High Levels Of DNA Fragmentation Research Articles

Reduced SIRT1 and SIRT3 and Lower Antioxidant Capacity of Seminal Plasma Is Associated with Shorter Sperm Telomere Length in Oligospermic Men.

Infertility affects millions of couples worldwide and has a profound impact not only on their families, but also on communities. Telomere attrition has been associated with infertility, DNA damage and fragmentation. Oxidative stress has been shown to affect sperm DNA integrity and telomere length. Sirtuins such as SIRT1 and SIRT3 are involved in aging and oxidative stress response. The aim of the present study is to determine the role of SIRT1 and SIRT3 in regulating oxidative stress, telomere shortening, and their association with oligospermia. Therefore, we assessed the protein levels of SIRT1 and SIRT3, total antioxidant capacity (TAC), superoxide dismutase (SOD), malondialdehyde (MDA) and catalase activity (CAT) in the seminal plasma of 272 patients with oligospermia and 251 fertile men. We also measured sperm telomere length (STL) and leukocyte telomere length (LTL) using a standard real-time quantitative PCR assay. Sperm chromatin and protamine deficiency were also measured as per standard methods. Our results for oligospermic patients demonstrate significant reductions in semen parameters, shorter STL and LTL, lower levels of SOD, TAC, CAT, SIRT1 and SIRT3 levels, and also significant protamine deficiency and higher levels of MDA and DNA fragmentation. We conclude that a shorter TL in sperms and leukocytes is associated with increased oxidative stress that also accounts for high levels of DNA fragmentation in sperms. Our results support the hypothesis that various sperm parameters in the state of oligospermia are associated with or caused by reduced levels of SIRT1 and SIRT3 proteins.

Separating the chaff from the wheat: antibody-based removal of DNA-fragmented sperm.

Is it possible to remove sperm with damaged DNA from a semen sample? By using immunomagnetic cell sorting that targets the sperm head-bound epididymal sperm-binding protein 1 (ELSPBP1), it was possible to produce an ELSPBP1(-) sperm fraction characterized by consistently lower levels of sperm DNA fragmentation (SDF). In bovines, ELSPBP1 is bound to dead spermatozoa. Human ejaculates with high SDF have increased detected levels of sperm ELSPBP1 when compared to ejaculates with low native SDF. We recruited 267 patients who were referred to the clinic for conjugal infertility. After applying exclusion criteria, such as fever within 90 days of the study, history of systemic diseases, alterations or surgical interventions to the genital tract and use of cigarette or drugs, a total of 133 patients were included. A total of 52 samples were used for the evaluation of sperm ELSPBP1 levels (Sub-study 1), 41 samples for determination of ELSPBP1 location in human sperm (Sub-study 2), and 40 samples for immunomagnetic cell sorting targeting ELSPBP1, to produce ELSPBP1(-) (without ELSPBP1) and ELSPBP1(+) (with ELSPBP1) fractions (Sub-study 3). Samples were collected between July 2016 and September 2019. In Sub-study 1, sperm ELSPBP1 levels were assessed by western blotting. For Sub-study 2, ELSPBP1 was localized in sperm by immunocytochemistry. Finally, for Sub-study 3, sperm were selected based on incubation of semen samples with antibody-coated magnetic microspheres targeting ELSPBP1. Two fractions were produced (with or without ELSPBP1), and these sub-populations were submitted to an alkaline Comet assay for determination of SDF. Men with high SDF presented higher sperm ELSPBP1 levels when compared to the control group (low SDF), while no difference between groups was observed in seminal plasma. ELSPBP1 was located in the head region of human sperm. The ELSPBP1(+) fractions presented high and variable levels of SDF, while their paired ELSPBP(-) fractions presented consistently low SDF. This work did not validate the levels of ELSPBP1 in other functional alterations of sperm, such as acrosome integrity or mitochondrial activity. Moreover, this is still a pre-clinical study, intended to demonstrate proof-of-concept that ELSPBP1 selects sperm with low DNA fragmentation; further investigation is warranted to demonstrate safety for use in ART. Sperm fractions were not assessed for sperm vitality. A clinical trial is still necessary for these findings to be extrapolated to outcomes in ART. Our findings demonstrate that ELSPBP1 is associated with sperm with higher levels of DNA fragmentation. The finding that the sperm membrane can reflect alterations in DNA integrity could give rise to a novel molecular method for sperm preparation prior to use of assisted reproductive procedures. Moreover, the detection of sperm-bound ELSPBP1 could serve as an indirect method for the determination of DNA fragmentation. L.B.B. was a recipient of a Ph.D. scholarship from the Sao Paulo Research Foundation-FAPESP (process number 2016/05487-3). R.P.B. is a recipient of a Scientific Productivity scholarship from the Brazilian National Council for Scientific and Technological Development-CNPq (process number 306705/2017-6). The authors have no conflict of interest to disclose. N/A.

Influence of Non-Thermal Plasma (DBD) On Infertility Male Semen with Low Sperm Motility and Dna Damage

Non-thermal plasma(Dielectric barrier discharge) has many uses including living tissue sterilization, inactivation of the bacteria, excimer formation, angiogenesis, and surface treatment. This research aim is to use cold plasma as a tool to search the effect of the dielectric barrier discharge system at room temperature on human sperm motility and DNA integrity. This work was performed on 60 human semen samples suffering from low motility; each sample was prepared by centrifugation method, then each semen sample was divided into two sections, the first section is before significant exposure to the plasma system (DBD) and the second section is after treatment with the DBD system at ambient temperature. Before and after exposure to non-thermal Plasma (DBD), DNA integrity and sperm motility were assessed, patients suffered from asthenospermia has a high level of DNA fragmentation than fertile male, ( 24.16±4.14) p<0.001 for untreated and after treatment the semen slide with dielectric barrier discharge the percentage decreases to (9.16±1.76) p>0.006, and the comet slide was (35.44±4.15) then the percentage decline to (19.86 ±2.44) these results have shown that cold plasma improves sperm motility and decreases from DNA damage in patients with medium and high level of DNA damage.

Effect of season on germinal vesicle stage, quality, and subsequent in vitro developmental competence in bovine cumulus-oocyte complexes.

Although the reproductive performance of grazing cattle is lower in summer compared to winter, the effect of season on oocyte developmental competence has not been thoroughly examined. We measured the effect of season on oocyte chromatin compaction, cumulus cell quality, and embryonic development after in vitro fertilization. Cumulus oocytes-complexes (COCs) were collected from abattoir cows' ovaries during the winter and summer months. First, we evaluated the degree of chromatin compaction in germinal vesicle (GV) oocytes (GV1 through GV3), which is associated with different degrees of developmental competence. Then, we determined the apoptotic index in cumulus cells from immature and in vitro matured COCs. Finally, in vitro matured oocytes were fertilized to determine blastocyst rate and embryo quality. During the summer months, we observed a significantly lower proportion of oocytes reaching the GV3 stage and higher levels of DNA fragmentation in cumulus cell. As a result, blastocyst yield and quality were reduced during the summer months. In conclusion, summer negatively affected oocyte GV stage progression, cumulus cell quality, and embryo development. Increased cumulus cell DNA fragmentation during summer, may partially explain the reduced oocyte maturation capacity, considering the relevance of cumulus-oocyte communication during this stage.

DNA fragmentation and membrane integrity in sperm of farmed Arctic charr (Salvelinus alpinus)

Despite the relatively long history of captive breeding, the Arctic charr still exhibits a generally low, but highly variable reproductive performance in aquaculture. A recent publication exposed potential paternal factors influencing the reproductive outcome of Arctic charr broodstock from the Swedish breeding program. Interestingly, the paternal factor appeared to be more closely connected to embryo survival than to fertilisation rates. This lead to speculations on whether e.g. chromatin related issues, potentially related to oxidative stress could be involved. In order to investigate this hypothesis the present study assessed the levels of DNA fragmentation, using the SCD-method, and membrane integrity, using flow cytometry, in sperm of farmed Arctic charr. Moreover, the existence of associations was tested between DNA fragmentation and membrane integrity in individual semen samples and viability of their resulting progeny. We found high levels of DNA fragmentation in sperm from the Arctic charr sires, ranging from 24% to 86% with a median of 67%. Membrane integrity values were high, with individual levels of 93.1% to 99.6% viable sperm cells, median 98.8%. DNA fragmentation and membrane integrity values were moderately correlated (r = 0.304, p < 0.05). Fertilisation rates and proportions of eyed eggs showed substantial individual variation and were correlated (r = 0.497, p < 0.05). However, large differences between proportion of eyed eggs and fertilisation rate, median 52% and 81.6% respectively, highlight that the main loss occurred due to embryo mortality rather than failed fertilisation. No correlation was found between either DNA fragmentation or membrane integrity and the resulting reproductive outcome (fertilisation rate and eyed eggs) of the individual Arctic charr sires. Overall, our study identified very high levels of DNA fragmentation, which could influence the fertility of the broodstock in question and thereby be a mitigating mechanism involved in the low reproductive success most often observed in farmed Arctic charr. Further exploration of this relationship would be needed, though.

P–086 High level of sperm DNA breaks in infertile men with varicocele: its association with sperm cells death, seminal oxidative stress, and spermatic parameters

Abstract Study question Our objectives were to determine the extent of nuclear sperm injury in varicocele patients with and without altered spermatic parameters and to investigate its relationship with apoptosis and oxidative stress. Summary answer Oxidative stress (OS) in the varicocele patients may play a role in the etiology of nuclear sperm DNA damage associated with apoptosis. What is known already Varicocele is associated with high level of DNA Breaks. Study design, size, duration Ejaculated sperm samples from 51 patients diagnosed with varicocele and 29 fertile men were examined. According to the guidelines, the patient’s sperm samples were classified into varicocele with normal semen parameters (n = 11) and varicocele with abnormal semen parameters (n = 40). Participants/materials, setting, methods Sperm DNA breaks was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The proportion of both viable and dead spermatozoa with externalized phosphatidylserine was detected by the bivariate annexin V cy3/6-CFDA staining method. Seminal malondialdehyde (MDA) amounts and antioxidant enzymes activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured spectrophotometrically. Main results and the role of chance Sperm DNA Breaks, viable spermatozoa with externalized PS, and MDA levels were significantly higher in studied subgroups of patients with varicocele, either with normal or with abnormal semen parameters than controls. The seminal antioxidant enzymes activities were significantly reduced in both subgroups of patients with varicocele compared to the controls. The percentage of spermatozoa with fragmented DNA was positively correlated to the MDA level as well as the proportion of viable spermatozoa with externalized PS. However, the decreased seminal antioxidant status was negatively correlated with the increased proportion of sperm DNA fragmentation and apoptotic spermatozoa. Limitations, reasons for caution We suggest further comparative studies connecting the varicocele patients with and without altered spermatic parameters representing high level of DNA fragmentation with more apoptotic and oxidative stress markers. Wider implications of the findings: This study reveals that impaired seminal antioxidant profile and increased seminal level of lipid peroxidation may be involved in the pathophysiological mechanisms of cell death-mediated DNA breaks in patients with varicocele. Trial registration number Not applicable

P–043 Six years’ retrospective statistical study (2013 – 2018) investigating the impact of Sperm DNA fragmentation on sperm analysis parameters

Abstract Study question What is the relationship between Sperm DNA fragmentation (SDF) levels and sperm analysis (Spermocytogramme) parameters results? Summary answer SDF level of patients with pathological spermocytogramme presents negative correlations to total spermatozoa mobility, vitality and concentration, and positive correlation to sperm morphology defects. What is known already The relationship between SDF and Sperm analysis parameters and especially sperm morphology needs to be more studied since few studies over the last years were focused on this relationship. However, abnormalities in these two parameters are considered as the most important biological indicators of male infertility. The pathogenesis of Teratozoospermia (&amp;lt;4% morphologically normal sperm cells according to WHO 2010) is continuously increasing over the last decade according to several studies. In addition, SDF is also increasing over the years because of several factors such as pollution, stress and lifestyle changing. Study design, size, duration Retrospective study including 331 infertile patients undergoing SDF-index testing with Spermocytogramme from January 2013 – December 2018. Patients divided into two groups: 143 patients with normal-Spermocytogramme and 188 patients with pathological-Spermocytogramme. Each group includes patients with abnormal SDF levels (&amp;gt;30%). Statistical analyzes were performed using SPSS22.0 for Windows-software. Kolmogorov–Smirnov-test for normality analysis and comparisons by Student-t-test or Mann–Whitney U-test, as appropriate. Pearson/Spearman’ tests for correlations were used as appropriate, P-value&amp;lt;0.05 was considered as significant. Participants/materials, setting, methods 143 patients with normal Spermocytogramme (2.8% abnormal-SDF) vs 188 patients with pathological Spermocytogramme (10.6% abnormal-SDF). WHO–2010 instructions for sperm-analysis were used through Makler®-counting-chamber (Sefi-Medical Instrument Ltd) for sperm-concentration and motility-determination using Sperm-class-analyzer-software (CASA-system (Microptic®)) to detect sperm abnormalities. Normozoospermia was determined when sperm progressive-motility is ≥ 32%, sperm-concentration ≥15x106/mL, and sperm-morphology ≥4%. “Diff-Quick” staining-method for the coloration of the fixed-sperm-slides was used for Sperm-morphology analysis. GoldCyto Sperm®Kit (Goldcyto Biotech corp.) was used to analyze SDF. Main results and the role of chance SDF is significantly higher in pathological spermocytogramme’ patients than in normal spermocytogramme’ patients (17.02 ± 11.88 vs 12.16 ± 9.58 respectively). In patients with pathological spermocytogramme, SDF is negatively correlated to Progressive sperm motility (r= –0.137; p = 0.042), Total sperm motility (r= –0.153; p = 0.036), vitality (r=–0.140; p = 0.048) and concentration (r=–0.195; p = 0.007). In the other hand, SDF presented positive correlation with teratozoospermia and especially with sperm midpiece defects (r = 0.171; p = 0.02). However, SDF did not present any correlation with age, testosterone levels and total ejaculated sperm volume. However the latter was positively correlated to spermatozoa midpiece and head defects (r = 0.156; p = 0.034; r = 0.203; p = 0.006, respectively). These results are in accordance with García-Ferreyra et al. (2014) who found that men with abnormal spermatozoa morphology showed high levels of DNA fragmentation, Sá et al. (2015) who confirmed that semen with lower concentration, motility and morphology have higher levels of SDF and showed that sperm head staining patterns are correlated with the degree of SDF. In addition, recently the study of Jakubik-Uljaszstudy et al. (2020) could confirms our results when it concluded that detailed sperm structural defects coexist with abnormal nuclear sperm DNA dispersion and that men with teratozoospermia may have a higher risk for sperm DNA damage. Limitations, reasons for caution Our study is a retrospective statistical investigation that included patients attending to the laboratory for fertility diagnosis after a period of infertility. Meta-analyzes studies in addition to more prospective-randomized-controlled-trials with couples undergoing assisted-reproductive-treatments and in comparison with fertile men are needed to confirm the relationship between SDF and spermocytogramme defects. Wider implications of the findings: These results should interest andrologists, reproductive science fundamentalists and embryologists who want to improve the investigations on the origin of infertility especially when it comes from male side. Trial registration number Not applicable

Impact of metabolic syndrome on sex hormones and reproductive function: a meta-analysis of 2923 cases and 14062 controls.

Current evidence is inconsistent regarding the impact of metabolic syndrome (MetS) on sex hormones and reproductive function, and this meta-analysis aimed to illuminate the association. A literature search was conducted in public databases to identify all relevant studies, and study-specific standardized mean differences (SMD) and 95% confidence intervals (CI) were pooled using a random-effects model. Finally, 21 studies were identified with a total of 2923 MetS cases and 14062 controls. In males, MetS cases had a lower level of testosterone, inhibin B, total sperm count, sperm concentration, sperm normal morphology, sperm total motility, sperm progressive motility and sperm vitality, and a higher level of DNA fragmentation and mitochondrial membrane potential. In females, MetS cases had a higher level of testosterone. No significant difference was detected for follicle-stimulating hormone, luteinizing hormone, oestradiol, prolactin, anti-Müllerian hormone and semen volume in males, and for oestradiol, follicle-stimulating hormone, luteinizing hormone and progesterone in females. In conclusion, this meta-analysis indicated the impact of MetS on sex hormones and reproductive function, and MetS cases had a potential risk of infertility.

Differential nitric oxide induced by Mycobacterium bovis and BCG leading to dendritic cells apoptosis in a caspase dependent manner

Dendritic cells (DCs) are critical for both innate and adaptive immunity. Meanwhile, nitric oxide (NO) is a member of reactive nitrogen species (RNS) generally considered to play a key role in the bactericidal process in innate immunity against Mycobacterium tuberculosis complex infection. The present study therefore investigated the mechanism of NO production in murine DCs induced by Mycobacterium bovis (M.bovis) and its attenuated strain Bacillus Calmette-Guérin (BCG) infection. The expression of genes Slc7A1, Slc7A2, iNOS, and ArgI essential to NO synthesis was up-regulated in M.bovis/BCG infected DCs. IFN-γ addition further increased, while the iNOS inhibitor L-NMMA significantly inhibited their expression. Accordingly, the end products of arginine metabolism, NO and urea, were found to be significantly increased. In addition, BCG induced significantly higher levels of apoptosis in DCs compared to M.bovis shown by higher levels of DNA fragmentation using flow cytometry and release of mitochondrial Cytochrome C, and up-regulation of the genes caspase-3, caspase-8, caspase-9 and dffa critical to apoptosis by qRT-PCR detection and western blot analysis. Furthermore, IFN-γ increased, but L-NMMA decreased apoptosis of M.bovis/BCG infected DCs. In addition, mycobacterial intracellular survival was significantly reduced by IFN-γ treatment in BCG infected DCs, while slightly increased by L-NMMA treatment. Taken altogether, our data show that NO synthesis was differentially increased and associated with apoptosis in M.bovis/BCG infected DCs. These findings may significantly contribute to elucidate the pathogenesis of M.bovis.

Features of structure and dynamics in damages of fertile properties in ejaculate under the influence of toxocariasis invasion

Purpose of the study. To study the features of changes of spermiologic values and degree of spermatozoa DNA fragmentation depending on the presence/absence of toxocariasis invasion.&#x0D; Materials and methods. 89 men aged 20 to 45 years were examined, which were divided into 5 groups. The first (control) group was 12 fertile men; the second group (comparison group) – 27 infertility patients with normal level of DNA fragmentation of sperm and without antibodies to toxocariasis; The third group – 20 infertility men with normal level of DNA fragmentation of sperm and presence of antibodies to toxocariasis. By the fourth and fifth groups included 15 infertility men with high levels of DNA fragmentation of sperm and the presence of antibodies to toxocariasis and lack of respectively. A comprehensive research was conducted for all men that included analysis of spermogram according to WHO recommendations, determination of sperm DNA fragmentation and presence of toxocariasis invasion.&#x0D; Results. As a result of spermograms in the studied groups of men were identified the violations of ejaculate fertility (asteno-, teratoand oligozoospermia). It has been established that the degree disturbance of spermatogenesis (including significant oligoand teratozoospermia) is depending on the fragmentation of DNA in male gametes and the presence of antibodies to toxocara. At the same time more severe patozoospermia observed in the presence of elevated levels of fragmented sperms and the absence of antibodies to toxocara antigens, which is the basis for further research.&#x0D; Conclusions. Thus, the determination of sperm DNA fragmentation and availability of toxocariasis invasion in men with reproductive disorders is a necessary component of a comprehensive examination, which may allow to improve the diagnostics of male infertility and to promote the development of the right tactics and optimal treatment regimens.

The Male Is Significantly Implicated as the Cause of Unexplained Infertility.

Male infertility is recognized as a relatively common, complex condition, generated by a broad array of environmental and genetic factors. Historical reliance on the conventional semen profile has tended to underestimate the true contribution of "the male factor" to human infertility. This review highlights the importance of genetic and epigenetic factors in the etiology of male infertility, identifying a range of mutations responsible for primary testicular failure and impaired fertilizing potential. More than three quarters of all de novo mutations arise in the male germline via mechanisms that involve the inefficient or defective repair of DNA damage. Understanding the range of factors capable of creating genetic turmoil in the paternal germline is essential, if we are to gain a deep understanding of the causes of male infertility, rather than just the symptoms that characterize its presence. High levels of DNA fragmentation induced by oxidative stress are part of this equation. Oxidative stress is, in turn, driven by biological (age, ejaculation frequency, varicocele, infection), lifestyle (smoking, obesity), and environmental factors (heat, other forms of electromagnetic radiation, and toxins) that can impair the fertilizing potential of the spermatozoa and influence the incidence of spontaneous mutations that may cause infertility in the offspring.

In vitro assessment of murine melanoma cells sensitivity to non-thermal atmospheric plasma

Melanoma is a dangerous skin cancer incidence of which has been increasing over the past years, so is important the search for new treatments. The purpose of the present work is to evaluate the relative sensitivity of a melanoma cell line to helium-generated non-thermal plasma. For that, three cell types were used (murine melanoma B16 cells, mouse embryo fibroblasts, and peripherical blood lymphocytes) and both cytotoxicity and genotoxicity were evaluated. The lethality produced by no-thermal plasma was higher in melanoma mouse cells compared with lymphocytes and fibroblasts. Accordingly, B16 cells showed higher levels of DNA fragmentation by this agent. Overall, the results suggest that non-thermal plasma has the potential to become a good alternative for treating skin cancer.

Incidence of high sperm DNA fragmentation in a targeted population of subfertile men

ABSTRACTSperm DNA integrity is important for fertility, however the incidence of high levels of DNA fragmentation (DNA fragmentation index (DFI) >30%) is not well described. In 2011, our clinics implemented guidelines for sperm DNA fragmentation (SDF) testing based on risk factors using the sperm chromatin structure assay (SCSA). The aim of this retrospective study is to characterise SDF and associated factors (age, semen parameters, smoking status and BMI) for sub-fertile males (n = 1082) and sperm donors (n = 234). The average DFI was 12.1 ± 9.8%. The distribution of men with low, moderate and high SDF (<15, 15–30 and >30%) was 74.8%, 19.4% and 5.8%, respectively. Men with high DFI were older (45 ± 9.5 vs 38 ± 6.7) and had lower percentage of motile sperm (38.8 ± 16.1% vs 55.3 ± 15.8%) than men with normal DFI. Over 17% of the men in the quartile with the highest age and lowest motility had a high DFI (>30%), compared to a high DFI rate of 2-4% for the other 3 quartiles. Repeat testing following lifestyle interventions was available for 29 couples where the men had high initial DFI (35 ± 9.5%). Of these men, 71.4% had a decrease of DFI into the moderate or low range. This study shows that SDF testing can be targeted based on age and sperm motility, thereby reducing unnecessary testing. Furthermore, we provide evidence that lifestyle modifications can reduce DNA fragmentation in men with high DFI.

Effect of medical comorbidities over seminal parameters and sperm DNA fragmentation

Several publications have been shown that it could be a relationship between male infertility and general health status. The aims of this study were to investigate the prevalence and effect of some medical comorbidities over sperm parameters and DNA fragmentation in an Argentinian population. Retrospective controlled cohort study. Under the approval of the institutional ethics committee, a retrospective study was performed for 1,092 men who were examined due to infertility between August 2017 and April 2019. The initial evaluations were comprised of a complete medical history, a physical examination, endocrine assessment, and at least two semen analyses. Sperm parameters and DNA fragmentation were compared between men with and without medical comorbidities. Significant medical comorbidities were found in 112 of 1092 (10.3%) men, including 3.6% with hypertension, 2,3% with hypothyroidism, 2% with mental, 2% with diabetes/dyslipemia and 0.6% with respiratory disease. Semen volume, sperm count and progressive motility were significantly lower in men with comorbidities that in men without comorbidities (p=0.045, p=0.036 and p=0.025, respectively). Regarding sperm DNA fragmentation, it was higher in patients with comorbidities (p=0.018). Sperm vitality and strict morphology were not significantly different. Within patients with comorbidities, patients with diabetes/dyslipemia and anxiety disorders presented significantly higher levels of DNA fragmentation (p=0.001). After this preliminary study, we can conclude that medical comorbidities are associated with the impairment of sperm production and function. It has been published that obesity and metabolic disorders could be associated with impair sperm function by altering physical and molecular structure of germ cells. A complete male infertility evaluation, including an exhaustive anamnesis, could offer the possibility of specific therapy in order to improve some semen parameters. We didn’t assess this theoretical benefit, however it would be very interesting to evaluate if that therapy, despite the improvement of the general health status, could improve the spermatogenesis.

Condition of a cellular link of innate immunity in infertile men against the background of toxocariasis invasion

Aim. Investigating the role of various factors in the development of pathospermia, such as: the presence of toxocariasis invasion, the level of DNA fragmentation of sperm, indices of the cellular link of innate immunity, could complement existing methods and will allow the determination of the share of influence of each factor on the ultrastructure of spermatozoa for a more in-depth understanding of the pathogenesis of violations of male reproductive function. That is why the aim of our research was to study the peculiarities of disorders of the cellular link of innate immunity in men with infertility against the background of toxocariasis invasion. Materials and methods. 89 men aged from 20 to 45 years, which were divided into 5 groups, were examined. The first (control) group consisted of 12 fertile men; the second group (comparison group) – 27 infertile patients with normal level of DNA fragmentation of sperm and without antibodies to toxocariasis; the third group – 20 infertile men with normal level of DNA fragmentation of sperm and presence of antibodies to toxocariasis. The fourth and fifth groups included 15 infertile men with high levels of DNA fragmentation of sperm and the presence and absence of antibodies to toxocariasis respectively. A comprehensive research was conducted for all the men that included determination of sperm DNA fragmentation, presence of toxocariasis invasion and the study of the state of the cellular link of innate immunity as well as assessment of the degree of immune system disorders. Results. The incompleteness of phagocytosis in neutrophilic and monocytic elements was observed in all studied groups, in the second and fifth groups with the preservation of the functional-metabolic reserve and the depletion of it in the third and fourth groups. The results of the assessment of the degree of immune system disorders have shown that most immunologically compromised are the groups without the presence of toxocariasis invasion, in which there was an immune failure of 2–3 degree. In groups with the presence of toxocariasis, on the contrary, changes from activation to immune failure of 2 degree (due to the growth of absorbing capacity of monocytes) were determined, which may be due to the presence of toxocariasis invasion, which, in turn, obviously causes the activation of the monocytic link. Conclusions. The state of the cellular link of innate immunity in infertile men is characterized by incompleteness of phagocytosis of both neutrophilic and monocytic elements. The presence of toxocariasis invasion in this contingent of patients, in turn, introduces certain changes to the phagocytic system, which manifests itself as an increase in absorbing capacity of monocytes, which are key immune factors in the protection against helminths.

Influence of DNA fragmentation in the sperm on the development of embryos in vitro fertilization program

The effect of the level of DNA fragmentation in the sperm on the development of early embryos in IVF programs for men with low fertility was investigated. A significant negative association between the level of DNA fragmentation in the sperm and the total blastocyst formation rates and a percent of blastocysts with high quality morphology in patients with impaired fertility is proved (rs = -0,59; rs= -0,56). Evaluation of semen quality in men with low fertility is a necessary step before the use of assisted reproductive technologies. The connection between the levels of sperm DNA fragmentation and the peculiarities of the early development of embryos obtained in programs of extracorporal fertilization has been proved. Assessing the quality of sperm in men with reduced fertility is a necessary measure before using auxiliary reproductive technologies. According to literature, the morphology of the blastocyst does not always depend on chromosomal changes in the embryos. Consequently, it is promising to carry out pre-implantation genetic diagnostics of embryos obtained in programs of extracorporal fertilization in patients with high levels of DNA fragmentation in spermatozoa to study the effect of this violation on the karyotype of embryos.

Re: Reproductive Outcomes of Testicular Versus Ejaculated Sperm for Intracytoplasmic Sperm Injection Among Men with High Levels of DNA Fragmentation in Semen: Systematic Review and Meta-Analysis

Re: Reproductive Outcomes of Testicular Versus Ejaculated Sperm for Intracytoplasmic Sperm Injection Among Men with High Levels of DNA Fragmentation in Semen: Systematic Review and Meta-Analysis

DNA Damage Observed in Unaffected Individuals with Family History of T2DM

Diabetes has been documented to cause high levels of DNA fragmentation in some cases. As diabetes is inheritable and influenced by both genetic and environmental factors, an investigation into the genomic stability of individuals who are strongly at risk of inheriting diabetes was conducted by inducing oxidative stress, as DNA damage in unaffected individuals could be a sign of onset of the disease or the presence of genetic alterations that reduce cellular defences against reactive oxygen species. In this study, alkaline comet assay was performed on isolated human leukocytes to determine whether individuals with a family history of Type 2 Diabetes Mellitus (T2DM) are more prone to DNA damage under oxidative stress. Visual scoring of comets showed that these individuals have higher degree of DNA damage compared to a control individual with no family history of Type 2 Diabetes Mellitus. Further studies with large sample could determine the presence of disabled cellular defences against oxidative stress in unaffected individuals and intervention with antioxidants could prevent or manage Type 2 Diabetes Mellitus and its complications.

Measuring the response of human head and neck squamous cell carcinoma to irradiation in a microfluidic model allowing customized therapy.

Radiotherapy is the standard treatment for head and neck squamous cell carcinoma (HNSCC), however, radioresistance remains a major clinical problem despite significant improvements in treatment protocols. Therapeutic outcome could potentially be improved if a patient's tumour response to irradiation could be predicted ex vivo before clinical application. The present study employed a bespoke microfluidic device to maintain HNSCC tissue whilst subjecting it to external beam irradiation and measured the responses using a panel of cell death and proliferation markers. HNSCC biopsies from five newly-presenting patients [2lymph node(LN); 3primary tumour (PT)] were divided into parallel microfluidic devices and replicates of each tumour were subjected to single-dose irradiation (0, 5, 10, 15 and 20Gy). Lactate dehydrogenase(LDH) release was measured and tissue sections were stained for cytokeratin (CK), cleaved-CK18 (cCK18), phosphorylated-H2AX (γH2AX) and Ki‑67 by immunohistochemistry. In addition, fragmented DNA was detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Compared with non‑irradiated controls, higher irradiation doses resulted in elevated CK18-labelling index in two lymph nodes [15Gy; 34.8% on LN1 and 31.7% on LN2 (p=0.006)] and a single laryngeal primary tumour (20Gy; 31.5%; p=0.014). Significantly higher levels of DNA fragmentation were also detected in both lymph node samples and one primary tumour but at varying doses of irradiation, i.e.,LN1 (20Gy; 27.6%; p=0.047), LN2 (15Gy; 15.3%; p=0.038) and PT3 (10Gy; 35.2%; p=0.01). The γH2AX expression was raised but not significantly in the majority of samples. The percentage of Ki‑67 positive nuclei reduced dose-dependently following irradiation. In contrast no significant difference in LDH release was observed between irradiated groups and controls. There is clear inter- and intra-patient variability in response to irradiation when measuring a variety of parameters, which offers the potential for the approach to provide clinically valuable information.

Reproductive outcomes of testicular versus ejaculated sperm for intracytoplasmic sperm injection among men with high levels of DNA fragmentation in semen: systematic review and meta-analysis

To compare sperm DNA fragmentation (SDF) levels between testicular and ejaculated sperm and to evaluate outcomes of intracytoplasmic sperm injection (ICSI) with the use of testicular (Testi-ICSI) versus ejaculated (Ejac-ICSI) sperm in nonazoospermic men with high SDF. Systematic review and meta-analysis. Not applicable. Normo- and oligozoospermic men with high levels of SDF in semen subjected to Testi-ICSI or Ejac-ICSI. Summary mean difference (MD) and odds ratio (OR) were calculated with the use of an inverse variance model and fixed- or random-effects models, respectively. Primary outcomes were SDF levels, clinical pregnancy rates (CPRs), and live birth rates (LBRs). Secondary outcomes were fertilization and miscarriage rates. Five studies involving 143 patients provided paired SDF rates for testicular and ejaculated sperm, revealing lower SDF in testicular sperm (MD -24.58%). Four studies involving 507 cycles and 3,840 oocytes reported clinical outcomes of Testi-ICSI and Ejac-ICSI. Fertilization rates were not different between sperm sources, but a trend to lower rates was observed with Testi-ICSI. CPRs were higher for Testi-ICSI than for Ejac-ICSI, as were LBRs, whereas miscarriage rates were reduced with Testi-ICSI. Testicular sperm have lower levels of SDF than ejaculated sperm, with Testi-ICSI for high post-testicular SDF men improving reproductive outcomes compared with Ejac-ICSI. Infertile couples may benefit from Testi-ICSI if male partners have confirmed high SDF in the ejaculate.