determine the prevalence of MetS in men from infertile couples and evaluate its effect on semen analysis (SA).Participants and MethodsA cross-sectional descriptive study was performed in men from infertile couples diagnosed based on the World Health Organization 2010 criteria and treated at the Hue Center for Reproductive Endocrinology and Infertility, Vietnam. General information included medical history, lifestyle, MetS factors, SA, and sperm DNA fragmentation test were collected. Based on the diagnostic criteria of the American Heart Association and the National Heart, Lung, and Blood Institute for Asian men, the study population was divided into two groups: MetS and non-MetS groups. The outcomes were analyzed for any relationship between MetS and the SA index and the DNA fragmentation index (DFI).ResultsA total of 534 men from infertile couples were included in this study. The prevalence of MetS was 23.4%, and abnormal semen analysis accounted for 93.8%. Age, body mass index (BMI), waist-to-hip ratio (WHR), waist-to-height ratio (WHtR), hepatitis B and total cholesterol were related to the occurrence of MetS in infertile men (p <0.05). MetS did not reveal any impact on the parameters of SA. There was a positive correlation between waist circumference (WC), WHR, WHtR, and systolic blood pressure (BP) with abnormal sperm head and DFI (p <0.05).ConclusionAlthough the prevalence of MetS was remarkable in men from infertile couples, there was no association between MetS and semen quality. However, WC, WHR, WHtR, and systolic BP were found to be significantly associated with abnormal sperm head and DFI.

Susceptibility of boar spermatozoa to heat stress using in vivo and in vitro experimental models.

Induction of heat stress as an experimental procedure in animals is commonly used to examine heat-related impacts on sperm quality. This study aimed to develop potential heat stress models that could be used at any time of the year, to advance the study of seasonal infertility in the pig under controlled conditions. Heat stress was induced by either housing boars (n = 6) at 30°C inside a hot room for 42days (55-65% humidity; LD 12:12h; in vivo), or by heating boar semen (n = 7) for 30min at various temperatures (35.5, 38.8, 40, 42, 46, 50, 54 and 60°C; in vitro). Sperm motility was then characterized by computer-assisted sperm analysis (CASA; IVOS version 10: Hamilton Thorne, USA), and DNA integrity was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) and flow cytometry. Our in vivo hot room model induced biologically meaningful levels of DNA damage in boar spermatozoa (10.1 ± 1.9 hot room vs. 6.7 ± 1.7% control; P > 0.05), although not statistically significant from controls. Moreover, sperm concentration and motility parameters did not differ between treatments (P > 0.05). Compared to the 38.8°C control, our in vitro heat shock model significantly increased sperm DNA damage after incubation at 54 and 60°C (3.0 ± 1.0, 2.9 ± 1.0, 1.2 ± 0.3, 2.5 ± 0.7, 9.0 ± 3.7, 16.2 ± 7.1, 14.2 ± 5.8 and 41.8 ± 18.6% respectively; P ≤ 0.05). However, these temperatures rendered sperm completely immotile or dead, with most motility parameters declining rapidly to zero above 40 or 42°C. In conclusion, our results suggest that temperature combined with individual factors may contribute to a boar's overall susceptibility to heat stress. Refinement of these models particularly of the in vitro heat shock model could be further pursued to overcome environmental variability, reduce whole animal experiments and provide a putative diagnostic fertility screening tool to evaluate heat tolerance in the boar.

Sperm source does not affect the ICSI outcome of patients with severely compromised spermatogenesis.

Patients with spermatogenic dysfunction may display sperm parameters ranging from extremely severe oligozoospermia (sperm count lower than 2 million/ml) to azoospermia. It has been proposed that, since these patients may have increased sperm DNA damage that could affect their ICSI outcome, the use of surgically retrieved testicular spermatozoa should be preferred to improve their chance of fathering their biological offspring. However, studies in this field have yielded conflicting results. The present study provides an updated assessment of this subject by comparing the ICSI outcome of 762 patients with nonobstructive azoospermia and 419 with sperm count lower than 2 million/ml (median sperm count 300,000/ml). Both groups were homogeneous for the number of retrieved and injected MII oocytes. No difference was seen in terms of fertilisation, clinical pregnancy and cumulative live birth rates. Only the number of injected MII oocytes was found to independently predict the live birth rate, even when adjusted for the number of transferred embryos (OR 1.10 (1.0-1.2, p=0.038)). The results of the present study stand against the use of testicular spermatozoa in patients with extremely severe spermatogenic dysfunction with available spermatozoa in their ejaculate.

A novel pathway for the induction of DNA damage in human spermatozoa involving extracellular cell-free DNA

DNA damage is a common feature of human spermatozoa associated with an impaired capacity to fertilize the oocyte and an increased mutational load in the offspring. However, the etiology of this damage remains poorly defined. In this study we demonstrate that a major pathway for the induction of DNA damage in mammalian spermatozoa is triggered by exposure to exogenous cell free DNA (cfDNA). Exposure of human and mouse spermatozoa to cfDNA (calf thymus, mouse liver and salmon testes) in vitro induced a dose-dependent increase in sperm DNA damage that could be effectively suppressed by the concomitant presence of DNase. The induction of such damage was not accompanied by any concomitant change in sperm motility or vitality and was not directly associated with the induction of oxidative stress. In vivo the injection of exogenous DNA again precipitated an increase in sperm DNA fragmentation that could be reversed by the prior administration of DNase. Similarly, the induction of a transient unilateral testicular ischemia induced an increase in DNA fragmentation that was evident within 24 h and sustained for at least 14 days via mechanisms that could be completely suppressed by the prior administration of DNase. We conclude that exogenous cfDNA activates a defensive response in human spermatozoa associated with the nuclease-mediated induction of DNA fragmentation, possibly involving the participation of TLR9 and CD4. These novel insights have significant implications for our understanding of DNA fragmentation in the male germ line and open up new pathways for the remediation of this condition.

Evaluation of Sperm DNA Fragmentation via Halosperm Technique and TUNEL Assay Before and After Cryopreservation.

Human sperm DNA fragmentation is one of the factors suggested for male infertility. The ratio of sperm DNA damage in semen may adversely affect both the fertilization rate and the embryo development of in vitro fertilization/ intracytoplasmic sperm injection cycles. Sperm cryopreservation both increases the success rates in assisted reproductive techniques (ARTs) and contributes to the preservation of fertility before testis surgery, chemotherapy, and radiotherapy. The aim of the current study is to determine sperm DNA fragmentation, following cryopreservation. A cross-sectional, observational study was conducted at a university hospital infertility clinic. One hundred (n = 100) volunteer fertile men (ages between 21 and 39 years) with normozoospermic sperm parameters were involved in the current study. Sperm DNA damage was evaluated with the Halosperm technique and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Fresh samples were studied in liquid form. The remaining samples were kept frozen and then thawed after 1 month and reevaluated with the Halosperm technique and TUNEL assay. Results were then compared between the fresh and frozen samples. Sperm DNA fragmentation results with the Halosperm technique both before and after cryopreservation were 25% (5%-65%) and 40% (6%-89%), respectively, with a statistically significant increase (15%; P < .001). Sperm DNA fragmentation results by TUNEL assay before and after cryopreservation were 17% (3%-43%) and 36% (7%-94%), respectively, with a statistically significant increase (19%; P <.001). The current data demonstrate increased sperm DNA damage after cryopreservation. Further studies may contribute to development of less harmful techniques and cryoprotectants in order to improve the results of ART.

Detection of chlamydia infection within human testicular biopsies.

STUDY QUESTIONCan Chlamydia be found in the testes of infertile men?SUMMARY ANSWERChlamydia can be found in 16.7% of fresh testicular biopsies and 45.3% of fixed testicular biopsies taken from a selection of infertile men.WHAT IS KNOWN ALREADYMale chlamydial infection has been understudied despite male and female infections occurring at similar rates. This is particularly true of asymptomatic infections, which occur in 50% of cases. Chlamydial infection has also been associated with increased sperm DNA damage and reduced male fertility.STUDY DESIGN, SIZE, DURATIONWe collected diagnostic (fixed, n = 100) and therapeutic (fresh, n = 18) human testicular biopsies during sperm recovery procedures from moderately to severely infertile men in a cross-sectional approach to sampling.PARTICIPANTS/MATERIALS, SETTING, METHODSThe diagnostic and therapeutic biopsies were tested for Chlamydia-specific DNA and protein, using real-time PCR and immunohistochemical approaches, respectively. Serum samples matched to the fresh biopsies were also assayed for the presence of Chlamydia-specific antibodies using immunoblotting techniques.MAIN RESULTS AND THE ROLE OF CHANCEChlamydial major outer membrane protein was detected in fixed biopsies at a rate of 45.3%. This was confirmed by detection of chlamydial DNA and TC0500 protein (replication marker). C. trachomatis DNA was detected in fresh biopsies at a rate of 16.7%, and the sera from each of these three positive patients contained C. trachomatis-specific antibodies. Overall, C. trachomatis-specific antibodies were detected in 72.2% of the serum samples from the patients providing fresh biopsies, although none of the patients were symptomatic nor had they reported a previous sexually transmitted infection diagnosis including Chlamydia.LIMITATIONS, REASONS FOR CAUTIONNo reproductively healthy male testicular biopsies were tested for the presence of Chlamydia DNA or proteins or Chlamydia-specific antibodies due to the unavailability of these samples.WIDER IMPLICATIONS FOR THE FINDINGSApplication of Chlamydia-specific PCR and immunohistochemistry in this human male infertility context of testicular biopsies reveals evidence of a high prevalence of previously unrecognised infection, which may potentially have a pathogenic role in spermatogenic failure.STUDY FUNDING/COMPETING INTEREST(S)Funding for this project was provided by the Australian NHMRC under project grant number APP1062198. We also acknowledge assistance from the Monash IVF Group and Queensland Fertility Group in the collection of fresh biopsies, and the Monash Health and co-author McLachlan (declared equity interest) in retrieval and sectioning of fixed biopsies. E.M. declares an equity interest in the study due to financing of fixed biopsy sectioning. All other authors declare no conflicts of interest.TRIAL REGISTRATION NUMBERN/A