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Numerous studies have demonstrated the detrimental effects of sperm DNA fragmentation (SDF) on reproductive outcomes, whether under natural or assisted conditions. The sperm DNA fragmentation (SDF) test has emerged as a valuable supplement in the evaluation of infertile men, and the rapid development of various SDF tests has enhanced the perception of its clinical utility. Consequently, the latest manual from the World Health Organization has determined that this test should be described as a specialized analysis (and thus applicable in many laboratories worldwide) rather than a research analysis. However, the normal reference ranges for these tests to evaluate male fertility potential remain controversial. This article addresses the pathophysiological aspects of sperm chromatin and the mechanisms underlying DNA fragmentation. The effects of this fragmentation on various reproductive outcomes are also reviewed. An analysis of the different available fragmentation tests is provided as well. Finally, we discuss preventive and therapeutic measures to reduce sperm DNA fragmentation and potential future research opportunities. [ABSTRACT FROM AUTHOR]

Today, plastic pollution is a widespread problem in all ecosystems and has a particularly severe impact on marine ecosystems and external fertilisers such as the mussel Mytilus galloprovincialis. The present study aims to assess the toxicological reproductive health effects in this organism following exposure to two concentrations of polystyrene microplastics (PS-MPs) (0.5 and 1 μg/L), representative of conditions in the Mediterranean Sea. After exposure, the electrophoretic pattern of protamine-like (PL) proteins, the major basic protein component of Mytilus galloprovincialis sperm chromatin, was analysed. Compared to the unexposed condition, differences were observed by SDS-PAGE and an increased ability of PL to bind and protect DNA from oxidative damage was then measured, particularly for PL from mussels exposed to 1 μg/L PS-MPs. At this dose of PS-MPs, a reduced release of all PLs from the sperm nuclei was also observed, whereas the digestion by micrococcal nuclease did not show any significant differences between the exposed and the unexposed conditions. Finally, the possibility of poly(ADP)-ribosylation of the PLs was investigated. PL-II showed an increase in poly(ADP)-ribosylation after PS-MPs exposure, which may account for the difference in the ability of the PLs to bind DNA. In conclusion, while all the results might suggest a molecular mechanism of gametic plasticity occurring upon exposure of mussels to PS-MPs 1 μg/L, they also indicate that this dose of exposure could be extremely detrimental to the reproductive health of Mytilus galloprovincialis because it could prevent the release of basic nuclear proteins from the sperm DNA at fertilisation. • Microplastics cause an increase of ɣH2AX a marker of DNA double strand break. • Microplastics produce changes in Mytilus galloprovincialis PL to DNA binding. • Microplastic exposure increased poly(ADP)-ribosylation in PL-II and H1 histone. • 1 μM/mL polystyrene microplastic reduces the release of PLs from the sperm nuclei. • Microplastics have toxicological effects and a potential reproductive risk. [ABSTRACT FROM AUTHOR]

Male fertility potential depends on physical, endocrine, and genetic factors responsible for producing functional male gametes. Although the main function of the male gamete, the spermatozoon, is to deliver its genetic material to the oocyte, this premise has been modified over the past few years. It is believed that the spermatozoon provides essential factors for fertilization and pre-implantation embryo development. A viable/healthy spermatozoon has functional subcellular compartments (nucleus, acrosome, midpiece, and flagellum) due to the actions of proteins, transcripts, and epigenetic marks in the organelles present in them that have important roles in reproductive biology. Male fertility potential reflects viable spermatozoa with proper function. Therefore, new approaches to functional sperm analysis are essential. Additionally, intrinsic factors and sperm molecules constitute potential biomarkers of viable spermatozoa and male fertility. Among these factors are proteins, the genome, and coding and non-coding RNAs, such as microRNAs, that act during fertilization and early embryo development. Research has been seeking increasingly efficient tools to predict fertility and functional studies of these molecules through gene and protein expression. Thus, analytical tools are essential to identify and classify viable and functional spermatozoa, to evaluate assisted reproductive male potential. • The evaluation of sperm function could involve more than usual analyses. • The knowledge of sperm morphology and function can assist in the interpretation and prediction of male fertility. • There is a direct relationship between the male subject and the fertility. • The sperm intrinsic factors such as microRNAs confer individual features that modulate male fertility. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Do defects in sperm chromatin protamination and condensation have an impact on ICSI outcomes? SUMMARY ANSWER Sperm protamination is related to fertilization rates in healthy donors, and the in vitro capacity of sperm to condense their chromatin is linked to blastocyst rates, both associations being more apparent in women <33 years of age. WHAT IS KNOWN ALREADY Previous data on how sperm chromatin damage affects ICSI outcomes are inconsistent. Revealing which sperm factors influence embryo development is necessary to understand the male contribution to ICSI success and to develop novel sperm selection techniques or male-based treatments. Sperm chromatin is mainly condensed in protamines, which are cross-linked through disulphide bridges. This study aimed to determine whether sperm protamination and the integrity of disulphide bonds (condensation) are related to embryo development after ICSI. STUDY DESIGN, SIZE, DURATION The design was a retrospective study with a blind analysis of sperm chromatin. Gametes were divided into two groups: double donation (DD) cohort and single donation (SD) cohort. Samples from 45 semen donors used in 55 ICSI cycles with oocyte donors (age range 19–33 years), generating 491 embryos, were included in the DD cohort. The SD cohort consisted of samples from 34 semen donors used in 41 ICSI cycles with oocytes from healthy females (single-parent families or lesbian couples, age range 20–44 years), generating a total of 378 embryos. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Donor sperm samples from DD and SD cohorts were used for standard ICSI, and embryo development was observed by time-lapse imaging. The incidence of thiol reduction (dibromobimane, DBB) and the degree of chromatin protamination (chromomycin A3, CMA3, indicating non-protaminated regions) in sperm were determined by flow cytometry at 0 and 4 h post-thawing. MAIN RESULTS AND THE ROLE OF CHANCE Percentages ± standard deviation of CMA3 were 21.08 ± 9.09 and 35.01 ± 14.68 at 0 and 4 h post-thawing, respectively, in the DD cohort and 22.57 ± 9.48 and 35.79 ± 12.58, at 0 and 4 h post-thawing, respectively, in the SD cohort. Percentages of DBB+ were 16.57 ± 11.10 and 10.51 ± 8.40 at 0 and 4 h post-thawing (P  <   0.0001), respectively, in the DD cohort and 17.98 ± 10.19 and 12.72 ± 8.76 at 0 and 4 h post-thawing (P  <   0.0001), respectively, in the SD cohort. Female age correlated with fertilization rates, and the relation between sperm chromatin and embryo development was determined through multiple linear regression. While CMA3 was associated with fertilization rates, with no influence of female age, in the DD cohort (β1 = −1.036, P  <   0.001 for CMA3; β2 = 0.667, P  =   0.304 for female age), this was not observed in the SD cohort, where female age had a significant effect, masking the effects of CMA3 (β1 = −0.066, P  =   0.804 for CMA3; β 2 = −1.451, P  =   0.003 for female age). The in vitro capacity of sperm to condense their chromatin after 4 h of incubation was associated with blastocyst rates, independent of female age (DD cohort: β1 = −0.238, P  =   0.008 for %DBB+ variation; β2 = 0.404, P  =   0.638 for female age; SD cohort: β1 = −0.278, P =  0.010 for %DBB+ variation; β2 = −0.292, P  =   0.594 for female age). The in vitro capacity of sperm to condense their chromatin was also related to the time required for the embryo to reach blastocyst stage in the DD cohort (P  =   0.007). Finally, multiple logistic regression showed that both chromatin protamination and condensation, together with the age of the oocyte donors and the embryo recipients, had an impact on pregnancy achievement (P  <   0.01) and on live birth rates (P  <   0.01). LIMITATIONS, REASONS FOR CAUTION The main limitation was the restrictive selection of couples, which led to a relatively small sample size and could influence the observed outcomes. For this reason, and to reduce Type I error, the level of significance was set at P ≤  0.01. On the other hand, the use of cryopreserved samples could also be a limitation. WIDER IMPLICATIONS OF THE FINDINGS This research demonstrated that protamination and condensation of sperm chromatin are related to embryo development after ICSI, but female age could be a confounding factor when oocytes from older females are used. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the European Union's Horizon 2020 Research and Innovation scheme under the Marie Skłodowska-Curie grant agreement No 801342 (Tecniospring INDUSTRY; TECSPR-19-1-0003); La Marató de TV3 Foundation (214/857-202039); the Ministry of Science and Innovation, Spain (IJC2019-039615-I); the Catalan Agency for Management of University and Research Grants, Regional Government of Catalonia, Spain (2017-SGR-1229); and the Catalan Institution for Research and Advanced Studies, Spain (ICREA). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Nickel is associated with reproductive toxicity, but little is known about the molecular mechanisms of nickel-induced effects on sperm chromatin and protamine-like proteins (PLs). In the present work, we analyzed PLs from Mytilus galloprovincialis by urea-acetic acid polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and assessed their binding to DNA by Electrophoretic Mobility Shift Assay (EMSA) after exposing mussels to 5, 15, and 35 µM NiCl2 for 24 h. In addition, a time course of digestion with MNase and release of PLs from sperm nuclei by the NaCl gradient was performed. For all exposure doses, in AU-PAGE, there was an additional migrating band between PL-III and PL-IV, corresponding to a fraction of PLs in the form of peptides detected by SDS-PAGE. Alterations in DNA binding of PLs were observed by EMSA after exposure to 5 and 15 µM NiCl2, while, at all NiCl2 doses, increased accessibility of MNase to sperm chromatin was found. The latter was particularly relevant at 15 µM NiCl2, a dose at which increased release of PLII and PLIII from sperm nuclei and the highest value of nickel accumulated in the gonads were also found. Finally, at all exposure doses, there was also an increase in PARP expression, but especially at 5 µM NiCl2. A possible molecular mechanism for the toxic reproductive effects of nickel in Mytilus galloprovincialis is discussed. [ABSTRACT FROM AUTHOR]

Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote. [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]

Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm chromatin protamination in three subpopulations of fertile normozoospermic controls and infertile patients with oligo-/oligoasthenozoospermia. For the first time, a sequential staining protocol was applied, which allowed researchers to analyse 5mC/5hmC levels by immunofluorescence staining, with a previously determined chromatin protamination status (aniline blue staining), using the same spermatozoa. TUNEL assay determined the sperm DNA fragmentation level. The 5mC/5hmC levels were diversified with respect to chromatin protamination status in both studied groups of males, with the highest values observed in protaminated spermatozoa. The linkage between chromatin protamination and 5mC/5hmC levels in control males disappeared in patients with deteriorated semen parameters. A relationship between 5mC/5hmC and sperm motility/morphology was identified in the patient group. Measuring the 5mC/5hmC status of sperm DNA according to sperm chromatin integrity provides evidence of correct spermatogenesis, and its disruption may represent a prognostic marker for reproductive failure. [ABSTRACT FROM AUTHOR]

The purpose of this study was to observe the effect of different thawing methods on semen parameters such as motility and morphology and sperm chromatin integrity as assessed by toluidine blue (TB) staining. A total of 20 frozen sperm straws from the same Holstein bull were used. While the 30 sec thawing protocol at 37°C, which is used for thawing frozen sperm straws, constitutes our slow thawing group (n=10), the 6 sec thawing protocol at 70°C constitutes our fast-thawing group (n=10). The motility, viability, morphology, plasma membrane integrity, and sperm chromatin condensation parameters of all thawed sperm were investigated. There was a significant difference (P<0.05) in sperm plasma membrane integrity, head defect, and total abnormal sperm morphology. The chromatin decondensation rate detected by TB in bull semen thawed in the slow thaw system, and the decondensation rate in the fast thaw system, differed significantly from each other in line with the literature data (P<0.05). According to the evaluations made in terms of chromatin decondensation rate, the rate obtained in slow thawing (7±0.39) shows an increase up to two times compared to the fast-thawing rate (3.3±0.33) (P<0.05). The TB staining procedure can be used to evaluate infertility and chromatin integrity, especially in cases that are suspicious and require rapid evaluation. [ABSTRACT FROM AUTHOR]

Chromium (VI) is the most dangerous oxidation state among the stable forms of chromium. In this work, we evaluated the effect of exposing Mytilus galloprovincialis for 24 h to 1, 10, and 100 nM chromium (VI) on the properties of Protamine-like (PLs) and their gene levels in the gonads. Specifically, we analyzed, by AU-PAGE and SDS-PAGE, PLs extracted from unexposed and exposed mussels. In addition, via EMSA, we evaluated the ability of PLs to bind DNA and also verified their potential to protect DNA from oxidative damage. Finally, we assessed possible alterations in gonadal expression of mt10, hsp70, and genes encoding for PLs-II/PL-IV and PL-III. We found that for all experimental approaches the most relevant alterations occurred after exposure to 1 nM Cr(VI). In particular, a comigration of PL-II with PL-III was observed by SDS-PAGE; and a reduced ability of PLs to bind and protect DNA from oxidative damage was recorded. This dose of chromium (VI) exposure was also the one that produced the greatest alterations in the expression of both mt10 and PL-II/PL-IV encoding genes. All of these changes suggest that this dose of chromium (VI) exposure could affect the reproductive health of Mytilus galloprovincialis. [ABSTRACT FROM AUTHOR]

Sperm chromatin is distinct from somatic cell chromatin, as a result of extensive remodeling during the final stages of spermatogenesis. In this process, the majority of histones is replaced with protamines. The chromatin is consequently highly condensed and inert, which facilitates protection of the DNA. The sperm epigenomic landscape is shaped by histone retention, histone and protamine modification, DNA methylation, and RNAs. In recent years, sperm chromatin integrity and its epigenetic marks have been increasingly studied, and the constitution of sperm chromatin is steadily being uncovered. This growing body of research prompts assessment of the frequently overlooked involvement of sperm in fertility and embryonic development. Moreover, numerous endogenous and exogenous factors are known to affect sperm chromatin, which may in turn impact the reproductive success. Concerns have been raised about the effects of assisted reproductive technology (ART) on the sperm epigenome, embryonic development and offspring health. This review examines the structure and epigenetic signatures of sperm chromatin in the context of fertility and early embryonic development. Additionally, sperm chromatin evaluation and causes of aberrant integrity are outlined. Building on the knowledge discussed in the current review, future research should aim to elucidate the intricate relationship between all aspects of sperm chromatin and embryo development. This could lead to the uncovering of new targets for treating infertility, as well as the acquisition of much needed insights into the possible reciprocal association between ART and sperm chromatin integrity. • Sperm chromatin is extensively remodeled during spermiogenesis. • Crosstalk between epigenetic signatures of DNA, histones, protamines and RNA shapes the sperm epigenome. • The relationship between sperm chromatin integrity and embryonic development requires further investigation. • Sperm chromatin protamination, condensation and integrity are crucial for reproductive success. [ABSTRACT FROM AUTHOR]

Background: Formalin is commonly applied as an antiseptic and tissue fixative. It has reactive molecules that lead to its cytotoxic effects. According to recent studies, formalin causes a change in the testicular and sperm structure and L-carnitine (LC) acts as an antioxidant to counteract its effects. Objective: This study aimed to investigate the protective effects of LC on the parameters, chromatin condensation and apoptosis of mice sperm exposed to formalin. Materials and Methods: In this experimental study, 24 balb/c mice (25-40 gr,10-12 wk) were divided into three groups (n = 8/each): group I without any injections or gavage; group II, received 10 mg/ kg formalin intraperitoneally (I.P); and group III was exposed to formalin and LC, where a dose of 10 mg/kg formalin was injected I.P daily and LC the dose of 100 mg/kg was kept in a solvent solution. After 31 days, the sperm examination was performed as follows: to evaluate chromatin and DNA quality of the sperm, we applied aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), and terminal transferase-mediated deoxy uridine triphosphate biotin end labeling (TUNEL) tests. Results: Sperm parameters such as count, motility, morphology, and viability displayed a significant decrease in the formalin group. While the data exhibited a considerable augment in sperm parameters in the formalin + LC than the formalin and control groups (p < 0.001), significant differences were detected between groups with respect to TB staining, TUNEL test, CMA3 test and AB staining in the formalin and formalin + LC groups. Conclusion: LC can reduce the negative effects of formalin on sperm parameters, chromatin stability, and percentage of apoptosis in an animal model. [ABSTRACT FROM AUTHOR]

Centrifugation of boar semen through one layer of 40% colloid (Porcicoll) was previously shown to separate spermatozoa from bacteria without having a detrimental effect on sperm quality. However, some spermatozoa were lost. The purpose of the present study was to determine whether 20% or 30% Porcicoll could be used to recover most of the spermatozoa without impacting on sperm quality. Insemination doses (n = 10) from a commercial boar station were sent to the laboratory at the Swedish University of Agricultural Sciences and processed by Single Layer Centrifugation with 20% and 30% Porcicoll approximately 7 hr after semen collection. The resulting sperm samples and controls were evaluated for sperm quality immediately and again after storage at 16–18°C for 4 and 7 days. Sperm recovery was 94 ± 18% and 87 ± 15% for 20% and 30% Porcicoll, respectively (p >.05). Sperm mitochondrial membrane potential and chromatin integrity were unaffected (p >.05). The proportion of live spermatozoa producing superoxide (9 ± 8%, 7 ± 6% and 3 ± 1%; p <.05), and the proportion of spermatozoa with high stainability DNA (0.68 ± 19%, 0.61 ± 0.22% and 0.96 ± 0.23%; p <.05‐ <0.01), were marginally increased whereas membrane integrity, although high, was lower in the centrifuged samples than in the controls (82 ± 8%, 83 ± 5% versus 92 ± 4%; p <.05). In conclusion, centrifugation through 20% or 30% Porcicoll enables most spermatozoa to be recovered, without having a major effect on sperm quality. These results are encouraging for further studies involving microbiological investigation of the processed samples, and scaling‐up to process larger volumes of boar ejaculates. [ABSTRACT FROM AUTHOR]

Background: Spermatogenesis is the process by which germ cells develop into spermatozoa in the testis. Sperm protamines are small, arginine-rich nuclear proteins which replace somatic histones during spermatogenesis, allowing a hypercondensed DNA state that leads to a smaller nucleus and facilitating sperm head formation. In eutherian mammals, the protamine-DNA complex is achieved through a combination of intra- and intermolecular cysteine cross-linking and possibly histidine-cysteine zinc ion binding. Most metatherian sperm protamines lack cysteine but perform the same function. This lack of dicysteine cross-linking has made the mechanism behind metatherian protamines folding unclear. Results: Protamine sequences from UniProt's databases were pulled down and sorted into homologous groups. Multiple sequence alignments were then generated and a gap weighted relative entropy score calculated for each position. For the eutherian alignments, the cysteine containing positions were the most highly conserved. For the metatherian alignment, the tyrosine containing positions were the most highly conserved and corresponded to the cysteine positions in the eutherian alignment. Conclusions: High conservation indicates likely functionally/structurally important residues at these positions in the metatherian protamines and the correspondence with cysteine positions within the eutherian alignment implies a similarity in function. One possible explanation is that the metatherian protamine structure relies upon dityrosine cross-linking between these highly conserved tyrosines. Also, the human protamine P1 sequence has a tyrosine substitution in a position expecting eutherian dicysteine cross-linking. Similarly, some members of the metatherian Planigales genus contain cysteine substitutions in positions expecting plausible metatherian dityrosine cross-linking. Rare cysteine-tyrosine cross-linking could explain both observations. [ABSTRACT FROM AUTHOR]

Background: Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa. Objectives: The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis. Materials and methods: For this purpose, absolute q‐RT‐PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry. Results: We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results. Conclusion: Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility. [ABSTRACT FROM AUTHOR]

A large number of heavy metals resulted toxic to the reproductive system, but invertebrate infertility has been poorly explored, and above all, there are limited molecular, cellular and toxicological studies. In the present work, we exposed Mytilus galloprovincialis to three individual metal chlorides (CuCl 2 15 μM, CdCl 2 1.5 μM, NiCl 2 15 μM) and their mixture for 24 h, to evaluate the effects on the protamine-like proteins (PLs), sperm DNA and on their interaction in the formation of sperm chromatin. Under all exposure conditions, but particularly after exposure to the metals mix, relevant changes in the electrophoretic pattern, by AU-PAGE and SDS-PAGE, and in fluorescence spectroscopy measurements of PLs were shown. In addition, alterations in DNA binding of these proteins were observed by Electrophoretic Mobility Shift Assay (EMSA) and through their release from sperm nuclei. Moreover, there was evidence of increased accessibility of micrococcal nuclease to sperm chromatin, which was also confirmed by toluidine blue staining. Furthermore, morphological analyses indicated severe gonadal impairments which was also corroborated by increased PARP expression, by Western blotting, and sperm DNA fragmentation, by comet assay. Finally, we investigated the expression of stress genes, gst , hsp70 and mt10 , in gonadal tissue. The latter investigations also showed that exposure to this metals mix was more harmful than exposure to the individual metals tested. The present results suggest that these metals and in particular their mixture could have a negative impact on the reproductive fitness of M. galloprovincialis. Based on these evidences, we propose a molecular mechanism. [Display omitted] • PLs-DNA binding is affected by mussels exposure to specific mixtures of heavy metals • Interaction between these heavy metals enhances DNA damage • Heavy metals and their mixture cause a disconnection of germ cells in gonads • Possible toxicological mechanisms of heavy metals action on M. galloprovincialis • Heavy metals cause a different chromatin condensation in spermatozoa [ABSTRACT FROM AUTHOR]

Objective: Fibronectin (FN) is a multifunctional diametric glycoprotein on the surface of the sperm that plays an important role in the sperm-oocyte interaction and fertilization process. The aim of the present study was to assess the FN levels as a sperm surface biomarker for sperm selection in assisted reproductive technology. Material and methods: Polyclonal antibody against human FN was produced in rabbit. Its quality, purity, and immune reactivity were assessed by SDS-PAGE and western blot. In addition, the presence of FN on the sperm surface was assessed through immunocytochemistry and flow cytometry. The amount of FN and the sperm quality were assessed in normozoospermia (N) (42 men) and asthenoteratozoospermia (AT) (72 men) groups through sperm chromatin dispersion (SCD), sperm chromatin structure assay (SCSA), and chromatin maturation index (CMI). Results: The results showed the distribution of FN protein on the equatorial region of the human sperm surface. In addition, the FN levels were found to have a significant difference between the two groups with 24.64±9.08% in N and 16.90±7.27% in AT (p≤0.0001). In addition, FN level negatively correlated with SCD (p≤0.0001), SCSA (p≤0.0001), and CMI (p≤0.001). Threshold values of FN level and DNA fragmentation index (DFI) percentage were 16 and 30 and were identified as cut-off values to determine the N group with a specificity of 83.3% and 81.0% and a sensitivity of 16.8% and 19.0%, respectively. The specificity and sensitivity of FN-DFI were 91.2% and 8.8%, respectively. Conclusion: It appears that FN can be used for the selection of sperm with suitable quality, although future studies are recommended. [ABSTRACT FROM AUTHOR]

Purpose: Advancing maternal and paternal age leads to a decrease in fertility, and hence, many infertile couples opt for assisted reproductive technologies [ART] to achieve biological parenthood. One of the key determinants of achieving a live outcome of ART, embryo quality, depends on both the quality of the oocyte and sperm that have created the embryo. Several studies have explored the effect of oocyte parameters on embryo quality, but the effects of sperm quality on the embryo have not been comprehensively evaluated.Method: In this review, we assess the effect of various genetic factors of paternal origin on the quality and development of the embryo.Results: The effects of sperm aneuploidy, sperm chromatin structure, deoxyribonucleic acid [DNA] fragmentation, role of protamines and histones, sperm epigenetic profile, and Y chromosome microdeletions were explored and found to negatively affect embryo quality.Conclusion: We propose that careful assessment of spermatozoal parameters is essential to achieve embryo development and a healthy live birth. However, the heterogeneity in test results and the different approaches of assessing a single sperm parameter highlight the need for more research and the development of standardized protocols to assess the role of sperm factors affecting embryo quality. [ABSTRACT FROM AUTHOR]

Background: Cryopreserved human sperm are used in assisted reproductive technology. However, the effect of cryopreservation on sperm DNA integrity is unclear. Objectives: The objectives of this study were to: (i) determine the impact of semen cryopreservation on human sperm DNA integrity and chromatin structure; (ii) test if parameters obtained from TUNEL and SCSA® correlate; and (iii) verify correlation between sperm motility, morphology and viability with TUNEL and SCSA® parameters. Materials and methods: Men attending a fertility clinic were recruited and grouped according to their sperm parameters (n = 9/group): normozoospermia, oligoasthenoteratozoospermia and teratozoospermia. Each semen sample was processed as follow: (i) directly frozen at −80 °C; (ii) diluted in Sperm Maintenance Medium, cooled for 30 min at 4 °C and frozen at −80 °C; (iii) diluted in Sperm Maintenance Medium; or (iv) in SpermFreeze. Each mixture from method (iii) and (iv) was then suspended for 30 min in liquid nitrogen vapor and plunged into liquid nitrogen. After at least two months of storage, samples were thawed at room temperature and analyzed for motility and viability, TUNEL and SCSA® assays. Results: Progressive motility and viability decreased after freeze‐thawing. TUNEL scores increased significantly in all samples after freezing‐thawing while no significant change in the DNA fragmentation index (DFI) from SCSA® was observed. No change in the percentage high DNA stainability (HDS) was observed in normozoospermic samples; however it was significantly increased in all the methods in oligoasthenoteratozoospermic and in the methods (ii)–(iv) in teratozoospermic samples. The DFI and TUNEL scores correlated significantly with each other and inversely with sperm motility, viability and morphology. Discussion and conclusion: Cryopreservation seems to be deleterious for the integrity of human sperm DNA and compaction. However, the sperm DFI was not affected during cryopreservation under the various methods of storage tested. Clinicians and investigators should take this information into consideration when using cryopreserved sperm for assisted reproduction. [ABSTRACT FROM AUTHOR]

Background: Prescribing antidepressant drugs is becoming common. These drugs are known to affect sexual functions. Objective: The study is aimed to assess the effects of amitriptyline and venlafaxine on sperm parameters and evaluate Malondialdehyde (MDA) and 1, 1-Diphenyl-2-picryl-hydrazyl values in BALB/mice spermatozoa. Materials and Methods: Forty adult male BALB/c mice were separated into five groups. Group I (control) received distilled water; group II amitriptyline (4 mg/kg); group III amitriptyline (4 mg/kg) +vitamin C (10 mg/kg); group IV venlafaxine (2 mg/kg); and group V received vitamin C (10 mg/kg) + venlafaxine (2 mg/kg). All drugs were administered by oral gavage for 35 days. After excision of caudal epididymis, it was located in 1 mL Ham's F10 medium at 37oC for 15 min and then analysis of sperm parameters was performed. To examine lipid peroxidation and total antioxidant capacity, the MDA and 1, 1-Diphenyl-2-picryl-hydrazyl were measured, respectively. Results: The mean sperm parameters in the group treated with amitriptyline were significantly lower than in the other groups. MDA tests showed a significant difference between amitriptyline and control groups (p=0.007). Conclusion: The results of this study showed that amitriptyline consumption can weaken sperm parameters, which can be attributed to the increased production of ROS and toxicity resulting from amitriptyline consumption. Moreover, venlafaxine improved sperm parameters in mice and the lipid peroxidation in this group did not change compared to the control group. [ABSTRACT FROM AUTHOR]

Summary: The purpose of this study was to investigate the relationship between the proportion of sperm chromatin linked to remaining histone and assisted reproductive technology (ART) outcome. A prospective cohort study was performed on couples undergoing ART process at the Department of Reproduction Medicine (HFME, Bron, France). The histone‐to‐protamine ratio (HPR) was measured using the method described by Wykes & Krawetz (2003) J Biol Chem 278, 29471. The correlations with sperm DFI, blastocyst formation, pregnancy rate, and delivery rate were investigated. A total of 291 ART cycles were included (42 c‐IVF and 249 ICSI procedures): 3870 oocytes were punctured and 2211 embryos were obtained, among which 507 were transferred and 336 frozen. The mean HPR was 18.9%. A significant negative correlation was found between HPR and DFI (r = −0.12, p < 0.05). Regarding the type of ART procedure (c‐IVF or ICSI), the same kind of relationship between HPR and ART parameters was observed. Regardless of the type of ART procedure used, when the HPR was within the range [6%; 26%], the blastocyst formation rate was higher: 87.8% vs. 71.2% (HPR<6%; p < 0.01) and 74.6% (HPR >26%; p < 0.01). The highest delivery rate (DR; 24.5%) was obtained for HPR within the range [6%; 26%]; DR was 21.9% for HPR<6% and 18.3% for HPR>26%; however, the differences were not statistically significant. The procedure described in this study seems to be a reliable evaluation of the HPR. The HPR parameter seems to be correlated to embryonic development up to the blastocyst stage, but its involvement in clinical pregnancy/delivery could not be confirmed. HPR should be further investigated for confirming the relationship with blastocyst formation. After this, the next step will be to investigate the etiologies of HPR alterations for improving the sperm nucleus quality for increasing the chance of pregnancy. [ABSTRACT FROM AUTHOR]

Summary: The present study was performed to investigate the relation of Chlamydia trachomatis infection to sperm chromatin/DNA integrity in a population of infertile men (male partner of infertile couples) from Iran. Blood, semen and first‐void urine samples were obtained from 250 infertile men. Data were analysed with regard to the results of (i) serological analysis for specific antibodies to C. trachomatis in serum; (ii) the presence of C. trachomatis and DNA in first‐void urine; and (iii) in the semen sample of the male partner, in addition to sperm analysis, four different tests (aniline blue, chromomycin A3, acridine orange and TUNEL) were used to detect sperm chromatin and DNA abnormalities. The main conclusions of the results were: (i) no evidence of C. trachomatis infection in semen samples was found; (ii) sperm DNA fragmentation and chromatin studies were not correlated with C. trachomatis diagnosis; (iii) the percentage of DNA fragmentation is positively correlated with the percentage of immotile sperm but negatively with semen volume, normal morphology; and (iv) in sperm chromatin evaluations, only the percentage of chromatin protamination was related to male age. [ABSTRACT FROM AUTHOR]

Summary: Globozoospermia is a severe form of teratozoospermia with low incidence in infertile patients, considered as one of the important causes of male infertility. The objective was to investigate the chromatin/DNA integrity as well as apoptosis in ejaculated spermatozoa of cases with partial or total globozoospermia. Fifty‐seven semen samples were divided into three groups of partial globozoospermia (n = 17), total globozoospermia (n = 10) and normozoospermia (control; n = 30). Sperm chromatin condensation, DNA integrity and apoptosis were assessed using cytochemical assays. The results showed significant differences in sperm parameters of count and motility between two case groups versus controls. The percentages of spermatozoa with abnormal chromatin packaging and protamine deficiency were significantly higher in total and partial globozoospermic men compared to normozoospermic samples. Also, the rates of TUNEL‐positive spermatozoa were significantly increased in both globozoospermic cases with respect to the control (18.3 ± 10.1 and 12.3 ± 9.2 versus 5.9 ± 3 respectively). However, no significant differences were noticed between two subgroups of patients with regard to sperm DNA denaturation, DNA fragmentation and apoptosis. Abnormal chromatin packaging, DNA damage and apoptosis were significantly higher in cases than controls. The sperm chromatin/DNA anomalies may be considered as one of the main aetiology of ART failure in globozoospermic patients. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to develop a novel one-step ICSI approach to select sperm with better chromatin maturity than the conventional method. Methods: This was a pilot diagnostic study, which prospectively recruited men during a 6-month period in a University-affiliated infertility centre. Forty consecutive semen samples were provided for analysis. The positive rheotaxis extended drop (PRED) was set up creating a pressure and viscosity gradient. Each semen sample was divided into four aliquots: one aliquot for density gradient centrifugation (DGC), two aliquots for PRED (fresh semen (PRED-FS) and processed semen (PRED-DGC)), and one aliquot as the control (FS). In PRED, a mean of 200 spermatozoa were collected consecutively without selection from the outlet reservoir. The aniline blue assay was used to assess chromatin immaturity. Results: The mean channel length, measured from inlet to outlet, was 32.55 ± 0.86 mm, with a mean width of 1.04 ± 0.21 mm. In 82.5% of cases (33/40), at least 50 spermatozoa were captured between 15 and 30 min. Improved chromatin maturity after the DGC preparation and the PRED approach was observed in all samples. This was reflected by a mean reduction from 28.65 ± 8.97% uncondensed chromatin in the native ejaculates to 17.29 ± 7.72% in DGC and 0.89 ± 1.31% in the PRED approach ( P < 0.01). Conclusions: The PRED method may improve the current ICSI technique by providing it with its own sperm selection process. ICSI would probably become an even more complete technique comprising selection, capture and injection of the male gamete. [ABSTRACT FROM AUTHOR]

In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca 2+ level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performed to assess mean DFI, SD of DFI and % DFI in raw frozen-thawed as well as in extended sperm after 24 and 48 h of storage. The month of semen collection affected (P < 0.05) all parameters evaluated in raw semen and all criteria except progressive motility as well as rapid cells in semen stored for 24 and 48 h, respectively. Ejaculate volume was higher and sperm concentration lower in summer compared to winter and motility lower in July than in any other month of the year (P < 0.05). In semen processed in April and stored for 24 h the percentage of rapid cells was improved compared to January and after 48 h of storage progressive motility (%) was higher in January and October than in July (P < 0.05). After 24 h of cold storage PMAI % was higher in October than in January and after 48 h values were higher in September compared to January and February as well as from April to July (P < 0.05). Regarding sperm with low intracellular Ca +2 level (%) after storage for 24 and 48 h, higher values were measured in winter and in October compared to April, June and July (P < 0.01). Seasonal changes in DNA fragmentation were most evident with respect to mean DFI. In raw frozen-thawed semen mean DFI was lower from August to November than in June and July (P < 0.001). Values were lower during winter compared to spring and early summer (P < 0.05) and lower in December than from April to September (P < 0.001). After 24 h of cold storage mean DFI was lower in September and October when compared to January, February, May, July and November (P < 0.05) and after 48 h storage mean DFI was reduced in spring and autumn compared to February, June and July (P < 0.05). In conclusion, a seasonal effect was evident on semen characteristics of raw and cold-stored sperm. Semen quality was impaired in midsummer when low sperm motility and viability were combined with an elevated DNA fragmentation and Ca 2+ level of sperm. [ABSTRACT FROM AUTHOR]

Background: Teratoasthenozoospermia (TA) is a severe form of male infertility with no clear etiology. Objective: To compare the level of intracellular anion superoxide (O2-), heat shock protein A2 (HSPA2) and protamine deficiencies in ejaculated spermatozoa between teratoasthenozoospermic and normozoospermic men. Materials and Methods: In this case- control study, semen samples of 20 infertile men, with TA (with normal morphology lower than 4%_ and total motility lower than 40% ) as the case group and 20 normozoospermic fertile men as the control group were evaluated for intracellular O2- and HSPA2 by flow cytometry and protamine deficiency by Chromomycin A3 (CMA3) test. Results: The rate of CMA3+ spermatozoa in the case group was higher than controls (p=0.001). The percentages of HSPA2+ spermatozoa in the cases were significantly lower than controls (p=0.001). Also, intracellular O2- levels in the case group were significantly higher than controls (p=0.001) and had positive correlations with sperm apoptosis (r=0.79, p=0.01) and CMA3 positive sperm (r=0.76, p=0.01), but negative correlations with normal morphology (r=-0.81, p=0.01) and motility (r=-0.81, p=0.01). There was no significant correlation between intracellular O2- and HSPA2 in the case group (r=0.041, p=0.79). Conclusion: We suggest that the increase in intracellular O2-, decrease in spermatozoa HSPA2+, and high percentages of spermatozoa with immature chromatin might be considered as etiologies of infertility in TA patients. [ABSTRACT FROM AUTHOR]

In this review we provide an overview of the components of the spermatozoa playing an important role in reproductive success beyond fertilization, showing the relationship between the integrity of the diverse elements and the development of a healthy offspring. The present knowledge about fish sperm chromatin organization, epigenetic modifications of DNA and histones and sperm-borne RNAs, essential in controlling embryo development, is summarized, pointing out the possibility of using specific genes or transcripts as biomarkers of sperm quality. Data about commercial species are reported when available and more detailed information about zebrafish sperm is presented. Considering the implications that the integrity of sperm genome and epigenome has on the preservation of a proper genotype and phenotype in the progeny, the methods applied for the study of chromatin damage and for the study of transcriptome are described. Moreover we discuss some injuring agents affecting paternal information, from the presence of contaminants in the aquatic environment, to the reproductive practices applied in fish farming. The consequences of fertilizing with damaged spermatozoa, as well as the zygotic ability to repair damage are also reviewed. Statement of relevance Role of the sperm on achieving high rates of healthy fries. [ABSTRACT FROM AUTHOR]

Background: Methamphetamine (MA) was shown to have harmful effects on male reproductive system. Objective: To investigate probable effects of daily administration of MA on sperm parameters and chromatin/DNA integrity in mouse. Material and Methods: Thirty-five NMRI male mice were divided into five groups including low, medium, and high dosage groups which were injected intraperitoneally with 4, 8 and 15 mg/kg/day for 35 days, respectively. Normal saline was injected in sham group and no medications were used in control group. Then, the mice were killed and caudal epididymis of each animal was cut and placed in Ham's F10 medium for sperm retrieval. To evaluate sperm chromatin abnormalities, the aniline blue, toluidine blue and chromomycine A3 were used. For sperm DNA integrity and apoptosis, the acridine orange, sperm chromatin dispersion, and TUNEL assay were applied. For sperm morphology, Papanicolaou staining was done. Results: Normal morphology and progressive motility of spermatozoa decreased in medium and high dosage groups in comparison with the control group (p=0.035). There was a significant increase in rate of aniline blue, toluidine blue, and chromomycine A3 positive spermatozoa in high dosage group. In a similar manner, there was an increase in rates of acridine orange, TUNEL and sperm chromatin dispersion positive sperm cells in high dosage group with respect to others. Conclusion: MA abuse in a dose-dependent manner could have detrimental effects on male reproductive indices including sperm parameters and sperm chromatin/DNA integrity in mice. [ABSTRACT FROM AUTHOR]

There is evidence to show that varicocele repair can improve conventional sperm parameters but the effects on sperm chromatin integrity have not been fully elucidated. We sought to examine the effects of varicocelectomy on sperm maturation, nuclear chromatin integrity and nuclear sulfhydryl groups. We conducted a prospective study of consecutive infertile men (n=29) that underwent a microsurgical sub-inguinal varicocelectomy for treatment of a clinically palpable varicocele and abnormal semen parameters. Six healthy sperm donors served as controls. We evaluated conventional sperm parameters and markers of sperm chromatin and DNA integrity (aniline blue (AB) staining, iodoacetamide fluorescein (IAF) fluorescence and, % DNA fragmentation index (%DFI) and percent high DNA stainability (%HDS) by sperm chromatin structure assay) before and 6 months after surgery. The sperm %DFI, %HDS, % 5-IAF staining (diffuse head staining) and % AB staining (dark blue) were all significantly lower in the control group compared to infertile men with varicocele (8 vs. 20%, 4.0 vs. 9.6%, 1.7 vs. 16.3%, and 2.5 vs. 13.5% respectively). The %HDS and %DFI decreased significantly after surgery (from 10% to 6% and from 20% to 13%, respectively). Similarly, the %5-IAF and %AB staining also decreased significantly after surgery (from 16.3% to 5.4%, and from 13.5% to 5.4%, respectively). We observed significant inversely relationships between sperm progressive motility and both %IAF staining and %DFI (r=–0.44 and –0.43, respectively). The data show that varicocelectomy is associated with an improvement in sperm DNA integrity and chromatin compaction using three different assays of sperm chromatin integrity. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate sperm chromatin maturity and integrity of that injected into good-quality oocytes in anin vitrofertilization-intra cytoplasmic sperm injection (IVF-ICSI) program. A cut-off value of sperm chromatin maturity and integrity was developed as a function of their correlation to the zygote development, i.e., embryo formation and cleavage rate. The study assessed sperm chromatin maturity using aniline blue (AB) staining, whereas toluidine blue (TB) staining was used to assess sperm chromatin integrity. Ejaculates from 59 patients undergoing ICSI and 46 fertile normozoospermic donors for determination of normal values of sperm chromatin status were used in this study. Embryo formation and cleavage rates were observed for the period of 3 days after ICSI. There was a significant difference in the percentage of sperm with mature chromatin between ejaculate from ICSI patients and fertile donor (p=0.020); while there was no significant difference in sperm chromatin integrity of both samples (p=0.120). There was no significant correlation between sperm chromatin maturity and either embryo formation or cleavage rate; as well as sperm chromatin integrity to both parameters of zygote development (p>0.05). Furthermore, we found that the cut-off value of sperm chromatin maturity and integrity of the fertile normozoospermic ejaculates were 87.2% and 80.2%, respectively. Using the cut-offs, we found that low sperm chromatin maturity at the level of <87% correlated significantly with the cleavage rate of the zygote (p=0.022; r=0.371); whereas poor sperm chromatin integrity at the level of <80% correlated with embryo formation (p=0.048; r=0,485). In conclusion, this study showed that poor maturity and integrity of sperm chromatin (AB<87% and TB<80%, respectively), could affect zygote development following ICSI. Abbreviations: AB: aniline blue; CMA3: chromomycin A3; ICSI: intra cytoplasmic sperm injection; IVF:in vitrofertilization; PBS: phosphate buffer saline; SPSS: Statistical Package for Social Science; TB: toluidine blue; WHO: World Health Organization [ABSTRACT FROM PUBLISHER]

This study is aimed at evaluating the association between occupational exposure to organophosphate (OP) and carbamate (CB) pesticides and semen quality as well as levels of reproductive and thyroid hormones of pesticide sprayers in Malihabad, Lucknow, Uttar Pradesh, India. Thirty-five healthy men (unexposed group) and 64 male pesticide sprayers (exposed group) were recruited for clinical evaluation of fertility status. Fresh semen samples were evaluated for sperm quality and analyzed for DNA fragmentation index (DFI) by flow cytometry. Pesticide exposure was assessed by measuring erythrocyte acetylcholinesterase and plasma butyrylcholinesterase (BuChE) with a Test-mate ChE field kit. Serum levels of total testosterone (Tt), prolactin (PRL), follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and free thyroxine (FT4) were analyzed using enzyme immunoassay kits. Evidence of pesticide exposure was found in 88.5% of sprayers and significant increments were observed in sperm DFI with significant decrease in some semen parameters. DFI was negatively correlated with BuChE, sperm concentration, morphology, and vitality in these pesticide sprayers. The levels of Tt, PRL, FT4, and TSH appeared to be normal; however, there was a tendency for increased LH and FSH levels in exposed workers. The results confirm the potential impact of chronic occupational exposure to OP and CB pesticides on male reproductive function, which may cause damage to sperm chromatin, decrease semen quality, and produce alterations in reproductive hormones, leading to adverse reproductive health outcomes. [ABSTRACT FROM AUTHOR]

Aim of the study The purpose of this study was to evaluate the dynamic of the sperm DNA integrity of spermatozoa from endangered Grey Steppe cattle breed bulls in order to establish the potential use of sperm DNA fragmentation (SDF) assay for improving the routine fertility screening in the conservation program. Aim of the study The purpose of this study was to evaluate the dynamic of the sperm DNA integrity of spermatozoa from endangered Grey Steppe cattle breed bulls in order to establish the potential use of sperm DNA fragmentation (SDF) assay for improving the routine fertility screening in the conservation program. Results Variability of percentages of spermatozoa with fragmented DNA was assessed in 2 Grey Steppe bulls. In the case of both bulls, the sperm DNA fragmentation index (DFI%) increased progresively during the 6 hours incubation, when the hightest DNA fragmentation index (DFI) was observed, 7.5% in bull 2, respectively, 4.5% in bull 1. At T0, the DFI% was greater for Bull 2, a value of 3.5% being registered as compared to bull 1, in which the DFI% was 2.5%. Conclusions The semen from the two Grey Steppe bulls analyzed in this study presented moderate DNA fragmentation index and may be used for AI and IVF in conservation program of the Grey Steppe breed. [ABSTRACT FROM AUTHOR]

A strategy to circumvent the poor polyphenols bioavailability is to load these compounds into liposomes. We evaluated the in vitro effects of quercetin (Q) and Q-loaded liposomes (QLL, 30, 50, 100 μM) on motility, viability and chromatin integrity of swim-up selected human sperm. Antioxidant power was assayed against tert-butylhydroperoxide induced lipid peroxidation (LPO) using C11-BODIPY581/591 fluorescent probe and transmission electron microscopy. QLL showed decreased toxicity for sperm motility and viability and increased DNA damage compared to Q. The percentage of sperm with fluorescence, marker of LPO, was decreased in samples incubated with Q vs QLL (P < 0.001). The ultrastructure of acrosomes and membranes was preserved with Q 30/100 μM, whereas QLL did not prevent membrane injury. Q alone appeared more effective than Q incorporated into liposomes; however liposomes could be considered as carriers that may convey different compounds inside sperm; they may therefore represent a field of research rich of many applications. [ABSTRACT FROM AUTHOR]

Moringa oleifera L. is a medicinal plant with potential antioxidant property. This study was aimed at investigating the chemoprotective effect of Moringa oleifera leaf extract ( MOE) on cyclophosphamide ( CP)-induced testicular toxicity. Two-week-old male Swiss albino mice were intraperitoneally injected with phosphate-buffered saline, 50 mg kg−1 of CP and 25 mg kg−1 of MOE. In combination treatment, mice were injected with 25 mg kg−1 of MOE 24 h prior to CP injection, 24 h prior and post- CP injection and 24 h post- CP injection for 5 consecutive days (10 mg kg−1). Six weeks later, mice were sacrificed to assess epididymal sperm parameters. MOE alone did not have any significant effect on sperm parameters. However, acute injection of CP resulted in significant decline in motility ( P < 0.001), increase in head abnormality ( P < 0.01) and DNA damage ( P < 0.05). Combining MOE with CP increased the sperm density, motility and reduced head defect and DNA damage, irrespective of the schedule and dosage of MOE. Administration of MOE prior to CP significantly elevated the level of superoxide dismutase and catalase with concomitant decrease in lipid peroxidation in the testicular tissue. In conclusion, MOE may have potential benefit in reducing the loss of male gonadal function following chemotherapy. [ABSTRACT FROM AUTHOR]

Study Question: Do the luminal fluids of the epididymis and the vas deferens contribute to sperm chromatin fragmentation (SCF) in mice?Summary Answer: The luminal fluids of both organs are required for activating SCF in mice, but the vas deferens luminal fluid does this more efficiently than that of the epididymis.What Is Known Already: Mice sperm have the ability to degrade their DNA in an apoptotic-like fashion when treated with divalent cations in a process termed SCF. SCF has two steps: the induction of reversible double-strand DNA breaks at the nuclear matrix attachment sites, followed by the irreversible degradation of DNA by nuclease. Single stranded DNA breaks accompany SCF.Study Design, Size, Duration: Luminal fluids from two reproductive organs of the mouse (B6D2F1 strain), the epididymis and vas deferens, were extracted and tested for SCF activation with divalent cations using four different combinations of the sperm and the surrounding luminal fluids: (i) in situ--sperm were kept in their luminal fluid and activated directly; (ii) reconstituted--sperm were centrifuged and resuspended in their luminal fluid before SCF activation; (iii) mixed--sperm were centrifuged and resuspended in the luminal fluid of the other organ; (iv) no luminal fluid--sperm were centrifuged and reconstituted in buffer. All four experiments were performed without (controls) and with divalent cations (resulting in SCF). For each experimental condition, two different mice were used and the analyses averaged.Participants/materials, Setting, Methods: DNA damage by SCF was analyzed by three different methods, the sperm chromatin structure assay (SCSA), terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) analysis and field inversion gel electrophoresis.Main Results and the Role Of Chance: In all three assays that we used, the vas deferens luminal fluid was much more efficient in stimulating SCF in the sperm from either source than that of the epididymis (P < 0.0001). Vas deferens sperm were capable of initiating lower levels of SCF in the absence of luminal fluid (P < 0.0001).Limitations, Reasons For Caution: Analyses were performed in only one species, the mouse, but we used three separate assays in our analysis.Wider Implications Of the Findings: The data suggest that the luminal fluid of the male reproductive tract interacts with sperm during their transit providing a mechanism to degrade the DNA. We hypothesize that this is part of an apoptotic-like mechanism that allows the reproductive tract to eliminate defective sperm. The SCF model also allowed us to identify differences in the types of DNA lesions that the three tests can identify, providing important background information for the use of these tests clinically. [ABSTRACT FROM AUTHOR]

Varicocele is one of the most common causes of primary male infertility. Pilea microphylla (PM) is being used as folk medicine. This study was aimed to investigate the effects of PM in a rat model of varicocele. A total of 30 male Wistar rats were divided into control, sham, varicocele, accessory varicocele and PM-treated groups. After 10 weeks of varicocele induction, sperm parameters and chromatin (Aniline blue, acridine orange and toluidine blue) were evaluated, except for the treated and accessory groups that received 50 mg/kg PM orally daily for 10 weeks and then were sacrificed. Sperm parameters significantly decreased in varicocele groups (P < 0.01). Moreover, there was a negative correlation between the DNA fragmentation and sperm parameters in varicocelized rats. Administration of PM led to significantly increased sperm parameters and AO staining (P < 0.05). These findings suggest that PM improves sperm parameters and DNA fragmentation in varicocelized rats. PM can reduce the damage to sperm DNA but not chromatin condensation. [ABSTRACT FROM AUTHOR]

Background: Currently, relation between reactive oxygen species (ROS) ROS concentration and semen quality was indicated. Saffron has traditionally been not only considered as a food additive but also as a medicinal herb, which has a good antioxidant properties. Objective: The aim of this study was to evaluate the protection potency of saffron and vitamin E on sperm chromatin integrity. Materials and Methods: Thirty adult male Wistar rats divided equally into saffron (100 mg/kg), vitamin E (100 mg/kg) and control (0.5cc distilled water /day) groups. After 60 days, cauda epididymis dissected and sperm cells were used for analysis of sperm chromatin packaging by chromomycin A3 (CMA3) staining, and sperm chromatin susceptibility to acid denaturation by acridine orange (AO) staining Results: The mean percentage of CMA3 positive sperm was significantly decreased in saffron and vitamin E groups relative to control group (p<0.001). Moreover, the AO staining results showed that the mean percentage of sperm with DNA damage was significantly decreased in saffron and vitamin E groups as compared with control group (p<0.001). Conclusion: Our results purposed that saffron can protect sperm against DNA damage and chromatin anomalies. [ABSTRACT FROM AUTHOR]

Background: Sperm DNA damage is associated with male infertility but whether normozoospermic infertile men also have DNA damage is unknown. Objective: To evaluate sperm DNA and chromatin integrity in men with mild male factor infertility. Design, setting and participants: Prospective study of 102 consecutive men (78 normozoospermic, 15 asthenozoospermic, 9 oligozoospermic) enrolled for intrauterine insemination (IUI) and 15 fertile controls. Outcome measurements and statistical analysis: Standard semen parameters and sperm chromatin and DNA integrity were assessed and compared between groups. Sperm chromatin quality was assessed by (1) aniline blue staining (AB is specific to histone lysines), (2) iodoacetamide fluorescein fluorescence (IAF targets free protamine sulfhydryl groups) and (3) sperm chromatin structure assay (SCSA) with the results expressed as % DNA fragmentation index (%DFI). Results and limitations: The mean (±SD) percentage of spermatozoa with positive IAF fluorescence was significantly higher in the IUI population compared to fertile controls (17 % ± 10 % vs. 8 % ± 6 %, P = 0.0011) and also in the normozoospermic subset ( n = 78) compared to controls (16 % ± 9 % vs. 8 % ± 6 %, P < 0.0001, ANOVA). We also observed a trend toward lower %progressive motility, and higher %AB staining and %DFI in the IUI group compared to controls. We observed significant relationships between sperm %DFI and progressive motility ( r = −0.40, P < 0.0001) and between positive AB staining and IAF fluorescence ( r = 0.58, P < 0.0001). Conclusions: The data indicate that sperm chromatin integrity may be abnormal in men enrolled in IUI treatment cycles, despite the fact that most of these men are normozoospermic. [ABSTRACT FROM AUTHOR]

Abstract: The objective of the present study was to explore the potential relationships of ovine sperm chromatin integrity, quantified using the sperm chromatin structure assay (SCSA), to the heat load of the scrotum and the discomfort felt by the animals because of fluctuations of microclimatic factors at different time periods before ejaculation. Ejaculates were collected once per week from five Chios rams and four East Friesian rams for 12 months and stored in liquid nitrogen. Frozen-thawed semen samples were analyzed using the SCSA, to determine the DNA fragmentation index (DFI) and the percentage of cells outside the main sperm population (%DFI) in each one of the samples. Scrotal surface temperature (SST) of each ram was measured using an infrared thermometer on a daily basis. Ambient air temperature and relative humidity were recorded at hourly intervals throughout the experimental period and temperature-humidity index (THI) was used to assess the discomfort felt by the rams. Mean values of SST (SSTmean) and THI (THImean) were computed for eight different time periods (up to 61 days) preceding each ejaculation day (Day 0). A linear mixed-effect model analysis was performed to describe the relation of SCSA parameters to collection month, SSTmean, and THImean of different time periods before ejaculation. The results of the statistical analysis revealed a relation of %DFI to the SSTmean of the last 12 days preceding ejaculation, namely the period that resembled the phase of epididymal maturation. On the contrary, the variation of DFI was most adequately described by the linear mixed-effect model applied for Days 54 to 48 before ejaculation, which resembled the phase of spermatogonial mitoses. The effect of collection month was significant for DFI and %DFI, with semen samples collected in September and February exhibiting the lowest DFI values; a less profound seasonal pattern was detected for %DFI. The effect of THImean on DFI and %DFI was proven nonsignificant in regard to all time periods. In conclusion, a relation of SCSA parameters to SSTmean of different periods before ejaculation was shown in the present study, implying an effect of scrotal microenvironment on intratesticular and epididymal sperm population. In contrast, we failed to detect any effect of microclimate-induced discomfort felt by the animals on the chromatin integrity of frozen-thawed ram spermatozoa. [Copyright &y& Elsevier]

Sperm chromatin integrity may affect the outcomes of assisted reproductive technology ( ART). Developing a clinically reliable strategy to enrich sperm samples with high chromatin quality spermatozoa prior to sperm banking or use in ART would thus be advantageous. The objectives of this study were to: (i) assess the sperm chromatin quality in men with different categories of semen parameters; and (ii) evaluate the extents of Annexin-V magnetic-activated cell sorting ( MACS) technology coupled with differential density gradient centrifugation ( DGC) in improving sperm chromatin quality. Three categories of men from couples attending a university-based fertility clinic were recruited based on their semen parameters: normozoospermic ( n = 13), asthenoteratozoospermic ( n = 17) and teratozoospermic ( n = 12). For each patient, spermatozoa in semen samples were processed first by DGC to enrich the motility and further by MACS to remove spermatozoa showing apoptotic features. The yield and enrichment of sperm quality was evaluated at each step with conventional semen parameters in conjunction with a combination of five complementary assays, to assess sperm maturity, chromatin structure, compaction and DNA integrity (Hyaluronic Binding Assay, SCSA, chromomycine A3 staining and TUNEL and COMET assays). Our results demonstrated that, compared with normozoospermic samples, raw asthenoteratozoospermic and teratozoospermic samples had a higher proportion of spermatozoa containing DNA breaks, but only asthenoteratozoospermic exhibited altered chromatin structure and decreased binding to hyaluronic acid. Interestingly, the DGC appeared to select for more mature spermatozoa with high DNA compaction. More importantly, in all categories of semen samples, Annexin-V MACS allows enrichment of spermatozoa with good chromatin quality as measured by the TUNEL and SCSA. Because effective treatment modalities to improve sperm DNA damage are limited, our results suggest a potential clinical value of MACS as a mean to enhance sperm quality that may improve assisted reproductive outcomes. [ABSTRACT FROM AUTHOR]

Abstract: Sperm chromatin status was assessed in 565 Zebu and Zebu crossbred beef bulls in extensive tropical environments using the sperm chromatin structure assay (SCSA). The SCSA involved exposure of sperm to acid hydrolysis for 0.5 or 5.0 minutes, followed by flow cytometry to ascertain relative amounts of double-stranded (normal) and single-stranded (denatured) DNA, which was used to generate a DNA fragmentation index (%DFI). With conventional SCSA (0.5-minute SCSA), 513 bulls (91%) had <15 %DFI, 24 bulls (4%) had 15 to 27 %DFI, and 28 bulls (5%) had >27 %DFI. In 5.0-minute SCSA, 432 bulls (76%) had <15 %DFI, 68 bulls (12%) had 15 to 27 %DFI and 65 bulls (12%) had >27 %DFI. For most bulls, the SCSA was repeatable on two to four occasions; however, because most bulls had <15 %DFI, repeatability of the SCSA will need to be determined in a larger number of bulls in the 15 to 27 %DFI and >27 %DFI categories. The %DFI was negatively correlated with several bull semen parameters and the strongest negative correlation was with normal sperm. There was a strong positive correlation between %DFI and sperm head abnormalities. Based on these findings, most Zebu beef bulls in extensive tropical environments had relatively stable sperm chromatin. Based on the apparent negative correlations with conventional semen parameters, we inferred that the SCSA measured a unique feature of sperm quality, which has also been suggested for other species. Further studies on the relationships between sperm chromatin stability and fertility are required in beef bulls before chromatin status can be used as an additional predictor of the siring capacity of individual bulls in extensive multiple-sire herds. [Copyright &y& Elsevier]

Background: Diabetes mellitus (DM), primary or idiopathic is a chronic disorder of the carbohydrate, lipid and protein metabolism. DM may impact male reproductive function at several levels. It is shown that DM has detrimental effects on sperm parameters in human and experimental animals. Objective: The aim of this study was to observe the effects of diabetes on sperm parameters (viability, count, morphology and motility) and evaluation of sperm chromatin quality in mice. Materials and Methods: Totally twenty adult male Syrian mice were divided randomly into 2 groups (n=10). The animals of group A were considered as controls while group B mice were diabetic that received a single dose (200 mg/kg) streptozotocin (STZ) intra peritoneally. After 35 days, the cauda epididymis of each diabetic mouse was dissected and placed in culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability. The sperm chromatin quality and DNA integrity, was evaluated with Aniline Blue (AB), Toluidine blue (TB), Acridine orange (AO) and Chromomycin A3 (CMA3) staining. Results: In sperm analysis, the diabetic mice had poor parameters in comparison with control animals (p=0.000). Regarding sperm chromatin quality, the results of TB and AO tests showed statically significant differences between two groups, but in AB and CMA3 staining, we didn't see any differences between them. Conclusion: The results showed that STZ-induced diabetes mellitus may influence the male fertility potential via affecting sperm parameters and DNA integrity in mice. However, according to our data, the diabetes doesn't have any detrimental effects on histone-protamines replacement during the testicular phase of sperm chromatin packaging. [ABSTRACT FROM AUTHOR]

Background: It has been claimed that by using different washing methods, the sperms can be separated according to size, motility, density, chromosomal content and surface markings and charge. These methods also reduce sperm chromatin deficiencies and screen the sperms before applying in assisted reproduction techniques. Objective: This study compared simple density gradient methods and a combined method with albumin density gradient and PureSperm separation (alb/PureSperm) for sex preselection by double fluorescence in situ hybridization (FISH) versus chromomycin A3 staining to determine chromatin integrity. Materials and Methods: 30 normal semen samples were prepared with PureSperm, albumin gradients and alb/PureSperm. All samples were then stained by FISH and chromomycin A3. The results were compared with SPSS 11.5 and the Kruskal- Wallis test. Results: The proportion of X-bearing spermatozoa by PureSperm separation (47.58±5.67) and Y-bearing spermatozoa by albumin gradient (46.13±3.83) methods were slightly higher than in putative normal sperm samples (1:1), but there were no significant differences in the X- or Y- bearing spermatozoa counts among the three methods. Albumin gradient separation tended to underestimate abnormal spermatozoa compared to PureSperm and combined alb/PureSperm. Conclusion: Routine separation methods slightly enriched X- or Y- bearing spermatozoa, but the differences were not significant for clinical purposes. The combined alb/PureSperm method had no advantages for assessing sex ratio or chromatin integrity compared to simpler gradient methods. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects of a mild induced testicular and epididymal hyperthermia (+2°C) on sperm chromatin integrity in men. Design: Experimental prospective study. Setting: University hospital. Patient(s): Five healthy fertile volunteers. Intervention(s): Testicular and epididymal hyperthermia was induced by maintaining the testes at inguinal position with the support of specially designed underwear 15 ± 1 hours daily for 120 consecutive days. Main Outcome Measure(s): Classic semen characteristics. Sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) were analyzed by sperm chromatin structure assay. Result(s): Compared with baseline values, sperm DFI and HDS were significantly increased as early as day (D) 20 and D34, respectively, and remained elevated during the entire period of hyperthermia. Percentages of motile and viable spermatozoa decreased as early as D20 and D34, respectively, and total sperm count decreased at D34 during hyperthermia and remained low during the entire hyperthermia period. All studied parameters returned to respective baseline values at D73 after cessation of hyperthermia. Conclusion(s): Mild induced testicular and epididymal hyperthermia largely impaired sperm chromatin integrity, which appeared before any changes in sperm output. These findings may have clinical implications in male contraception, infertility, and assisted reproductive technology. [ABSTRACT FROM AUTHOR]

Background and purpose: The sperm of infertile men with varicocele exhibit markedly high DNA damage that appears to be related to high oxidative stress (OS). Aminoguanidine (AG) is a specific inhibitor of the nitric oxide synthase (NOS) isoforms iNOS and an antioxidant, the effects of which decrease NO and peroxynitrite production. The aim of this study was to determine the effects of AG on sperm chromatin in varicocelized rats. Methods: Thirty male Wistar rats were divided into 5 groups: control, sham, varicocele, and AG and placebo-treated groups. At 10 weeks after varicocele induction, sperm chromatin was evaluated in all groups, except in the treated groups. The treated groups received intraperitoneal injections of 50 mg/kg AG or placebo daily for 10 weeks and then were killed for chromatin assessment. Sperm chromatin was evaluated by aniline blue, acridine orange, toluidine blue, and chromomycin A3 staining. Results: The results of the 4 above tests were significantly increased between varicocele and control (and sham) groups (P < .05). Conclusion: The findings of this study suggest that AG improves sperm DNA fragmentation that is associated with infertility in varicocelized rats, and treatment with AG can reduce the damage to sperm DNA. [ABSTRACT FROM PUBLISHER]

This article reports the results of the most recent in a series of EHSRE workshops designed to synthesize the current state of the field in Andrology and provide recommendations for future work (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential application of new knowledge about sperm chromatin organization, vulnerability and repair to improve the diagnosis and treatment of clinical infertility associated with that damage. Equally important is the use and reliability of these tests to identify the extent to which environmental contaminants or pharmaceutical agents may contribute to the incidence of sperm DNA damage and male fertility problems. A working group (for workshop details, see Appendix) under the auspices of ESHRE met in May 2009 to assess the current knowledgebase and suggest future basic and clinical research directions. This document presents a synthesis of the working group's understanding of the recent literature and collective discussions on the current state of knowledge of sperm chromatin structure and function during fertilization. It highlights the biological, assay and clinical uncertainties that require further research and ends with a series of 5 key recommendations. [ABSTRACT FROM PUBLISHER]

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 °C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 μm Fe2+/1 mm ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA®) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 ± 2.9%) and OXI (11.6 ± 7.6%) ( p < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI ( p < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 ± 0.8% OXI vs. 17.4 ± 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa. [ABSTRACT FROM AUTHOR]

To assess the influence of structural differences in sperm nuclei on embryo development in intracytoplasmic sperm injection (ICSI). Semen obtained from forty-four infertile patients who underwent ICSI was examined. In assessing blastocyst development, only those patients who had successfully obtained over five fertilized eggs were included to exclude any oocyte factors ( n = 22). Spermatozoa were assessed using excitation fluorescence flow cytometry with acridine orange and the sperm chromatin dispersion (SCD) test. There was a significant positive correlation between the COMP values obtained from flow cytometry and blastocyst formation. ( r = 0.477, p = 0.025). There was a significant negative correlation between the SCD values representing DNA fragmentation and blastocyst formation. ( r = 0.796, p < 0.001). COMP values and SCD values were independent parameters to assess sperm nuclear quality regarding embryo development in vitro ( r = 0.224, p = 0.080). Results suggest that injection of spermatozoa with fewer disulfide bonds and less nuclear DNA fragmentation could achieve better blastocyst formation in human ICSI. Assessment of sperm chromatin should help to predict embryo development after ICSI. [ABSTRACT FROM AUTHOR]

Different methods have been used to evaluate the beneficial effect of varicocelectomy; these include semen parameters and pregnancy rate. Because of high biological variability of semen parameters, sperm functional tests have been considered as an efficient end point in assessment of fertility. Therefore, the aim of this study was to evaluate the effect of varicocelectomy on semen parameters and sperm protamine deficiency in 192 patients. The results of the present study show that all the three semen parameters and percentage of sperms with normal protamine content have improved post-surgery. The cumulative pregnancy rate was 34.6%. Comparing the results of the semen parameters and protamine content between patients whose partner became pregnant to those who did not benefit from varicocelectomy before and 6 months after surgery, show that patients may benefit from varicocelectomy that had higher initial semen density and better sperm morphology prior to surgery. Detailed analyses of sperm morphology, along with aforementioned results reveal that the factors which account for pregnancy difference are: (i) improvement in early events of spermatogenesis, possibly during spermatocytogensis and reduction division; and (ii) late spermiogenesis events. Thus, it can be suggested that patients with low initial sperm count may benefit more from assisted reproductive techniques or varicocelectomy followed by assisted reproduction. [ABSTRACT FROM AUTHOR]

Over the years, the development of assisted reproductive technology to bypass male factor infertility has improved drastically. Considered one of the most perplexing disorders in the reproductive field, male factor infertility is prevalent and may be on the rise. Unfortunately, its aetiology remains elusive. One of the main reasons lies in the complex machinery and structure of the hydrodynamic sperm cell. Its polyunsaturated fatty acid cell membrane, the protamines in its genetic material and the absence of antioxidants in its cytoplasm ensure that the spermatozoon is highly susceptible to environmental effects. The spermatozoon's genesis, storage, and transport through the male reproductive tract are also susceptible, genetically and pathologically, to environmental effects. This review aims to include all the possible causes of disruption to this unique cell and their probable solutions, in the hope of clearing up the ambiguity that surrounds male factor infertility. [ABSTRACT FROM AUTHOR]