DNA Damage In Human Spermatozoa Research Articles

The effect of myo-inositol antioxidant activity on human sperm parameters and DNA damage in ultra-rapid and conventional freezing methods

Male fertility preservation is still challenged by cell damage induced during sperm cryopreservation and impaired sperm structure and function. Sperm ultra-rapid freezing, despite a higher protective effect compared to conventional freezing method, is still associated with suboptimal sperm cryosurvival and needs to be modified to increase its efficiency in sperm protection. Sperm freezing media supplemented with antioxidants can improve sperm parameters following freezing-warming process. In this study, we aimed to investigate the effect of employing ultra-rapid freezing and myo-inositol on sperm cryosurvival. Thirty semen samples with normal sperm parameters were collected and each one was divided into four portions to cryopreserve by conventional freezing, ultra-rapid freezing, conventional freezing + myo-inositol 2 mg/ml, and ultra-rapid freezing + myo-inositol 2 mg/ml. Sperm samples warmed after at least 24 h of freezing and sperm cryosurvival were analyzed by evaluation of sperm motility, viability, morphology and DNA fragmentation index (DFI). Freezing method had a significant influence on post-thaw sperm DFI and morphology (p < 0.05) and the interaction between freezing method and antioxidant supplementation significantly affected sperm morphology (p < 0.05). The highest percentage of sperm normal morphology and minimal DFI was achieved using ultra-rapid freezing supplemented by myo-inositol antioxidant compared to other groups (P < 0.05). The highest sperm DNA damage after freezing-warming was observed following the conventional freezing method. In conclusion, sperm freezing method was identified as factor strongly influencing sperm DFI and morphology after thawing/warming. Sperm samples can be rapidly frozen using the modified freezing media supplemented by myo-inositol without impacting sperm DNA and morphology.

Perfluorooctane sulfonate and perfluorooctanoic acid inhibit progesterone-responsive capacitation through cAMP/PKA signaling pathway and induce DNA damage in human sperm

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are two persistent organic pollutants harmful to human health. They induce negative effects on male reproduction by influencing male hormones, spermatogenesis, and sperm quality. However, their effects and mechanisms on human sperm capacitation and fertilization remain unclear. Here, human sperm were incubated with different concentrations of PFOS or PFOA with progesterone during capacitation. Both PFOS and PFOA inhibited human sperm hyperactivation, sperm acrosome reaction, and protein tyrosine phosphorylation levels. PFOS and PFOA decreased intracellular Ca2+ concentration in the presence of progesterone, and subsequently decreased cAMP level, and PKA activity. PFOS and PFOA increased reactive oxygen species production and sperm DNA fragmentation during the only 3 h capacitation incubation. Conclusively, PFOA and PFOS may inhibit human sperm capacitation via the Ca2+-mediated cAMP/PKA signaling pathway in the presence of progesterone, and induce sperm DNA damage through increased oxidative stress, which is not conducive to fertilization.

Investigation on the mechanisms of human sperm DNA damage based on the proteomics analysis by SWATH-MS

BackgroundSpermatozoa have the task of delivering an intact paternal genome to the oocyte and supporting successful embryo development. The detection of sperm DNA fragmentation (SDF) has been emerging as a complementary test to conventional semen analysis for male infertility evaluation, but the mechanism leading to SDF and its impact on assisted reproduction remain unclear. Therefore, the study identified and analyzed the differentially expressed proteins of sperm with high and low SDF.MethodsSemen samples from men attended the infertility clinic during June 2020 and August 2020 were analyzed, and sperm DNA fragmentation index (DFI) was detected by the sperm chromatin structure assay. Semen samples with low DFI (< 30%, control group) and high DFI (≥ 30%, experimental group) were optimized by density gradient centrifugation (DGC), and the differentially expressed proteins of obtained sperm were identified by the Sequential Window Acquisition of All Theoretical Mass Spectra Mass Spectrometry (SWATH-MS) and performed GO and KEGG analysis.ResultsA total of 2186 proteins were identified and 1591 proteins were quantified, of which 252 proteins were identified as differentially expressed proteins, including 124 upregulated and 128 downregulated. These differentially expressed proteins were involved in metabolic pathways, replication/recombination/repair, acrosomal vesicles, kinase regulators, fertilization, tyrosine metabolism, etc. Western blotting results showed that the expression levels of RAD23B and DFFA proteins and the levels of posttranslational ubiquitination and acetylation modifications in the experimental group were significantly higher than those in the control group, which was consistent with the results of proteomics analysis.ConclusionsProteomic markers of sperm with high DNA fragmentation can be identified by the SWATH-MS and bioinformatic analysis, and new protein markers and posttranslational modifications related to sperm DNA damage are expected to be intensively explored. Our findings may improve our understanding of the basic molecular mechanism of sperm DNA damage.

Investigation of the mechanisms leading to human sperm DNA damage based on transcriptome analysis by RNA-seq techniques

Research questionWhat are the molecular mechanisms leading to human sperm DNA damage? DesignSemen samples were collected and the sperm DNA fragmentation index (DFI) was assessed. Differentially expressed RNA in spermatozoa with a high (DFI ≥30%, experimental group) or normal (DFI <30%, control group) DFI were identified by RNA-sequencing (RNA-seq) technology, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed. Three differentially expressed RNA related to sperm DNA damage and repair, namely PMS1, TP53BP1 and TLK2, were validated using real-time quantitative (RT-qPCR). ResultsA total of 19,970 expressed RNA were detected in the two groups. Compared with the control group, the expression levels of 189 RNA in the experimental group were significantly increased and those of 163 genes decreased. Gene Ontology enrichment analysis showed that these RNA were mainly concentrated in the ATPase-dependent transmembrane transport complex, extracellular exosome, somatic cell DNA recombination, protein binding, cytoplasm and regulation of localization. KEGG pathway analysis showed that these RNA were mainly related to the PI3K-Akt signalling pathway, endocytosis, p53 signalling pathway and cGMP-PKG signalling pathway. The RT-qPCR results showed that the expression levels of PMS1, TP53BP1 and TLK2 in the experimental group were significantly lower than in the control group (P = 0.01, 0.015 and 0.004, respectively), which was identical to the results of RNA sequencing. ConclusionsDifferentially expressed RNA related to sperm DNA damage and repair may be identified by RNA-seq technology, which provides new insights into the understanding of sperm DNA damage and repair, and will help to discover new biomarkers related to sperm DNA damage.

DNA damage in human sperm: The sperm chromosome assay.

BackgroundSperm DNA damage is a major cause of pre‐ and post‐implantation embryonic loss in humans. However, the factors that control how and when such DNA damage occurs in human sperm are poorly understood.MethodsHere, I review information relating to sperm DNA damage that can be obtained from the sperm chromosome assays described in the existing literature.Main findingsThe sperm chromosome assays, which consist of interspecific in vitro fertilization or intracytoplasmic sperm injection using murine oocytes and subsequent chromosome analysis, indicate that the proportion of sperm showing DNA damage is initially low and there are larger numbers of sperm with potential membrane and DNA damage that are induced after ejaculation and separation from the seminal plasma. Other assays that directly detect sperm DNA (e.g., TUNEL assays, Comet assays, and acridine orange test) are not able to distinguish and detect the initial and potential DNA damage. Furthermore, the positive values in these direct assays are influenced by the frequency of immotile sperm and amorphous sperm populations.ConclusionThe findings in the sperm chromosome assays show that further improvements in sperm preparation protocols may result in the reduction of sperm DNA damage, followed by more successful outcomes in infertility treatment.

P–033 In vitro protective effect of α -tocopherol and anthocyanin against TiO2-NPs induced genotoxicity on human spermatozoa

Abstract Study question Do α -tocopherol and anthocyanin counteract human sperm DNA damage provoked by titanium dioxide nanoparticles (TiO2-NPs)? Summary answer: ↑-tocopherol and anthocyanin are able to counteract TiO2-NPs genotoxicity on human sperm cells reducing oxidative stress. What is known already The environmental release and the extensive use of TiO2-NPs have been implicated in poor human sperm functionality.TiO2-NPs is genotoxic on human sperm cells causing a loss of sperm DNA integrity, an increase of apoptotic process and a reduction of genomic stability related to an over production of intracellular ROS.Antioxidants are the substances that can scavenge free radicals. α -tocopherol, present in vegetables, is the most important lipophilic antioxidant involved in restore sperm parameters in several experimental models. Anthocyanin, present in Aronia melanocarpaand belonging to the flavonoid family, is able to prevent damage caused by varicocele-induced ROS in rats. Study design, size, duration Semen samples from 132 men were obtained by masturbation following 3–5 days sexual abstinence and were examined for sperm concentration, viability, motility and morphology according to WHO 2010. The sperm cells, after purification with 45–90% double density gradient, were exposed in vitro to 1 µg/L of TiO2-NPs, 1 µg/L of TiO2-NPs whit 1 mg/L of anthocyanin and 1 µg/L of TiO2-NPs plus 1 mg/L of α -tocopherolfor 15,30,45 and 90 minutes at 37 °C. Participants/materials, setting, methods Sperm motility and concentration were analyzed with Makler camber while sperm viability and morphology were evaluated by Eosin-Nigrosin Test and by Testsimplets® prestained slides respectively. Antigenotoxicity was evaluated by Comet assay, TUNEL test and RAPD-PCR technique and Genomic Template Stability (GTS,%) calculation. The intracellular ROS level was assessed by DFC Assay. The data were analyzed using ANOVA test by GraphPad Prism 6 and considered significant if p-value ≤ 0.05. Main results and the role of chance Sperm analyses showed none statistically significant changes in sperm viability and motility (progressive and non-progressive) for each treatment. Anthocyanin and α -tocopherol counteracted sperm DNA damage induced in vitro by TiO2-NPs neutralizing ROS in a time-dependent way. Comet assay displayed that both antioxidants reduced sperm DNA strand breaks produced by TiO2-NPs, in particular the damage was no longer statistically significant starting from 30 and 90 minutes of anthocyanin-TiO2-NPs and α-tocopherol-TiO2-NPs co-exposure respectively. The antioxidant supplementation induced a statistically decrease of sperm DNA fragmentation provoked by TiO2-NPs after 45 co-treatment minutes.The RAPD-PCR technique evidenced variations of bands number in the TiO2-NPs treated sperm compared to the negative control and anthocyanin and α -tocopherol-TiO2-NPs co-treated samples. Human sperm genomic stability increased after anthocyanin and α -tocopherol TiO2-NPs co-exposure respect to the TiO2-NPs single treatment, until it almost reaches the negative control at 90 minutes. Intracellular ROS percentage was significantly lower both in anthocyanin and α -tocopherol TiO2-NPs co-treated compared to TiO2-NPs alone starting from 45 minutes. Limitations, reasons for caution In vitro study. Wider implications of the findings: Our results showed a protective effect of anthocyanin and α -tocopherol on human DNA by neutralizing intracellular ROS induced by TiO2NPs. We suggest anthocyanin and α -tocopherol as suitable molecules to defend human sperm DNA from oxidative stress, with a potentially role in treatmentof male infertility due to environmental factors. Trial registration number None

Liquid chromatography-tandem mass spectrometry reveals an active response to DNA damage in human spermatozoa

To investigate how endogenously elevated DNA fragmentation alters the human sperm proteome, and whether this fragmentation contributes to genomic deletions. Research study. Commercial fertility clinic. Men with low (0%-4%, n = 7) or high (≥16%, n = 6) sperm DNA fragmentation, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. None. Global sperm proteome, single-nucleotide polymorphism genotyping array. A total of 78 significantly differentially abundant proteins (30 decreased, 48 increased) were observed in control vs. high DNA damage samples. DNA damage resulted in robust proteomic responses, including markers of oxidative stress and apoptosis, DNA damage repair proteins, and transcription/translation and protein turnover machinery. Several key sperm functional proteins were significantly decreased in ejaculates with high DNA damage. We were unable to substantiate a link between increased DNA fragmentation and genomic deletions in human spermatozoa. Developing human spermatozoa initiate an active transcriptional response to endogenous DNA damage, which manifests as alterations in the sperm proteome.

Should we be measuring DNA damage in human spermatozoa? New light on an old question

Assessments of sperm DNA damage are controversial because of perceived uncertainties over the relationship with pregnancy and the limited range of therapies available should positive results be returned. In this article, we highlight recent data supporting a chain of associations between oxidative stress in the male germ line, DNA damage in spermatozoa, defective DNA repair in the oocyte, the mutational load carried by the resulting embryo and the long-term health trajectory of the offspring. Any condition capable of generating oxidative damage in spermatozoa (age, obesity, smoking, prolonged abstinence, varicocele, chemical exposures, radiation etc.) is capable of influencing offspring health in this manner, creating a range of pathologies in the progeny including neuropsychiatric disorders and cancer. If sperm DNA damage is detected, there are several therapeutic interventions that can be introduced to improve DNA quality prior to the use of these cells in ART. We therefore argue that infertility specialists should be engaged in the diagnosis and remediation of sperm DNA damage as a matter of best practice, in order to minimize the risk of adverse health outcomes in children conceived using ART.

Effect of N-acetylcysteine on Human Sperm Parameters and DNA Damage in Frozen-thawed Sperm Samples of Asthenozoospermic Men

Effect of N-acetylcysteine on Human Sperm Parameters and DNA Damage in Frozen-thawed Sperm Samples of Asthenozoospermic Men

CeO2 Nanomaterials from Diesel Engine Exhaust Induce DNA Damage and Oxidative Stress in Human and Rat Sperm In Vitro.

Cerium dioxide nanomaterials (CeO2 NMs) are widely used in nano-based diesel additives to decrease the emission of toxic compounds, but they have been shown to increase the emission of ultrafine particles as well as the amount of released Ce. The Organization for Economic Cooperation and Development included CeO2 NMs in the priority list of nanomaterials that require urgent evaluation, and the potential hazard of aged CeO2 NM exposure remains unexplored. Herein, human and rat sperm cells were exposed in vitro to a CeO2 NM-based diesel additive (called EnviroxTM), burned at 850 °C to mimic its release after combustion in a diesel engine. We demonstrated significant DNA damage after in vitro exposure to the lowest tested concentration (1 µg·L−1) using the alkaline comet assay (ACA). We also showed a significant increase in oxidative stress in human sperm after in vitro exposure to 1 µg·L−1 aged CeO2 NMs evaluated by the H2DCF-DA probe. Electron microscopy showed no internalization of aged CeO2 NMs in human sperm but an affinity for the head plasma membrane. The results obtained in this study provide some insight on the complex cellular mechanisms by which aged CeO2 NMs could exert in vitro biological effects on human spermatozoa and generate ROS.

Dynamic assessment of human sperm DNA damage III: the effect of sperm freezing techniques.

Among the currently available strategies for sperm freezing, vitrification may be considered as the leading alternative to conventional cryopreservation. Nevertheless, a direct comparison of both techniques with respect to the iatrogenic sperm DNA damage has not been performed yet. As such, this study was focused to assess the static and dynamic behavior of human sperm DNA damage following thawing of cryopreserved or vitrified spermatozoa. Semen samples were obtained from fifty donors with a normal spermiogram, and divided into four aliquots. The first aliquot represented the neat sample. In the second aliquot the seminal plasma was discarded, and the resulting sperm pellet was resuspended in PBS. The third fraction was used for slow freezing and the fourth fraction was subjected to vitrification. Each set of samples was incubated at 37°C for 24h and sperm DNA damage (SDF) was assessed using the chromatin-dispersion test following 0h, 2h, 4h and 24h of incubation. When comparing the rate of DNA fragmentation (r-SDF) at 2h, significant differences were observed between the PBS group, cryopreserved (p .000) or vitrified semen (p .015). Furthermore, the sperm longevity comparison using Kaplan-Meier survival curves revealed significant differences between cryopreservation and vitrification (p .000). Our data suggest that exposure of spermatozoa to low temperatures, independently of the chosen freezing protocol, leads to a higher susceptibility of sperm DNA towards damage. This damage is nevertheless lower following vitrification in comparison to traditional cryopreservation. As vitrification leads to a smaller proportion of spermatozoa with DNA damage, we may recommend its use in reproductive techniques which rely on a longer sperm survival, such as artificial insemination.

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.

Use of antioxidant could ameliorate the negative impact of etoposide on human sperm DNA during chemotherapy.

A previous study showed that N-acetylcysteine (NAC), used after in-vitro exposure to the gonadotoxic chemotherapeutic drug etoposide, has the ability to decrease DNA damage in human spermatozoa; however, it showed no benefit when used before exposure. This study aimed to evaluate the impact of the NAC on the preservation of sperm quality during in-vitro exposure to etoposide. Twenty semen samples were submitted to four experimental conditions: control, NAC-only incubation, etoposide-only incubation, and concomitant etoposide and NAC incubation. After in-vitro incubation, semen parameters, sperm chromatin condensation, sperm DNA fragmentation, sperm oxidative stress and sperm metabolism were used to evaluate the role of NAC in protecting human spermatozoa from etoposide. Etoposide did not affect semen parameters, nor did it cause sperm oxidative damage or alterations in glycolytic profile. However, it induced chromatin decondensation and DNA fragmentation, which were fully prevented by NAC. NAC was able to protect sperm DNA integrity during etoposide treatment in vitro, suggesting that NAC may be useful as an adjuvant agent in preserving male fertility during chemotherapy treatments.

Protective Effects of Plants Against Nicotine Induced Reproductive Toxicity in Male Animal Models: A Literature Review

Nicotine the major ingredient in Cigarette is said to have many adverse effects on Spermatozoa and its parameters and affects various other fertility factors. The damages caused by cigarette smoking are due to the biological finding that Nicotine increases the level of free radicals, which results in oxidative stress. These increased the free radicals which also causes DNA damage and lipid peroxidation in human sperm, thus resulting in impairment of sperm quality. Nicotine administration in experimental animals affect spermatogenesis, epididymal sperm count, motility and the fertilizing potential of sperms. A huge number of medicinal herbs have been listed in siddha system that improve spermatogenesis and increase the quality of sperm. This review provides an overview on the association between male fertility induced by nicotine and protective effects of various herbal plants against sperm damages.

Paternal impacts on development: identification of genomic regions vulnerable to oxidative DNA damage in human spermatozoa.

Do all regions of the paternal genome within the gamete display equivalent vulnerability to oxidative DNA damage? Oxidative DNA damage is not randomly distributed in mature human spermatozoa but is instead targeted, with particular chromosomes being especially vulnerable to oxidative stress. Oxidative DNA damage is frequently encountered in the spermatozoa of male infertility patients. Such lesions can influence the incidence of de novo mutations in children, yet it remains to be established whether all regions of the sperm genome display equivalent susceptibility to attack by reactive oxygen species. Human spermatozoa obtained from normozoospermic males (n = 8) were split into equivalent samples and subjected to either hydrogen peroxide (H2O2) treatment or vehicle controls before extraction of oxidized DNA using a modified DNA immunoprecipitation (MoDIP) protocol. Specific regions of the genome susceptible to oxidative damage were identified by next-generation sequencing and validated in the spermatozoa of normozoospermic males (n = 18) and in patients undergoing infertility evaluation (n = 14). Human spermatozoa were obtained from normozoospermic males and divided into two identical samples prior to being incubated with either H2O2 (5mm, 1h) to elicit oxidative stress or an equal volume of vehicle (untreated controls). Alternatively, spermatozoa were obtained from fertility patients assessed as having high basal levels of oxidative stress within their spermatozoa. All semen samples were subjected to MoDIP to selectively isolate oxidized DNA, prior to sequencing of the resultant DNA fragments using a next-generation whole-genomic sequencing platform. Bioinformatic analysis was then employed to identify genomic regions vulnerable to oxidative damage, several of which were selected for real-time quantitative PCR (qPCR) validation. Approximately 9000 genomic regions, 150-1000bp in size, were identified as highly vulnerable to oxidative damage in human spermatozoa. Specific chromosomes showed differential susceptibility to damage, with chromosome 15 being particularly sensitive to oxidative attack while the sex chromosomes were protected. Susceptible regions generally lay outside protamine- and histone-packaged domains. Furthermore, we confirmed that these susceptible genomic sites experienced a dramatic (2-15-fold) increase in their burden of oxidative DNA damage in patients undergoing infertility evaluation compared to normal healthy donors. The limited number of samples analysed in this study warrants external validation, as do the implications of our findings. Selection of male fertility patients was based on high basal levels of oxidative stress within their spermatozoa as opposed to specific sub-classes of male factor infertility. The identification of genomic regions susceptible to oxidation in the male germ line will be of value in focusing future analyses into the mutational load carried by children in response to paternal factors such as age, the treatment of male infertility using ART and paternal exposure to environmental toxicants. Project support was provided by the University of Newcastle's (UoN) Priority Research Centre for Reproductive Science. M.J.X. was a recipient of a UoN International Postgraduate Research Scholarship. B.N. is the recipient of a National Health and Medical Research Council of Australia Senior Research Fellowship. Authors declare no conflict of interest.

Dynamic assessment of human sperm DNA damage II: the effect of sperm concentration adjustment during processing.

To evaluate the effect of sperm concentration adjustment in human ejaculates on the sperm DNA quality and longevity. Semen samples were obtained from 30 donors with a normal spermiogram. Following centrifugation, the sperm pellet was resuspended in PBS, and the sperm concentration adjusted to 200, 100, 50, 25, 12, and 6 × 106/mL. Each set of samples was incubated at 37°C for 24h, and the sperm DNA damage was assessed using the chromatin-dispersion test following 0h, 2h, 6h, and 24h of incubation. Sperm DNA fragmentation (SDF) did not differ between the selected experimental conditions at T0; however, Kaplan-Meier estimates for survival showed significant differences with respect to the dilution and time (all P values were smaller than .001). DNA fragmentation in semen samples adjusted to 200 × 106/mL was approximately 3.3 times higher when compared to samples containing 25 × 106/mL and 3.9 higher in comparison with samples adjusted to 12 × 106/mL following 2h of in vitro incubation. Although there was evidence of individual variation in SDF during the incubation period, the general finding was that lower sperm concentrations resulted in a slower rate of DNA fragmentation. Incubation of spermatozoa for ART purposes should be done following a concentration adjustment below 25 × 106/mL in order to avoid a higher susceptibility of the sperm DNA molecule towards fragmentation.

Human sperm DNA damage has an effect on immunological interaction between spermatozoa and fallopian tube.

Toll-like receptors play a crucial role in the immunological interaction between the spermatozoa and fallopian tube and contribute to the ovulation, sperm capacitation, fertilization, and pregnancy. To investigate the expression of toll-like receptors and their adaptor molecules and cytokines under the effect of spermatozoa with high DNA fragmentation (high DF) in human fallopian tube cell line (OE-E6/E7) and compare to those in normal spermatozoa. Fresh semen samples were obtained from 10 unexplained infertile males with high DF (more than 20%) and from 10 healthy donors with a DF less than 3%. After sperm preparation, samples were co-cultured with OE-E6/E7. Toll-like receptors, myeloid differentiation factor 88 (MyD88), TIR domain-containing adapter protein (TIRAP), TIR domain-containing adapter-inducing IFN-b (TRIF), TRIF-related adapter molecule as well as IL-6, IL-8, IFN-β, and TNFα mRNA expression were evaluated by quantitative real-time PCR. Protein levels of these cytokines and chemokines were measured using ELISA method. TLR 1-6 mRNA expression in OE-E6/E7 was significantly higher under the effect of spermatozoa with high DF compared to the spermatozoa with low DF. Furthermore, significantly increased mRNA expression of MyD88, TIRAP, and TRIF was observed in the high DF group compared to the low DF group, except TRIF-related adapter molecule. Moreover, the expression of IL-6 and IL-8 in the high DF group was significantly higher than low DF group, although there was no significant difference in IFN-β and TNFα expression between the groups. Damage-associated molecular patterns from DNA damage activate TLR signaling pathway in human fallopian tubes and result in the upregulation of inflammatory cytokines and chemokines. This situation may provide pathologic environment for capacitation, fertilization, embryo development, and implantation in female reproductive tract and can be one of the mechanisms of infertility in men with high DF.

Dynamic assessment of human sperm DNA damage I: the effect of seminal plasma-sperm co-incubation after ejaculation.

The purpose of the study was to evaluate the impact of seminal plasma in human ejaculates on the sperm DNA quality and DNA longevity. Semen samples for this study were obtained from 20 donors with a normal spermiogram. Following centrifugation, the sperm pellet was resuspended either in the seminal plasma proceeding from its respective donor, or in an equal amount of PBS, adjusting the concentration to 50 × 106/ml. Each set of samples was incubated at 37°C for 24h and the sperm DNA damage was assessed using the chromatin-dispersion test following 0, 2, 6, and 24h of incubation. Sperm DNA fragmentation (SDF) did not differ between the two experimental conditions at T0; however, Kaplan-Meier estimates to test for survival showed an statistical significant increase of SDF in the seminal plasma group when compared to the PBS group following all timeframes (p 0.000). With respect to sperm DNA longevity, the most dramatic loss of sperm DNA quality occurred during the first 2h of incubation, with the rate of SDF (rSDF) in the seminal plasma being 2.1 more intense than in PBS. Interestingly, the rSDF was found to vary significantly between individuals, which was confirmed with significant correlations in all rSDF timeframes (rSDF T0-2, p 0.049; rSDF T2-6, p 0.000; rSDF T6-24: p 0.000). Co-incubation of semen samples in seminal plasma after ejaculation increases iatrogenic sperm damage. This effect is statistically significant after 2h of co-incubation. Subsequently, for ART purposes seminal plasma must be quickly removed after ejaculation-liquefaction, to avoid a higher susceptibility of sperm DNA towards fragmentation.

Very low concentration of cerium dioxide nanoparticles induce DNA damage, but no loss of vitality, in human spermatozoa

Cerium dioxide nanoparticles (CeO2NP) are widely used for industrial purposes, as in diesel, paint, wood stain and as potential therapeutic applications. The Organization for Economic Cooperation and Development included CeO2NP in the priority list of nanomaterials requiring urgent evaluation. As metal nanoparticles can cross the blood-testis barrier, CeO2NP could interact with spermatozoa. The genotoxicity of CeO2NP was demonstrated in vitro on human cell lines and mouse gametes. However, the effects of CeO2NP on human spermatozoa DNA remain unknown. We showed significant DNA damage induced in vitro by CeO2NP on human spermatozoa using Comet assay. The genotoxicity was inversely proportional to the concentration (0.01 to 10 mg·L−1). TEM showed no internalization of CeO2NP into the spermatozoa. This study shows for the first time that in vitro exposure to very low concentrations of cerium dioxide nanoparticles can induce significant DNA damage in human spermatozoa. These results add new and important insights regarding the reproductive toxicity of priority nanomaterials, which require urgent evaluation.

DNA damage in human spermatozoa; important contributor to mutagenesis in the offspring.

Nuclear DNA damage in spermatozoa could potentially have a major impact on the fertilizing capacity of these cells, their ability to establish a normal pattern of embryonic development as well as the health and wellbeing of subsequent offspring. Many laboratory techniques have been developed to assess this damage focusing on strand breaks, chromatin stability following exposure to extreme pH conditions and the formation of DNA adducts. Of particular importance may be the dominant role played by oxidative stress in the etiology of defective sperm function and DNA damage. The oxidative base lesions created via this mechanism have the potential to generate genetic and epigenetic mutations in the offspring that could have a profound impact on the latter’s long-term health trajectory. Moreover if oxidative stress is the cause of DNA damage in spermatozoa then there is a potential role for antioxidant therapy in the resolution of this problem, which deserves investigation.