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To analyze the differential expression profiles of microRNAs (miRNAs) in spermatozoa of patients with sperm DNA damage and to investigate the role of miRNAs in sperm DNA damage. Male infertility patients with sperm DNA damage who attended the First Affiliated Hospital of Henan University of Chinese Medicine from October 2023 to December 2023 were selected and included in this study as a case group. Fertile healthy men who were seen at the health check-up center during the same period and diagnosed by examination were also included as a control group. Sperm miRNA expression was detected in patients with sperm DNA damage (case group, n = 5) and healthy medical check-ups (control group, n = 5) using high-throughput sequencing technology. The differentially expressed miRNAs between the two groups were bioinformatically analyzed to explore the main biological functions of the target genes. We found that 63 miRNAs were significantly changed in the spermatozoa of patients with sperm DNA damage,|log2 (foldchange)| ≥ 1, p <.05. Gene Ontology (GO) enrichment analysis indicated that these differential miRNAs might be involved in developmental process, anatomical structure development, cellular macromolecule metabolic process, multicellular organism development, system development, and so on. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that that they mainly affect the PI3K-AKT signaling pathway. The present study suggests that the altered expression of miR-1255a, miR-921, and miR-3156-5p may play an important role in the sperm DNA damage process, and the mechanism may involve the phosphatidylinositol-3'-kinase-AKT (PI3K-AKT) signaling pathway. [ABSTRACT FROM AUTHOR]

Background: While the kinetics of human sperm nuclear DNA fragmentation (SDF‐nDNA) following ejaculation have been described, the dynamics and relationships of mitochondrial DNA copy number per spermatozoon (mtDNAcn) and fragmentation (SDF‐mtDNA) remain unexplored. Objectives: To compare post‐ejaculatory kinetics of mtDNAcn, SDF‐mtDNA and SDF‐nDNA, global, single‐strand DNA breaks (SDF‐SSBs) and double‐strand DNA breaks (SDF‐DSBs) in normozoospermic and non‐normozoospermic samples. Materials and methods: 28 normozoospermic and 43 non‐normozoospermic ejaculates were evaluated at 0, 6, 24 and 48 h of incubation in vitro. SDF‐nDNA was determined by sperm chromatin dispersion (SCD) assays. mtDNAcn and SDF‐mtDNA were analysed by dPCR. Results: SDF‐nDNA‐global values increased as a consequence of quadratic SDF‐SSBs and linear SDF‐DSBs kinetics. Non‐normozoospermic samples showed a slower SDF‐global rate between 6–24 h, due to lesser SSBs production. Regarding SDF‐DSBs, non‐normozoospermic samples exhibited a faster initial increase rate, followed by a slower final increment. The mtDNAcn median value decreased linearly, being 3.2× higher in non‐normozoospermics at all time points; mtDNAcn in both cohorts reduced to half of the baseline by 48 h. mtDNAcn was identified as a risk factor for discriminating non‐normozoospermia, a finding that was further strengthen when combined with SDF‐Global or SDF‐DSBs values. SDF‐mtDNA frequencies were identical, increasing over time correspondingly in both cohorts. The mtDNA fragmentation rate was initially fast, decreasing progressively with time for both cohorts; half of the initially unfragmented copies were fragmented after 48 h. Rates of mtDNAcn loss and SDF‐mtDNA increase were only marginally correlated with the rates of nuclear fragmentation. Conclusion: mtDNA fragmentation and loss occur post ejaculation. Their dynamics are likely to be associated with different and/or uncoupled mechanisms to that which cause nuclear DNA fragmentation. Our results indicate that while mtDNA fragmentation is not influenced by the sperm quality, the number of copies of sperm mtDNAcn can potentially serve as a risk factor for predicting non‐normozoospermia. [ABSTRACT FROM AUTHOR]

Male fertility can be affected by oxidative stress (OS), which occurs when an imbalance between the production of reactive oxygen species (ROS) and the body's ability to neutralize them arises. OS can damage cells and influence sperm production. High levels of lipid peroxidation have been linked to reduced sperm motility and decreased fertilization ability. This literature review discusses the most commonly used biomarkers to measure sperm damage caused by ROS, such as the high level of OS in seminal plasma as an indicator of imbalance in antioxidant activity. The investigated biomarkers include 8-hydroxy-2-deoxyguanosine acid (8-OHdG), a marker of DNA damage caused by ROS, and F2 isoprostanoids (8-isoprostanes) produced by lipid peroxidation. Furthermore, this review focuses on recent methodologies including the NGS polymorphisms and differentially expressed gene (DEG) analysis, as well as the epigenetic mechanisms linked to ROS during spermatogenesis along with new methodologies developed to evaluate OS biomarkers. Finally, this review addresses a valuable insight into the mechanisms of male infertility provided by these advances and how they have led to new treatment possibilities. Overall, the use of biomarkers to evaluate OS in male infertility has supplied innovative diagnostic and therapeutic approaches, enhancing our understanding of male infertility mechanisms. [ABSTRACT FROM AUTHOR]

Background. Sperm motility is a fundamental factor for sperm penetration into the oocyte and fertilization. According to the World Health Organization (WHO) 2021, less than 42% of motility is called asthenozoospermia, which is one of the most common causes of male infertility. This retrospective cohort study aims to investigate sperm DNA damage with TUNEL and SCSA methods and its relationship with sperm parameters, age, and body mass index (BMI) in severe asthenospermia (

Aberrant sperm protamination is linked to sperm dysmorphology and nuclear chromatin condensation. Yet, its effects on sperm cytoplasmic maturation remain largely unexplored. The relationships of protamines, sperm morphology, DNA damage, and cytoplasmic remodeling were illustrated in this study to provide fresh perspectives on the mechanisms of male infertility. A total of 205 infertile males were allocated into 5 groups according to the percentage of spermatozoa exhibiting abnormal morphology within their samples. Sperm concentration, motility, abnormal sperm morphology, cytoplasmic droplets (CDs), and excess residual cytoplasm (ERC) were analyzed according to the World Health Organization manual (2010). Sperm nuclear vacuoles (NVs) were determined by propidium iodide (PI) staining. Sperm protamine expressions (P1 and P2) were detected by western blot. DNA damage was measured by acridine orange test (AOT) to calculate the proportion of sperm with single-strand DNA breaks (SSBs). Our data showed that sperm concentration and motility in infertile males significantly decreased with the severity of abnormal sperm morphology (both P < 0.01). P1 level, P1/P2 ratio, and SSB rate increased with the severity of sperm dysmorphology, whilst the P2 level decreased (all P < 0.01). NVs, CDs, and ERC were more common in males with sperm dysmorphology and positively correlated with the SSB rate (all P < 0.01). The relationships between the SSB rate and the P1/P2 ratio were also significant (P < 0.01). Aberrant protamination may cause sperm dysmorphology and compromise male fertility by impairing sperm's nucleus and cytoplasm maturation, with the P1/P2 ratio potentially serving as a valuable indicator of sperm quality and male fertility.

Background: Oxidative stress (OS) is considered one of the major factors affecting male fertility, and research in this field has seen constant growth year by year. Currently, around 700 relevant papers are published each year, with a trend of further growth. Therefore, this study systematically summarizes the literature published in the last decade from a bibliometric perspective, revealing the dynamic development of the field, identifying research hotspots, analyzing future trends, and providing reference for further research. Methods: Relevant literature on oxidative stress and male fertility was retrieved from the Web of Science Core Collection (WoSCC) database, covering the timespan from 2014 to 2023 and including two types, articles and reviews. CiteSpace and VOSviewer were used for bibliometric analysis, including cluster analysis, co-occurrence analysis, co-citation analysis, and burst analysis of countries/regions, institutions, journals, authors, references, and keywords. Results: This paper studied a total of 5,301 papers involving 107 countries/regions, with China having the highest number of publications (898 papers) and the United States having the highest centrality (0.62). Burst analysis of journal citations revealed the emergence of many new journals (e.g., Antioxidants-Basel, Front Endocrinol) after 2021, indicating continuous expansion and development in this field. Cluster analysis of co-cited references and cooccurring keywords divided the research into areas such as oxidative stress and male infertility, oxidative stress level detection, and antioxidants. The keywords associated with research hotspots shifted from oxidative stress detection, sperm DNA damage, apoptosis, and redox potential to DNA methylation, embryonic development, infection, polyunsaturated fatty acids, and antioxidants. Conclusion: Bibliometric methods provide an intuitive reflection of the development process in the field of oxidative stress and male fertility, as well as the analysis of research hotspots in different periods. Research on oxidative stress and embryonic development, as well as antioxidant health management, may become hotspots in future research. [ABSTRACT FROM AUTHOR]

Background. Sperm motility is a fundamental factor for sperm penetration into the oocyte and fertilization. According to the World Health Organization (WHO) 2021, less than 42 percent of motility is called asthenozoospermia, which is one of the most common causes of male infertility. This retrospective cohort study aims to investigate sperm DNA damage with TUNEL and SCSA methods and its relationship with sperm parameters, age, and body mass index (BMI) in severe asthenospermia (<2% sperm motility) and normozoospermia. Methods. The study parameters between 111 subjects with severe asthenozoospermia and 113 subjects with normozoospermia were investigated. The 2010 World Health Organization guidelines were used to evaluate sperm parameters, and TUNEL and SCSA methods were used to assess sperm DNA damage. The statistical analysis was done using the t-test of two independent samples. Pearson's correlation coefficient was used to investigate the relationship between sperm DNA damage and sperm parameters, age, and BMI P<0.05 was considered significant. Results. The mean seminal volume, sperm concentration and count, and the percentage of sperm total motility and progressive motility were significantly lower in severe asthenospermic subjects compared to normozoospermic subjects (P<0.001). In addition, the mean percentage of sperm DNA damage in severe asthenozoospermic individuals was significantly higher than in normozoospermic individuals (P<0.001). There was also a positive and significant relationship between sperm DNA damage and the age of severely asthenozoospermic men. Conclusion. Sperm motility, sperm DNA damage, and the father's age are essential in the successful conception, development, and health of the embryo. Therefore, the evaluation of sperm DNA damage is recommended in examining male fertility and selecting the appropriate treatment approach for men with severe asthenozoospermia. Practical Implications. Considering that the sperm genome constitutes 50 percent of the genetic material of the next generation, evaluating sperm DNA health to determine the appropriate treatment approach in infertile men can be influential in the success of assisted reproductive techniques and in maintaining the next generation’s health. [ABSTRACT FROM AUTHOR]

Intracytoplasmic sperm injection (ICSI) using surgically retrieved spermatozoa outside the classic context of azoospermia has been increasingly used to overcome infertility. The primary indications include high levels of sperm DNA damage in ejaculated spermatozoa and severe oligozoospermia or cryptozoospermia, particularly in couples with ICSI failure for no apparent reason. Current evidence suggests that surgically retrieved spermatozoa for ICSI in the above context improves outcomes, mainly concerning pregnancy and miscarriage rates. The reasons are not fully understood but may be related to the lower levels of DNA damage in spermatozoa retrieved from the testis compared with ejaculated counterparts. These findings are consistent with the notion that excessive sperm DNA damage can be a limiting factor responsible for the failure to conceive. Using testicular in preference of low‐quality ejaculated spermatozoa bypasses post‐testicular sperm DNA damage caused primarily by oxidative stress, thus increasing the likelihood of oocyte fertilization by genomically intact spermatozoa. Despite the overall favorable results, data remain limited, and mainly concern males with confirmed sperm DNA damage in the ejaculate. Additionally, information regarding the health of ICSI offspring resulting from the use of surgically retrieved spermatoa of non‐azoospermic males is still lacking. Efforts should be made to improve the male partner's reproductive health for safer ICSI utilization. A comprehensive andrological evaluation aiming to identify and treat the underlying male infertility factor contributing to sperm DNA damage is essential for achieving this goal. [ABSTRACT FROM AUTHOR]

To date, infertility affects 10% to 15% of couples worldwide. A male factor is estimated to account for up to 50% of cases. Oral supplementation with antioxidants could be helpful to improve sperm quality by reducing oxidative damage. At the same time, there is a growing interest in the literature on the use of testicular sperm in patients with high DNA fragmentation index (DFI). This narrative review aims to evaluate the effectiveness of supplementation of oral antioxidants in infertile men with high DFI compared to testicular sperm retrieval. The current evidence is non-conclusive because of serious risk of bias due to small sample sizes and statistical methods. Further large well-designed randomised placebo-controlled trials are still required to clarify the exact role of these to different therapeutic approaches. [ABSTRACT FROM AUTHOR]

This case-control study, conducted from July 2021 to August 2022 in Duhok, Kurdistan Region, Iraq, investigates the semen parameters and sperm DNA damage in male partners of couples experiencing recurrent pregnancy loss (RPL) compared to fertile couples without pregnancy loss. The study, with participants gathered from various gynaecological clinics, sheds light on the implications for mammalian reproductive health and wildlife conservation such as captive breeding programs. Semen indices, analyzed according to World Health Organization (WHO) standards, revealed that couples with RPL exhibited significantly lower rates of progressive motility (45.62 ± 16.01 vs. 53.08 ± 10.11, P=0.037) and normal morphology (2.88 ± 1.5 vs. 3.48 ± 1.2, P=0.048) compared to the control group. Furthermore, the study employed the alkaline comet assay to assess the magnitude of sperm DNA damage. Results indicated a significantly higher degree of sperm DNA damage in couples with RPL (21.99 ± 1.13 vs. 17.42 ± 1.39, P=0.018), along with a notable increase in DNA Fragmentation Index (DFI) (43.46 ± 20.39 vs. 29.83 ± 16.25, P=0.0048) compared to the control group. These findings not only contribute to our understanding of human reproductive health but also have broader implications for mammalian fertility, potentially influencing wildlife populations. Further research is warranted to unravel the underlying mechanisms and explore interventions for couples experiencing RPL, with potential applications in mammalian and wildlife conservation. The semen of male partners of couples with RPL have lower progressive motility and a higher percentage of abnormal morphology with a significantly higher degree of DNA damage when compared with their age-matched control group, therefor sperm DNA damage may be included as an important step in the evaluation of couples with RPL. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To date, infertility affects 10% to 15% of couples worldwide. A male factor is estimated to account for up to 50% of cases. Oral supplementation with antioxidants could be helpful to improve sperm quality by reducing oxidative damage. At the same time, there is a growing interest in the literature on the use of testicular sperm in patients with high DNA fragmentation index (DFI). This narrative review aims to evaluate the effectiveness of supplementation of oral antioxidants in infertile men with high DFI compared to testicular sperm retrieval. The current evidence is non-conclusive because of serious risk of bias due to small sample sizes and statistical methods. Further large well-designed randomised placebo-controlled trials are still required to clarify the exact role of these to different therapeutic approaches.

Simple Summary: This study analyzed the association of various alterations in sperm morphology with the level of DNA damage, PRM deficiency, and the abundance of the PRM1 gene and protein in sperm. We found in this study that each parameter is associated with one another. Thus, gene expression and PRM1 protein abundance may play an essential role in the stability of sperm chromatin, especially concerning DNA damage and alterations in sperm head morphology. In addition, although it is not a novelty, the CMA3 in this study proved effective for bulls in Indonesia. It can be used as an alternative test to detect PRM content in sperm without having to carry out assessments at the molecular level, such as gene or protein expression assessments. This study aimed to analyze various alterations in the morphology of the sperm head and its association with nucleus instability and insufficient sperm protamine. Frozen-thawed semen from twenty local Indonesian bulls was used for all stages in this study. The results of sperm head defect assessments are used for bull grouping, high (HD) and low (LD). Sperm DNA damage was assessed using Acridine Orange and Halomax. The PRM1 protein abundance was carried out using an enzyme immunoassay, while PRM1 gene expression was carried out using the RT-qPCR. PRM deficiency was performed using CMA3. Several kinds of sperm head defects in the HD were significantly higher (p < 0.05) than in the LD bulls. Sperm DNA damage showed a significant (p < 0.05) difference between the HD and LD bulls. PRM1 abundance was significantly (p < 0.05) decreased in HD bulls. PRM deficiency was significantly (p < 0.05) higher in HD bulls than in LD bulls. PRM deficiency in bulls correlated significantly (p < 0.01) with sperm head defects, DNA damage, and PRM1 abundance. The lack of sperm protamine might affect the sperm nucleus's stability and induce morphological alterations in the sperm head. [ABSTRACT FROM AUTHOR]

Abstract Background Altered sperm DNA methylation patterns of imprinted genes as well as certain spermatogenesis-related genes has been proposed as a possible mechanism of male subfertility. Some reports suggest that there is an elevated risk of congenital diseases, associated with imprinted genes, in children conceived via intra-cytoplasmic sperm injection, due to the involvement of spermatozoa with aberrant imprinted genes obtained from infertile men. Methods In this study, the DNA methylation status of the promoter regions of six imprinted genes, namely potassium voltage-gated channel subfamily Q member 1 (KCNQ1), maternally expressed gene 3 (MEG3), insulin-like growth factor 2 (IGF-2), KCNQ1 overlapping transcript 1 (KCNQ1OT1), mesoderm specific transcript (MEST), and paternally expressed gene 3 (PEG3), were detected by a next generation sequencing-based multiple methylation-specific polymerase chain reaction analysis of sperm samples obtained from 166 men who sought fertility evaluation in our Reproductive Medicine Center. Thereafter, the semen samples were classified into subgroups according to sperm motility and DNA integrity status. Results As compared to the normozoospermic group, the samples of the asthenospermic group exhibited significant hypermethylation in two CpG sites of IGF-2 and significant hypomethylation in one CpG site of KCNQ1 as well as three CpG sites of MEST (P 0.05). Additionally, we found that 111 of 323 CpG sites were hypomethylated in the group with DNA fragmentation index (DFI) ≥ 30% as compared to the group with DFI 0.05). Hence, aberrant methylation patterns of imprinted genes were more prevalent in males with poor sperm quality, especially in those with severe sperm DNA damage. Conclusion In conclusion, abnormal DNA methylation of some CpG sites of imprinted genes are associated with poor sperm quality, including asthenospermia and severe sperm DNA impairment.

Objective: Oral antioxidants are one of the options for treating male patients with idiopathic infertility associated with increased sperm DNA fragmentation. The aim of this study is to assess the contribution of antioxidant treatment duration to treatment success in this patient group. Materials and Methods: In this cross-sectional study (between 2014 and 2019), 637 patients who received antioxidant therapy for male infertility were retrospectively analyzed. The results of patients with 30% or more sperm DNA damage and who did not meet the exclusion criteria and who had at least 6 months of follow-up were evaluated. DNA damage, semen parameters, and laboratory results of the patients receiving antioxidant therapy were evaluated before the treatment and at the third and sixth months of treatment. Results: A total of 53 patients with follow-up data met the study criteria. Significant decreases were observed in sperm DNA fragmentation index (DFI) values in the third and sixth months of the treatment. The sperm DFI was a median of 44% (interquartile range, 13.7%) before the treatment and 33.3% (IQR, 20.9%) after the 3 months and 18% (IQR, 13.4%) after the 6 months. Additionally, during the antioxidant treatment, a statistically significant decrease was observed between the third and sixth month DFI values. Conclusion: In idiopathic infertility cases, antioxidant treatment may have positive effects on sperm DFI values, and the prolongation of the treatment period may make an additional contribution to treatment success for infertile men with increased sperm DNA fragmentation (SDF). Nevertheless, possible side effects cost of treatment, patient compliance, and the condition of the partner should be considered while planning the duration of treatment. [ABSTRACT FROM AUTHOR]

Cryopreservation is an expanding strategy to allow not only fertility preservation for individuals who need such procedures because of gonadotoxic treatments, active duty in dangerous occupations or social reasons and gamete donation for couples where conception is denied, but also for animal breeding and preservation of endangered animal species. Despite the improvement in semen cryopreservation techniques and the worldwide expansion of semen banks, damage to spermatozoa and the consequent impairment of its functions still remain unsolved problems, conditioning the choice of the technique in assisted reproduction procedures. Although many studies have attempted to find solutions to limit sperm damage following cryopreservation and identify possible markers of damage susceptibility, active research in this field is still required in order to optimize the process. Here, we review the available evidence regarding structural, molecular and functional damage occurring in cryopreserved human spermatozoa and the possible strategies to prevent it and optimize the procedures. Finally, we review the results on assisted reproduction technique (ARTs) outcomes following the use of cryopreserved spermatozoa. [ABSTRACT FROM AUTHOR]

Recurrent pregnancy loss is a distressing condition affecting 1–2% of couples. Traditionally investigations have focused on the female, however more recently researchers have started to explore the potential contribution of the male partner. Seminal reactive oxygen species have a physiological function in male reproduction but in excess are suspected to generate structural and functional damage to the sperm. Evidence is mounting to support an association between elevated seminal reaction oxygen species and recurrent pregnancy loss. Studies suggest that the rates of sperm DNA damage are higher in the male partners of women affected by recurrent pregnancy loss compared with unaffected men. However, the available pool of data is conflicting, and interpretation is limited by the recent change in nomenclature and the heterogeneity of study methodologies. Furthermore, investigation into the effects of oxidative stress on the epigenome show promise. The value of antioxidant therapy in the management of recurrent pregnancy loss currently remains unclear. [ABSTRACT FROM AUTHOR]

Summary: Background: Short- and long-term implications of SARS-CoV-2 on the quality of the sperm and the results of this on fertility remain largely unknown due to lack of longitudinal studies. In this longitudinal observational cohort study, we aimed to analyse the differential effect and the impact of SARS-CoV-2 infection on different semen quality parameters. Methods: Sperm quality was assessed using the World Health Organization criteria, DNA damage to sperm cells by quantifying the DNA fragmentation index (DFI) and the high-density stainability (HDS), IgA- and IgG-anti-sperm antibodies (ASA) were assessed with light microscopy. Findings: SARS-CoV-2 infection was associated with sperm parameters that were independent of spermatogenic cycle like progressive motility, morphology, DFI and HDS, as well as spermatogenic cycle dependent parameters such as sperm concentration. Detection of IgA- and IgG-ASA allowed classification of patients in three different groups according to its sequence of appearance in sperm during post-COVID-19 follow-up. The maximum progressive motility was lowest during follow-up in patients without ASA (41.9%), intermediate in patients with only IgA-ASA (46.2%) and highest inpatients who had both IgA- and IgG-ASA (54.9%). Interpretation: SARS-CoV-2 infection was associated with changes of all analysed sperm parameters to a different degree which is also observed in their return to normality and is suggestive of individual variations in the patient's immune system performance. Firstly, sperm production is decreased through temporal immune mediated arrest of active meiosis, and secondly immune induced sperm DNA damage prevents fertilization if transferred to the oocyte. Both mechanisms are temporal, and most sperm parameters return to baseline after infection. Funding: AML (R20-014), Femicare.

Background: Spermatozoa 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. Methods: Semen 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. Results: A 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. Conclusions: Proteomic 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. Highlights: Differentially expressed proteins in sperm could be identified by the SWATH-MS. The validation results of Western blotting were identical to the results of proteomics analysis. Proteomic markers of sperm with high DNA fragmentation could be identified by the bioinformatic analysis. New protein markers and posttranslational modifications related to sperm DNA damage are expected to be intensively explored. [ABSTRACT FROM AUTHOR]

• Density gradient centrifugation did not affect the change in trend of sperm DNA fragmentation index. • Differentially expressed RNA in sperm could be identified by the RNA-seq technology. • The validation results of real-time quantitative PCR were identical to the results of RNA sequencing. • Molecular mechanisms leading to human sperm DNA damage are expected to be clarified. • New biomarkers related to sperm DNA damage are expected to be discovered. What are the molecular mechanisms leading to human sperm DNA damage? Semen 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). A 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. Differentially 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. [ABSTRACT FROM AUTHOR]

Background: Infertility has increased in recent years. About half of all infertility cases are due to male factors. Objectives: The present study aimed to diagnose infertile males by examining sperm volume, density, motility, count, pH, luteum hormone (LH), follicle-stimulating hormone (FSH), percentage of sperm with abnormal morphology, and percentage of sperm DNA damage. Methods: This study was performed on semen samples of 96 males, of whom45 were fertile and 51 were infertile. Sperm parameters were evaluated based on theWorld Health Organization (WHO) criteria among fertile and infertile males. Results: Volume, concentration, and motility were significantly lower and LH, FSH, and the percentage of spermDNA damage were higher in infertile than in fertile mals. Conclusions: By examining the quality and analysis of semen in men, it is possible to identify infertile people and follow the necessary and appropriate treatment strategies. [ABSTRACT FROM AUTHOR]

Abstract Background The assessment of sperm DNA integrity has been proposed as a complementary test to conventional mammalian semen analysis. In this sense, single-strand (SSB) and double-strand (DSB) DNA breaks, the two types of sperm DNA fragmentation (SDF), have been reported to have different aetiologies and to be associated to different fertility outcomes in bovine and humans. Considering that no studies in porcine have addressed how SDF may affect sperm quality and fertility outcomes, the present work aimed to determine the impact of global DNA damage, SSB and DSB on sperm quality and in vitro fertilising ability. To this end, 24 ejaculates (one per boar) were split into three aliquots: the first was used to assess sperm quality parameters through a computer-assisted sperm analysis (CASA) system and flow cytometry; the second was used to perform in vitro fertilisation, and the third, to evaluate sperm DNA integrity using alkaline and neutral Comet assays. Results The results showed that global DNA damage negatively correlates (P 0.05). Conclusion Considering all these findings, this work sets a useful model to study how SDF negatively influences fertility.

Background: Microfluidics (MF), an advanced sperm sorting technology results in the extraction of spermatozoa with higher DNA integrity and lower DNA damage compared to existing conventional sperm sorting methods. Aims: The aim of the present study is to assess the efficiency of MF and to isolate the best spermatozoa for intracytoplasmic sperm injection (ICSI) over the density gradient (DG) technique. Settings and Design: We recruited couples who choose the oocyte donation programme for this study to eliminate confounding factors associated with oocyte quality. Materials and Methods: Sperm was processed by MF (n = 180) and DG (n = 151). ICSI was performed and positive pregnancy, miscarriage and clinical pregnancy rates were compared. Statistical Analysis Used: All variables were analysed using Graph Pad Prism 5. The unpaired two-tailed t-test was used to assess the significance. A value of P < 0.05 was considered statistically significant. Results: There was no significant difference in pregnancy rates between the groups. However, a clear demarcation is seen in terms of clinical pregnancy rates, where the DG group achieved higher clinical pregnancies (91.7%) compared to the MF group (80.7%). Further, we compared miscarriage rates and biochemical pregnancies, and found a significantly higher miscarriage and biochemical pregnancy rate in the MF group (14.5% and 4%, respectively) compared to the DG group (6% and 1%, respectively). Conclusions: Based on the available literature, we anticipated a higher clinical pregnancy rate with MF compared with conventional processing. Our results show MF does not have any add-on positive effect on clinical pregnancy rate.

Background: Altered sperm DNA methylation patterns of imprinted genes as well as certain spermatogenesis-related genes has been proposed as a possible mechanism of male subfertility. Some reports suggest that there is an elevated risk of congenital diseases, associated with imprinted genes, in children conceived via intra-cytoplasmic sperm injection, due to the involvement of spermatozoa with aberrant imprinted genes obtained from infertile men. Methods: In this study, the DNA methylation status of the promoter regions of six imprinted genes, namely potassium voltage-gated channel subfamily Q member 1 (KCNQ1), maternally expressed gene 3 (MEG3), insulin-like growth factor 2 (IGF-2), KCNQ1 overlapping transcript 1 (KCNQ1OT1), mesoderm specific transcript (MEST), and paternally expressed gene 3 (PEG3), were detected by a next generation sequencing-based multiple methylation-specific polymerase chain reaction analysis of sperm samples obtained from 166 men who sought fertility evaluation in our Reproductive Medicine Center. Thereafter, the semen samples were classified into subgroups according to sperm motility and DNA integrity status. Results: As compared to the normozoospermic group, the samples of the asthenospermic group exhibited significant hypermethylation in two CpG sites of IGF-2 and significant hypomethylation in one CpG site of KCNQ1 as well as three CpG sites of MEST (P < 0.05). However, we did not observe any significant differences in the overall methylation levels of these six imprinted genes (P > 0.05). Additionally, we found that 111 of 323 CpG sites were hypomethylated in the group with DNA fragmentation index (DFI) ≥ 30% as compared to the group with DFI < 30% (P < 0.05). In this case, there were significant differences in the overall methylation levels of MEG3, IGF-2, MEST, and PEG3 (P < 0.05), but not in that of KCNQ1OT1 and KCNQ1 (P > 0.05). Hence, aberrant methylation patterns of imprinted genes were more prevalent in males with poor sperm quality, especially in those with severe sperm DNA damage. Conclusion: In conclusion, abnormal DNA methylation of some CpG sites of imprinted genes are associated with poor sperm quality, including asthenospermia and severe sperm DNA impairment. [ABSTRACT FROM AUTHOR]

Background: Men with spinal cord injury (SCI) show a high proportion of sperm DNA damage in their ejaculate but the underlying pathology remains elusive. Objective: To investigate the relative incidence of single (SSBs) and double‐strand DNA breaks (DSBs) and DNase activity in men with SCI. Materials and methods: This study included ejaculates of 20 men with SCI and 27 normozoospermic (sperm donors). A TwoTails comet assay (TTComet) allowed visualization of three categories of sperm DNA damage corresponding to SSBs, DSBs and those with a combination of SSBs and DSBs, facilitating accurate calculation of the total proportion of SSBs and DSBs. A subset of 15 individuals (sperm donors and SCI patients) was used to test for DNase activity in the seminal plasma. Results: While the proportion of DSBs in men with SCI (median‐57.5%) was higher (P = 0.000) than normozoospermic samples (median‐4.6%), the proportion of SSBs was higher (P = 0.022) in the normozoospermic ejaculates (median‐6.0%) compared to men with SCI (median‐2.5%). The relative proportion of the total DSBs with respect to the total SSBs was 3.3× in men with SCI but 0.9× in normozoospermic samples. We further confirmed the high DNase activity in the seminal plasma of men with SCI. Discussion: The TTComet assay provided new insights to the pathology of sperm DNA in men with SCI and may have diagnostic value in developing sperm selection methodologies to reduce DSBs prior to ART. Conclusion: Men with SCI are characterized by a high proportion of sperm with DSBs and high levels of DNase activity in the seminal plasma compared to normozoospermic men. [ABSTRACT FROM AUTHOR]

Background: No association between the length of ejaculatory abstinence (LEA) and semen characteristics has been confirmed. A short LEA has been linked to improved sperm characteristics and a higher pregnancy rate, but its negative influence on sperm chromatin maturity and longevity may adversely affect reproductive outcomes. Objectives: We sought to determine the influence of LEA on (i) semen parameters in normozoospermic and abnormal ejaculates; and (ii) the outcomes of sperm‐preparation methods in a large number of subfertile men undergoing infertility workups. Materials and methods: This retrospective registry‐based cohort study analyzed the data of 10,674 ejaculates from 7972 subfertile men, who were then segregated into normozoospermic, oligozoospermic, asthenozoospermic, and oligo‐asthenozoospermic cohorts. Variations in semen characteristics and post‐wash outcomes were studied between four LEA intervals across 0–15 days. Results: An age‐adjusted analysis of covariance (ANCOVA) model linked significant increases in ejaculate volume, sperm concentration (except in the oligozoospermic cohort), and total sperm count to an increased LEA (p < 0.05). LEA was negatively associated with motility (except in the asthenozoospermic cohort) and vitality (p < 0.05). Large‐headed spermatozoa were less common with an increased LEA only in the oligo‐asthenozoospermic cohort (p < 0.05). In the normozoospermic cohort, a longer LEA led to fewer spermatozoa with amorphous heads but more spermatozoa with tapered heads and cytoplasmic droplets (p < 0.05). LEA extension resulted in greater sperm DNA fragmentation in the abnormal cohort (p < 0.01). The post‐wash sperm concentration and total motile sperm count were significantly improved with a longer LEA in the normozoospermic cohort (p < 0.05). Discussion and conclusion: Considering the findings in this study and existing literature, a generalized recommendation for long LEA has no clinical value. The LEA should be individualized based on the ejaculate profile and the need for specific clinical intervention. [ABSTRACT FROM AUTHOR]

In this study, we compared the effects of sperm parameters and sperm DNA damage on pregnancy outcomes of women undergoing intrauterine insemination (IUI) because of an ovulatory dysfunction (OD) and unexplained infertility (UEI). This study was retrospective, and semen samples were collected from records of 88 infertile couples referred to private clinic for infertility treatment from December 2019 to March 2020. The study has two groups: Groups 1: couples with UEI, and Group 2: fertile males with their partners having OD. The participants' age and body mass index (BMI) were 28.20 ± 3.08 and 25.45±2.25, respectively. In the control group, the pregnancy rate was 9/41 (%21.9), and one out of nine patients had a miscarriage. The pregnancy rate in the study group (UEI) was 8/47 (17%), and half of the pregnancies ended as miscarriage. Our results showed that sperm DNA damage increases the abortion rate but has not influenced the pregnancy rate in IUI. [ABSTRACT FROM AUTHOR]

More than forty years after the first birth following in vitro fertilization (IVF), the success rates of IVF and of IVF-derived assisted reproduction techniques (ART) still remain relatively low. Interindividual differences between infertile couples and the nature of the problems underlying their infertility appear to be underestimated nowadays. Consequently, the molecular basis of each couple's reproductive function and of its disturbances is needed to offer an individualized diagnostic and therapeutic approaches to each couple, instead of applying a standard or minimally adapted protocols to everybody. Interindividual differences include sperm and oocyte function and health status, early (preimplantation) embryonic development, the optimal window of uterine receptivity for the implanting embryo, the function of the corpus luteum as the main source of progesterone production during the first days of pregnancy, the timing of the subsequent luteoplacental shift in progesterone production, and aberrant reactions of the uterine immune cells to the implanting and recently implanted embryos. In this article, the molecular basis that underlies each of these abnormalities is reviewed and discussed, with the aim to design specific treatment options to be used for each of them. [ABSTRACT FROM AUTHOR]

This Directions article examines the mechanisms by which a father's age impacts the health and wellbeing of his children. Such impacts are significant and include adverse birth outcomes, dominant genetic conditions, neuropsychiatric disorders, and a variety of congenital developmental defects. As well as age, a wide variety of environmental and lifestyle factors are also known to impact offspring health via changes mediated by the male germ line. This picture of a dynamic germ line responsive to a wide range of intrinsic and extrinsic factors contrasts with the results of trio studies indicating that the incidence of mutations in the male germ line is low and exhibits a linear, monotonic increase with paternal age (∼two new mutations per year). While the traditional explanation for this pattern of mutation has been the metronomic plod of replication errors, an alternative model pivots around the 'faulty male' hypothesis. According to this concept, the genetic integrity of the male germ line can be dynamically impacted by age and a variety of other factors, and it is the aberrant repair of such damage that drives mutagenesis. Fortunately, DNA proofreading during spermatogenesis is extremely effective and these mutant cells are either repaired or deleted by apoptosis/ferroptosis. There appear to be only two mechanisms by which mutant germ cells can escape this apoptotic fate: (i) if the germ cells acquire a mutation that by enhancing proliferation or suppressing apoptosis, permits their clonal expansion (selfish selection hypothesis) or (ii) if a genetically damaged spermatozoon manages to fertilize an oocyte, which then fixes the damage as a mutation (or epimutation) as a result of defective DNA repair (oocyte collusion hypothesis). Exploration of these proposed mechanisms should not only help us better understand the aetiology of paternal age effects but also inform potential avenues of remediation., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Purpose: To investigate the impact of metabolic syndrome factors on sperm DNA fragmentation (sDF) in males from infertile couples., Methods: A systematic literature search was performed across ten databases for literature published from January 1, 2013 until September 13, 2023. The protocol has been registered on PROSPERO (CRD42023458359), and the literature search strategy is adhered to the PRISMA framework. Studies that evaluated sDF, as indicated by DNA fragmentation index (%DFI), in males from infertile couples in relation to metabolic syndrome factors were included. Meta-analysis, using random effects model and Bayesian framework network, was performed, and data were presented as Standardized Mean Differences (SMD) with corresponding 95 % Confidence Interval (CI)., Results: Of the 2579 citations identified, eleven studies were included in this meta-analysis. The findings revealed that the %DFI was not associated with overall metabolic syndrome factors (p-tot = 0.235; SMD = 0.57 [95 %CI: -0.37, 1.52]), metabolic syndrome status (p-tot = 0.337; SMD = 0.08 [95 %CI: -0.08, 0.24), increased body mass index (p-tot = 0.237; SMD = 0.71 [95 %CI: -0.47, 1.89]), or glycaemic profile (p-tot = 0.93; SMD = 0.13 [95 %CI: -2.72, 2.98]). High levels of heterogeneity were observed (p < 0.01) in all subgroups, except for metabolic syndrome status., Conclusion: The association between metabolic syndrome factors and sDF is conflicting. However, interpreting the association requires caution, as confounding factors, indicated by high heterogeneity, may conceal the outcome. Metabolic syndrome may influence other factors contributing to male infertility, highlighting the importance of promoting a healthy lifestyle., Competing Interests: Declaration of competing interest All authors declare no competing interests that could influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

This study aimed to analyze various alterations in the morphology of the sperm head and its association with nucleus instability and insufficient sperm protamine. Frozen-thawed semen from twenty local Indonesian bulls was used for all stages in this study. The results of sperm head defect assessments are used for bull grouping, high (HD) and low (LD). Sperm DNA damage was assessed using Acridine Orange and Halomax. The PRM1 protein abundance was carried out using an enzyme immunoassay, while PRM1 gene expression was carried out using the RT-qPCR. PRM deficiency was performed using CMA3. Several kinds of sperm head defects in the HD were significantly higher (p < 0.05) than in the LD bulls. Sperm DNA damage showed a significant (p < 0.05) difference between the HD and LD bulls. PRM1 abundance was significantly (p < 0.05) decreased in HD bulls. PRM deficiency was significantly (p < 0.05) higher in HD bulls than in LD bulls. PRM deficiency in bulls correlated significantly (p < 0.01) with sperm head defects, DNA damage, and PRM1 abundance. The lack of sperm protamine might affect the sperm nucleus’s stability and induce morphological alterations in the sperm head.

There was a marker of high DNA stainability (HDS) in the detection of sperm DNA damage, which was defined as the sperm with high green stainability (HIGRN). The sperm with normal double‐stranded DNA was stained green by acridine orange (AO). However, the sperm with high green fluorescence or HDS were thought of as immature sperm or the sperm with poor chromatin condensation. Some previous literature reported that the proportion of sperm with HDS increased with age, and had a certain correlation with the poor outcome of assisted reproductive technology. Recently, several articles reported that the marker of HDS decreased linearly with age, which was obviously inconsistent with that reported by the previous literature. In this case, what kind of marker is HDS? Is it worth studying? After extensively reading the literature related to HDS and flow cytometry related books and performing a series of studies related to the detection of sperm DNA damage, we believe that the establishment of HDS in the detection of sperm DNA damage has no theoretical basis and also no support of evidence‐based medicine and that using HDS as a marker in the detection of sperm DNA integrity is inappropriate. [ABSTRACT FROM AUTHOR]

Present research aim was to identify functional tests in semen associated with DNA damage and chromatin maturity (protamination) which predict the outcome in assisted reproduction. Couples were grouped according to male partner semen parameters, into normozoospermia (NZs), severe male factor (SMF) and mild male factor (MMF). DNA fragmentation index (DFI) in spermatozoa was analysed by sperms chromatin dispersion (SCD), sperm chromatin structure assay (SCSA) and acridine orange testing (AOT). Chromomycin A3 (CMA3) and toluidine blue (TB) staining to measure sperm chromatin maturity (CM). DFI and chromatin decondensation were significantly lower in N compared to male factor categories (MMF and SMF). Aneuploidy embryos were significantly higher in couples with male factor infertility (MMF and SMF). A positive correlation was observed between fertilization rate (FR) and live birth rate (LBR) with sperm concentration, motility, vitality, normal sperm morphology and negative correlation between sperm DFI and sperm CM. No correlation was observed between embryo aneuploidy and sperm DFI or CM. Lower percentage of spermatozoa chromatin integrity are associated with low fertilization and live birth rate. Male factor infertility, due to impaired semen parameters and chromatin defects could be regarded in future as an indication of IVF/ICSI, and predictor of assisted reproductive techniques outcome. [ABSTRACT FROM AUTHOR]

BACKGROUND: Semen analysis is a routine predictor of male fertility, and however, measurements of sperm morphology, motility, and concentration do not always evince genomic defects. OBJECTIVE: To investigate sperm parameters of renal transplant patients and to evaluate sperm DNA defects. METHODS: Seminal samples from 25 healthy controls and 56 transplantation patients were analyzed to evaluate DNA fragmentation by TUNEL. The differences in TUNEL-assay results and seminal parameters were compared between kidney transplant patients and controls. RESULTS: Among the azoospermic patients, 37.5% had fathered children before the disease. Three patients receiving sirolimus treatment had oligoasthenoteratozoospermia and infertility. In kidney transplant patients, DNA fragmentation was slightly higher than controls. Total motility (%) of the spermatozoa from the kidney transplant patients (42.2 ± 21.9) was significantly lower (P<0.05) than those of the control group (64.3 ± 11.9). Moreover, control individuals had significantly higher (P<0.05) normal morphology (23.2%) compared to the patient group (20.3%). Concerning sirolimus treatment, three patients had severe oligoasthenoteratozoospermia in their ejaculate, and however, DNA fragmentation rates were not significantly higher than those in the remaining individuals of the transplant group. CONCLUSIONS: The sperm DNA fragmentation rate in kidney transplant patients was slightly higher than in the control group (P = 0.09). However, the amount of spermatozoa DNA damage may lead to infertility in kidney transplant patients. [ABSTRACT FROM AUTHOR]

In men from the general population, BMI has been associated with a lower sperm DNA fragmentation index (DFI). We wondered whether this could be due to estradiol, which is associated with BMI and reported important for sperm function. Our objective was to investigate the association between estradiol and DFI. In 2008–2010, we recruited 284 young men from the general population to deliver samples of semen and blood and answer questionnaires. Serum concentrations of reproductive hormones and DFI were analyzed, the latter using the Sperm Chromatin Structure Assay. Associations were studied using general linear models. The first model utilized metric values of estradiol, whereas the second model compared men with high and low levels, dichotomized by the median value. A possible interaction between estradiol and testosterone was also examined. When investigating metric estradiol levels and DFI, an inverse association was seen without adjustments (p =.02), but the statistical significance was lost at adjustments for potential confounders (p =.08). Men with lower estradiol levels (<88 pmol/L, mean 71 pmol/L) had a statistically significantly higher DFI than men with higher levels of estradiol (≥88 pmol/L, mean 110 pmol/L). Mean ratio difference was 1.21 (p =.002) without adjustments and 1.18 (p =.01) with adjustments. A statistically significant difference in DFI was observed in men with testosterone levels below median when comparing high and low estradiol (p <.001). This study supports the idea that serum estradiol levels are protective for sperm DNA integrity, at least at lower testosterone levels. [ABSTRACT FROM AUTHOR]

The development of new biomarkers for human male infertility is crucial to improve the diagnosis and the prognosis of this disease. Recently, seminal microbiota was shown to be related to sperm quality parameters, suggesting an effect in human fertility and postulating it as a biomarker candidate. However, its relationship to sperm DNA integrity has not been studied yet. The aim of the present study is to characterize the seminal microbiota of a western Mediterranean population and to evaluate its relationship to sperm chromatin integrity parameters, and oxidative stress. For that purpose, 14 samples from sperm donors and 42 samples from infertile idiopathic patients were obtained and were analyzed to assess the composition of the microbiota through full-length 16S rRNA gene sequencing (Illumina MiSeq platform). Microbial diversity and relative abundances were compared to classic sperm quality parameters (macroscopic semen parameters, motility, morphology and concentration), chromatin integrity (global DNA damage, double-stranded DNA breaks and DNA protamination status) and oxidative stress levels (oxidation-reduction potential). The seminal microbiota observed of these samples belonged to the phyla Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. The most abundant genera were Finegoldia, Peptoniphilus, Anaerococcus, Campylobacter, Streptococcus, Staphylococcus, Moraxella, Prevotella, Ezakiella, Corynebacterium and Lactobacillus. To our knowledge, this is the first detection of Ezakiella genus in seminal samples. Two clusters of microbial profiles were built based on a clustering analysis, and specific genera were found with different frequencies in relation to seminal quality defects. The abundances of several bacteria negatively correlate with the sperm global DNA fragmentation, most notably Moraxella, Brevundimonas and Flavobacterium. The latter two were also associated with higher sperm motility and Brevundimonas additionally with lower oxidative-reduction potential. Actinomycetaceae, Ralstonia and Paenibacillus correlated with reduced chromatin protamination status and increased double-stranded DNA fragmentation. These effects on DNA integrity coincide in many cases with the metabolism or enzymatic activities of these genera. Significant differences between fertile and infertile men were found in the relative presence of the Propionibacteriaceae family and the Cutibacterium, Rhodopseudomonas and Oligotropha genera, which supports its possible involvement in male fertility. Our findings sustain the hypothesis that the seminal microbiome has an effect on male fertility.

Recurrent pregnancy loss is a distressing condition affecting 1–2% of couples. Traditionally investigations have focused on the female, however more recently researchers have started to explore the potential contribution of the male partner. Seminal reactive oxygen species have a physiological function in male reproduction but in excess are suspected to generate structural and functional damage to the sperm. Evidence is mounting to support an association between elevated seminal reaction oxygen species and recurrent pregnancy loss. Studies suggest that the rates of sperm DNA damage are higher in the male partners of women affected by recurrent pregnancy loss compared with unaffected men. However, the available pool of data is conflicting, and interpretation is limited by the recent change in nomenclature and the heterogeneity of study methodologies. Furthermore, investigation into the effects of oxidative stress on the epigenome show promise. The value of antioxidant therapy in the management of recurrent pregnancy loss currently remains unclear.

Objectives: To identify the DNA comethylation patterns associated with sperm DNA fragmentation (SDF) and to explore the potential associations of hub genes with SDF.Design: Prospective study.Setting: University-affiliated reproductive medicine center.Patient(s): A total of 300 male patients consulting for couple infertility were recruited from the First Affiliated Hospital of Wenzhou Medical University.Intervention(s): None.Main Outcome Measure(s): Comethylation network analysis based on the genome-wide methylation profile of spermatozoal DNA from 20 men was performed to identify hub modules and genes involved in SDF. Human spermatozoa were used for targeted bisulfite amplicon sequencing (267 men) or droplet digital polymerase chain reaction (45 men). The potential role of Brca1 in DNA damage was explored in mouse GC2 spermatocyte cells. Oxidative damage to spermatocytes was modeled by incubating GC2 cells with H2O2 (25 mM) for 90 minutes.Result(s): BRCA1 was identified as a hub gene in SDF. Promoter hypermethylation of BRCA1 was observed in those samples with a high DNA fragmentation index (DFI) compared to those with a low DFI. Concomitantly, BRCA1 mRNA expression was lower in samples with a high DFI than with a low DFI. In the GC2 cell model, Brca1 knockdown reduced cell proliferation and increased sensitivity to oxidative stress. Moreover, it increased double-strand breaks and decreased the protein levels of the DNA repair genes MRE11 and RAD51.Conclusion(s): A prominent cluster of comethylated patterns associated with SDF was identified. BRCA1 may be the hub gene involved in sperm DNA damage. [ABSTRACT FROM AUTHOR]

Background: Microfluidics (MF), an advanced sperm sorting technology results in the extraction of spermatozoa with higher DNA integrity and lower DNA damage compared to existing conventional sperm sorting methods. Aims: The aim of the present study is to assess the efficiency of MF and to isolate the best spermatozoa for intracytoplasmic sperm injection (ICSI) over the density gradient (DG) technique. Settings and Design: We recruited couples who choose the oocyte donation programme for this study to eliminate confounding factors associated with oocyte quality. Materials and Methods: Sperm was processed by MF (n = 180) and DG (n = 151). ICSI was performed and positive pregnancy, miscarriage and clinical pregnancy rates were compared. Statistical Analysis Used: All variables were analysed using Graph Pad Prism 5. The unpaired two-tailed t-test was used to assess the significance. A value of P < 0.05 was considered statistically significant. Results: There was no significant difference in pregnancy rates between the groups. However, a clear demarcation is seen in terms of clinical pregnancy rates, where the DG group achieved higher clinical pregnancies (91.7%) compared to the MF group (80.7%). Further, we compared miscarriage rates and biochemical pregnancies, and found a significantly higher miscarriage and biochemical pregnancy rate in the MF group (14.5% and 4%, respectively) compared to the DG group (6% and 1%, respectively). Conclusions: Based on the available literature, we anticipated a higher clinical pregnancy rate with MF compared with conventional processing. Our results show MF does not have any add-on positive effect on clinical pregnancy rate. [ABSTRACT FROM AUTHOR]

Background: The assessment of sperm DNA integrity has been proposed as a complementary test to conventional mammalian semen analysis. In this sense, single-strand (SSB) and double-strand (DSB) DNA breaks, the two types of sperm DNA fragmentation (SDF), have been reported to have different aetiologies and to be associated to different fertility outcomes in bovine and humans. Considering that no studies in porcine have addressed how SDF may affect sperm quality and fertility outcomes, the present work aimed to determine the impact of global DNA damage, SSB and DSB on sperm quality and in vitro fertilising ability. To this end, 24 ejaculates (one per boar) were split into three aliquots: the first was used to assess sperm quality parameters through a computer-assisted sperm analysis (CASA) system and flow cytometry; the second was used to perform in vitro fertilisation, and the third, to evaluate sperm DNA integrity using alkaline and neutral Comet assays. Results: The results showed that global DNA damage negatively correlates (P < 0.05) with normal sperm morphology (R = − 0.460) and progressive motility (R = − 0.419), and positively with the percentage of non-viable sperm (R = 0.507). Multiple regression analyses showed that non-viable sperm were related to SSB (β = − 0.754). In addition, while fertilisation did not seem to be affected by sperm DNA integrity, global DNA damage, DSB and SSB were found to be correlated to embryo development outcomes. Specifically, whereas global DNA damage and DSB negatively affected (P < 0.05) the later preimplantation embryo stages (percentage of early blastocyst/blastocyst D6: for global DNA damage, R = − 0.458, and for DSB, R = − 0.551; and percentage of hatching/hatched blastocyst D6: for global DNA damage, R = − 0.505, and for DSB, R = − 0.447), global DNA damage and SSB had a negative impact (P < 0.05) on the developmental competency of fertilised embryos (R = − 0.532 and R = − 0.515, respectively). Remarkably, multiple regression analyses supported the associations found in correlation analyses. Finally, the present work also found that the inclusion of Comet assays to the conventional sperm quality tests improves the prediction of blastocyst formation (AUC = 0.9021, P < 0.05), but not fertilisation rates (P > 0.05). Conclusion: Considering all these findings, this work sets a useful model to study how SDF negatively influences fertility. [ABSTRACT FROM AUTHOR]

Purpose: The aims of this study were to associate sperm kinematics and standard semen parameters with sperm DNA damage and to evaluate whether the addition of sperm kinematics improve the multivariable prediction of sperm DNA fragmentation compared to standard semen parameters alone. Materials and Methods: We evaluated sperm kinematics, standard semen parameters, and DNA fragmentation index (DFI) in 122 men. Univariate and multivariate logistic regression models were fitted to evaluate the association of sperm kinematics and standard semen parameters with pathologically damaged sperm DNA (DFI≥26%), and receiver operating characteristics (ROC) curves were calculated for these models. Results: On univariate analyses, average velocity, curvilinear velocity, straight-line velocity, straightness (STR), beat-cross frequency (BCF), and the percentage of progressive motile sperm cells (PPMS) were significantly associated with pathologically damaged sperm DNA. Likewise, among standard semen parameters, sperm concentration, progressive motility, normal morphology, and vitality were found to be linked with sperm DNA damage. On the multivariate analysis, vitality was the strongest predictor of pathologically damaged sperm DNA with an area under the ROC curve (AUROC) of 88.3%. Adding STR, BCF, and PPMS to vitality increased the AUROC to the significant extent of 91.5%. Conclusions: Sperm vitality is the most accurate routine-based laboratory test for the prediction of pathologically damaged sperm DNA, but the addition of sperm kinematics increases its accuracy. Both standard semen parameters and sperm kinematics are complementary in predicting pathologically damaged sperm DNA, and might serve as a new tool to screen for fertile men.

Objective To investigate the association between exposure to polycyclic aromatic hydrocarbons(PAHs) in atmospheric fine particulate matter(PM2.5) and sperm DNA damage in Chongqing. Methods The subjects were sampled from our previously established cohort of the male reproductive health in Chongqing college students(MARHCS) study. The concentrations of PAHs(collected during the sampling periods) with low fused aromatic rings(2~4 rings) and with high fused aromatic rings(5~6 rings) in the air PM2.5 filter membranes were detected. Single cell gel electrophoresis(Comet assay) was used to evaluate DNA damage. Finally, the mixed-effects model was used to investigate the relationships between the exposure concentrations of PAHs components in PM2.5 and severity of sperm DNA damage. Results In 2013, the low molecular weight(LMW) PAHs accounted for a large proportion in atmospheric fine particles, mainly phenanthrene, fluorene and pyrene. While, the high molecular weight(HMW) PAHs dominated in 2014, such as indeno[1, 2, 3-cd]pyrene, benzo[g, h, i]perylene and benzo[a]pyrene. Sperm DNA damage indicators, such as tail length, tail moment and Olive tail moment were more common in the cohort in the year of 2014 than in 2013(all P < 0.05). The results of the mixed model showed that the concentrations of fluoranthene, acenaphthene, pyrene and ∑LMW PAHs were negatively correlated with tail length and tail moment. The concentrations of benzo[k]fluoranthene, benzo[b]fluoranthene and dibenz[a, h]anthracene were positively correlated with tail length [β(95%CI): 108.987(43.754~203.820), 158.700(65.957~303.272), 185.708(67.350~387.775), respectively; all corrected P < 0.05] and with tail moment [β(95%CI): 449.204(260.066~737.695), 683.108(374.515~1192.387), 683.108(374.515~1 192.387), respectively; all corrected P < 0.05]. Conclusion The exposure of HMW PAHs in Chongqing atmospheric fine particles may lead to the enhancement of sperm DNA damage.

Dimethoate is an organophosphorus pesticide used against agricultural insects, which causes oxidative stress and damage in many organs, including the reproductive ones. Cherry laurel (Laurocerasus officinalis Roem.) fruit is rich in vitamins and phenolic compounds with antioxidant effect. The aim of this study was to investigate how effective its extract would be against dimethoate-induced testis and sperm damage in rats. Sixty animals were divided in six groups of 10. Group 1 (control) received only 1 mL of saline (0.9 % NaCl). Group 2 received 7 mg/kg of dimethoate in 1 mL of saline. Group 3 received 4 mg/kg of extract in 1 mL of saline. Group 4 received the extract 30 min before dimethoate administration. Group 5 received vitamin C (positive control, 100 mg/kg in 1 mL of saline) 30 min before dimethoate administration. Group 6 received only dimethoate for the first four weeks and then a combination of dimethoate and extract for another four weeks. All doses were administered daily by oral gavage. After eight weeks of treatment, the rats were euthanised and their reproductive organs removed. We took their body and reproductive organ weights and evaluated testicular oxidative stress, semen characteristics, sperm DNA damage, testicular apoptosis, and histopathological changes. Dimethoate significantly decreased body and reproductive organ weights, sperm motility and concentration, testicular superoxide dismutase, and glutathione-peroxidase activities and significantly increased lipid peroxidation, abnormal sperm rate, sperm DNA damage, testicular apoptosis, and caused histopathological lesions. Cherry laurel extract significantly countered many dimethoate-induced adverse effects, both as pre- and post-treatment, including reproductive organ weight, semen parameters, oxidant-antioxidant balance, sperm DNA integrity, testicular apoptosis, and histological structure. Our findings clearly suggest that the beneficial effects of the extract are associated with countering oxidative stress, lipid peroxidation in particular.

Maintaining genome integrity in germ cells is essential not only for successful fertilization and embryo development, but also to ensure proper transmission of genetic information across generations. However, unlike oocytes, sperm are incapable of repairing DNA damage. Therefore, sperm DNA damage is repaired after fertilization in zygotes using maternal DNA repair factors. In this study, we found that zygotic repair of paternal DNA damage is enhanced by inhibiting WIP1 activity. Oxidative stress induced DNA damage in sperm and severely impaired motility. Although DNA damage in sperm did not compromise fertilization, it increased DNA damage in the paternal pronucleus of zygotes. However, WIP1 inhibition during fertilization reduced DNA damage in the paternal pronucleus, improving the rate of two-cell development, and subsequent zygotic genome activation. Therefore, our results suggest that WIP1 inhibition could enhance maternal DNA repair capacity and thereby decrease paternal DNA damage in zygotes.