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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]

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]

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]

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]

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]

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]

Summary The formation of motile spermatozoa involves the highly conserved formation of protamine-rich, tightly packed chromatin. However, genetic loss of protamine function in Drosophila and mice does not lead to significant decompaction of sperm chromatin. This indicates that other proteins act redundantly or together with protamines. Here, we identify Prtl99C as a Drosophila sperm chromatin-associated protein that is essential for male fertility. Whereas the loss of protamines results in modest elongation of sperm nuclei, knockdown of Prtl99C has a much stronger effect on sperm nuclei. Loss of protamines and Prtl99C indicates an additive effect of these proteins on chromatin compaction, in agreement with independent loading of these factors into sperm chromatin. These data reveal that at least three chromatin-binding proteins act together in chromatin reorganization to compact the paternal chromatin. [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]

Abstract: The objective of this study was to improve the quality of cryopreserved–thawed equine sperm using single-layer density centrifugation (SLC). Sperm quality was assessed by DNA integrity, motility, morphology, mitochondrial membrane potential, viability, and plasma membrane alteration. The percentage of DNA-damaged sperm (expressed in % COMP) was lower (P = .001) after SLC (1.6 ± 0.5% vs. 6.8 ± 0.5%). Total sperm motility (80 ± 2.4% vs. 41.7 ± 2.4%) and progressive sperm motility (69.5 ± 2.9% vs. 31.5 ± 2.9%) (P < .001), as well as the percentage of morphologically normal sperm (45 ± 3.9% vs. 27.7 ± 3.9%), increased after SLC compared with control sample. In addition, the proportion of sperm with high mitochondrial membrane potential increased (81.6 ± 1.8% vs. 42.1 ± 1.8%), as did the viability of sperm (71.1 ± 2.4% vs. 39.5 ± 2.4%), after SLC compared with the control sperm. The proportion of sperm with alteration in plasma membrane structure was lower after SLC compared with control sample (6.4 ± 1.1% vs. 18.9 ± 1.1%). Overall, sperm recovery was 72.7 ± 3.6% in the control sample compared with 14.8 ± 3.6% after SLC (P < .001). We conclude that based on the sperm parameters evaluated, SLC improves the quality of cryopreserved–thawed equine spermatozoa. [Copyright &y& Elsevier]

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]

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]

Abstract: Extensive use of organophosphorous pesticides (OP) by young men represents a public health problem. Toxicity of OP mainly results in neurotoxicity due to their oxygen analogues (oxons), formed during the OP oxidative activation. OP alter semen quality and sperm chromatin and DNA at different stages of spermatogenesis. Oxons are more toxic than the parent compounds; however, their toxicity to spermatogenic cells has not been reported. We evaluated sperm DNA damage by several OP compounds and their oxons in human spermatozoa from healthy volunteers incubated with 50–750μM of methyl-parathion (MePA), methyl-paraoxon (MePO), chlorpyrifos (CPF), chlorpyrifos-oxon (CPO), diazinon (DZN) or diazoxon (DZO). All concentrations were not cytotoxic (evaluated by eosin-Y exclusion), except 750μM MePO. Oxons were 15% to 10 times more toxic to sperm DNA (evaluated by the SCSA parameter, %DFI) than their corresponding parent compounds, at the following order: MePO>CPO=MePA>CPF>DZO>DZN, suggesting that oxon metabolites participate in OP sperm genotoxicity. [Copyright &y& Elsevier]

Abstract: Embryonic mortality in mammals is typically thought to result from ‘female factor’ infertility. There is growing evidence, however, that the status of sperm chromatin (DNA) at the time of fertilisation can also influence embryonic survival. During the final stages of spermatogenesis (spermiogenesis) a number of unique biochemical, morphological and physiological processes take place that are associated with marked changes in the structure of sperm chromatin. In early stages of spermatogenesis, sperm DNA is associated with histone nucleoproteins and structured into classical nucleosome core particles similar to other somatic cells. As spermiogenesis proceeds, the histone nucleoproteins are replaced by transition proteins which are subsequently replaced by protamines. At the completion of spermiogenesis the chromatin of mature sperm has a toroidal structure that is tightly compacted and resistant to denaturation. The compaction is necessary to protect sperm chromatin during transit through the epididymis and female reproductive tract. Disruption to chromatin remodelling during spermiogenesis results in chromatin that is susceptible to denaturation. Inappropriate chromatin structure has been shown in a number of mammalian species to be related to male infertility, and specifically the failure of embryonic development. A range of techniques are available to assess chromatin status in sperm but arguably the most informative is the sperm chromatin structure assay (SCSA). The SCSA is a flow cytometric assay that uses the metachromatic properties of acridine orange to measure the susceptibility of sperm chromatin to acid-induced denaturation. A relationship has been demonstrated, primarily in men, between the SCSA outcome and the probability of continued embryonic development and the establishment of pregnancy after fertilisation. The contribution of sperm chromatin instability to reproductive wastage in both natural mating and assisted reproduction warrants further investigation as it may prove valuable as a means of decreasing the incidence of embryonic mortality. In this regard, it is possible that ‘male factor’ infertility may emerge as an even more important component in embryonic development. [Copyright &y& Elsevier]

Abstract: The semen quality of seven young adult boars was assessed for percentages of sperm motility, normal acrosomes, abnormal sperm, cells positive to sHOST (short Hipoosmotic Swelling Test), HPNA cells (sHOST Positive with Normal Acrosome cells) and the percentage of sperm heads, which exhibited DNA fragmentation using the Sperm Chromatin Dispersion test (SCD). These parameters were analysed in sperm samples both undiluted and diluted using a commercial extender and stored at 15°C for 21 days. Results showed that semen quality decreases faster in the undiluted semen samples from day 0 to day 7 compared to diluted semen samples that remained with a high quality up to day 11. The undiluted semen exhibited a low DNA fragmentation index (DFI) during the first days and then a significant increase from day 7 up to day 21. This increase in the DFI coincided with the lowest levels of the other semen quality parameters. On the contrary, the samples diluted in the commercial extender showed very low levels of DNA fragmentation in all boars during the preservation period. When the evolution of DNA fragmentation was analysed in the undiluted samples, differences were found among boars. These differences were not shown in the samples diluted in the extender where the basal DFI remained stable during the 21 days. The main conclusion of this study was that some sperm extenders delay or partially prevent sperm DNA fragmentation. [Copyright &y& Elsevier]

Abstract: The effect of two different thawing temperatures on frozen boar semen viability, in vitro fertilizing capacity and chromatin condensation and stability was studied. Freeze-thaw motility, normal apical ridge (NAR), in vitro fertilizing (IVF) capacity and chromatin condensation and stability were evaluated after thawing at 42°C, 40s and 50°C, 40s. Chromatin condensation degree was determined by flow cytometry, using propidium iodide as fluorochrome intercalating agent, and chromatin stability was evaluated by the same procedure after inducing sperm chromatin decondensation with ethylene diamine tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS). The results showed that thawing straws at 42°C, 40s significantly reduced motility compared to straws thawed at 50°C, 40s. NAR, penetration, monospermy and polyspermy were not different between the two groups of samples thawed at different temperatures. Chromatin was significantly more compact when thawing was performed at 50°C, but its stability did not show any difference relative to thawing at 42°C. It is suggested that the interactions involved in chromatin overcondensation had a non-covalent nature. [Copyright &y& Elsevier]

Abstract: Lead (Pb) alters sperm chromatin condensation (CC) and the mechanisms are investigated. During spermatogenesis, protamines replace histones and disulfide bonds formation during epididymal maturation condense the chromatin. We evaluated sperm Pb uptake in testis and epididymis and the effects on CC in mice (0.06% Pb2+/16 weeks/drinking water). Spermatozoa from caput epididymis (CP) and cauda epididymis–vas deferens (CE–VD) were obtained and CC was measured by SCSA. Lead levels in spermatozoa from CP were lower than those from CE–VD, and correlated with a decreased CC, while Pb in CE–VD spermatozoa correlated with an increased CC. Lead accumulation into the nucleus was observed and Pb binding to nuclear sulfhydryl groups decreased chromatin decondensation in vitro. Our results suggest that spermatozoa take up Pb during testicular development and epididymal transport and alter CC, depending of the timing of Pb incorporation into the sperm nucleus, which finally may interfere with the chromatin decondensation process after fertilization. [Copyright &y& Elsevier]

Abstract: We present a new, rapid and simple method to study DNA Fragmentation Index (DFI) in sperm samples from boar under bright-field and fluorescence microscopy. Discrimination of sperm cells containing fragmented DNA relies on the extreme peripheral diffusion of their chromatin fragments, whereas those sperm nuclei without DNA fragmentation do not disperse or show very restricted spreading of DNA loops close to the flagellum. The basic methodology provided in the commercial kit Sperm-Sus-Halomax® allows, in addition to a direct estimation of DFI in a sperm sample under bright field microscopy, a direct visualization of DNA breaks by incorporation of labelled nucleotides using the DNA polymerase I following the in situ nick translation assay (ISNT methodology not provided in the kit). An external control using DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) on human and boar sperm samples was used in this experiment. The results obtained show (i) low levels of background DNA fragmentation (from 0.7 to 10%), (ii) no significant differences for DFI after the application of Sperm-Sus-Halomax and ISNT, with a tendency to be underestimated after using DBD-FISH and (iii) a characteristic chromatin organization in boar sperm nucleus, with a particular response to chromatin loop relaxation and preferential DNA labelling by ISNT at the proximal nuclear area, close to the flagellum. This methodology allows the routine assessment of boar sperm samples for DFI, as well as basic and clinical research on this relevant topic in any laboratory of semen analysis. [Copyright &y& Elsevier]

Abstract: The sperm chromatin structure assay (SCSA) was introduced by as a method to determine the susceptibility of sperm DNA to denaturation and how those results related to fertility. This initial study used human sperm and was followed by studies in bulls and boars . This assay was one of the first to introduce the technique of flow cytometry, which has the ability to evaluate specific sperm compartments of large numbers of sperm in a short time, as a methodology to evaluate sperm quality and further define the relationship of sperm quality to fertility. For any assay to be of use clinically, it must not only be validated and adapted for the species of interest, but guidelines that associate specific levels of fertility with assay results must be defined. This review will describe how our laboratory uses the SCSA for clinical diagnosis of reduced fertility in the stallion. [Copyright &y& Elsevier]

Objective: To improve the sperm chromatin dispersion (SCD) test and develop it as a simple kit (Halosperm® kit) for the accurate determination of sperm DNA fragmentation using conventional bright-field microscopy. Design: Method development, comparison, and validation. Setting: Medical genetics laboratory, academic biology center, and reproductive medicine centers. Patient(s): Male infertility patients attending the Reproductive Medicine Center. A varicocele patient and a group of nine fertile subjects. Intervention(s): None. Main Outcome Measure(s): [1] The quality of chromatin staining in relaxed sperm nuclear halos and tail preservation; [2] SCD scoring reproducibility; [3] comparison with the sperm chromatin structure assay in 45 samples; [4] frequency of sperm with DNA fragmentation after incubation with increasing doses of the nitric oxide donor sodium nitroprusside and in sperm samples for 9 fertile men, 46 normozoospermic patients, 23 oligoasthenoteratozoospermic patients, and a subject with varicocele. Result(s): The sperm nuclei with DNA fragmentation, either spontaneous or induced, do not produce or show very small halos of DNA loop dispersion after sequential incubation in acid and lysis solution. The improved SCD protocol (Halosperm® kit) results in better chromatin preservation, therefore highly contrasted halo images can be accurately assessed using conventional bright-field microscopy after Wright staining. Moreover, unlike in the original SCD procedure, the sperm tails are now preserved, making it possible to unequivocally discriminate sperm from other cell types. The χ2 test did not detect significant differences in the mean number of sperm cells with fragmented DNA as scored by four different observers. The intraobserver coefficient of variation for the estimated percentage of spermatozoa with fragmented DNA ranged from 6% to 12%. There was good correlation between the SCD and the sperm chromatin structure assay DNA fragmentation index (intraclass correlation coefficient R: 0.85; percent DNA fragmentation index mean difference: 2.16 significantly higher for SCD). Using the Halosperm®® kit, a dose-dependent increase in sperm DNA damage after sodium nitroprusside incubation was detected. The percentage of sperm cells with fragmented DNA in the fertile group was 16.3 ± 6.0, in the normozoospermic group, 27.3 ± 11.7, and in the oligoasthenoteratozoospermic group, 47.3 ± 17.3. In the varicocele sample, an extremely high degree of nuclear disruption was detected in the population of sperm cells with fragmented DNA. Conclusion(s): The improved SCD test, developed as the Halosperm® kit, is a simple, cost effective, rapid, reliable, and accurate procedure, for routinely assessing human sperm DNA fragmentation in the clinical andrology laboratory. [Copyright &y& Elsevier]

Abstract: We evaluated environmental-lead (Pb) effects on semen quality and sperm chromatin, considering Pb in seminal fluid (PbSF), spermatozoa (PbSpz), and blood (PbB) as exposure biomarkers in urban men (9.3μg/dL PbB). Several individuals (44%) showed decreases in sperm quality; sperm concentration, motility, morphology and viability associated negatively with PbSpz, whereas semen volume associated negatively with PbSF. Multiple linear regression estimated PbSF and PbSpz thresholds for alterations in semen quality. Forty-eight percent of samples showed high values of nuclear chromatin condensation (NCD) positively associated with PbSF and zinc in spermatozoa (ZnSpz). ZnSpz values were higher than in fertile men. These results suggest that Pb may affect sperm chromatin by altering sperm Zn availability. PbB was not associated with semen quality or NCD, suggesting that Pb in semen compartments assesses better the amount of Pb in the reproductive tract; therefore, these are better biomarkers to evaluate toxicity at low Pb-exposure levels. [Copyright &y& Elsevier]

: ObjectiveTo evaluate the association between chromatin structure and sperm motility.: DesignCross-sectional prospective study.: SettingScanian Andrology Centre, Malmo¨, Sweden; ENEA Casaccia, Rome, Italy; and Department of Occupational Medicine, Aarhus University, Aarhus, Denmark.: Patient(s)One hundred seventy-one males from Danish first pregnancy planner couples (group 1) and 278 Swedish military conscripts (group 2).: Main outcome measure(s)Sperm chromatin structure assay (SCSA) parameters, DNA fragmentation index (DFI), high DNA stainable (HDS), and sperm motility, which was evaluated manually and by use of computer-aided sperm analysis (CASA).: Result(s)A statistically significant negative correlation between DFI and the CASA percentage of motile sperms (group 1: r = −0.53; group 2: r = −0.38) was found. For the manual motility assessment, the correlation coefficients were slightly lower. Furthermore, HDS correlated negatively with CASA sperm motility (group 1: r = −0.39; group 2: r = −0.36) and percentage of World Health Organization category A motile sperm. In multiple linear regression analysis, concentration and SCSA parameters, but not the time of abstinence, were statistically significant predictors of sperm motility.: Conclusion(s)There is a moderate correlation between sperm motility and SCSA parameters. The study supports the assumption that both SCSA and motility can be relatively independent predictors of male fertility. [Copyright &y& Elsevier]

The effect of the straw volume employed for semen freezing was studied in 14 ejaculates from seven boars, by evaluating the viability, IVF capacity and chromatin state of spermatozoa. Frozen–thawed semen from 0.5 and 5 ml straws was compared to fresh semen. The chromatin condensation degree was determined by flow cytometry, using propidium iodide as fluorochrome, and the chromatin stability was evaluated by inducing its decondensation with SDS and EDTA. The results obtained for IVF, motility and normal apical ridge (NAR) were: 91.64, 78.14 and 81.47% sperm penetration, 80.78, 68.38 and 70.83% monospermy, 10.86, 9.76 and 10.64% polyspermy, 87.14, 50.71 and 47.86% motility, 79.14, 56.14 and 53.36% NAR, for fresh semen, thawed semen in 0.5 and 5 ml straws, respectively. Frozen–thawed spermatozoa showed significantly increased (P<0.05) chromatin compactness compared to fresh spermatozoa (55.42, 48.41 and 47.08 fluorescence units (MIFU), for fresh semen, thawed semen in 0.5 and 5 ml straws, respectively). Chromatin was significantly more unstable (P<0.05) in spermatozoa frozen in 0.5 ml straws (174.7 MIFU) compared to those frozen in 5 ml straws (155.53 MIFU) or to those in fresh semen (149.74 MIFU). [Copyright &y& Elsevier]

We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 °C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 °C showed a significant (P<0.05) rise in the SCSA measures (Mean(αt), S.D.(αt), and percent cells outside the main population—COMP(αt)) over time, with the degree of rise being more dramatic at 37 °C. Over all stallions, samples stored at 5 °C showed no significant (P>0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP(αt) from stallions classified as subfertile showed an increased susceptibility to denaturation or decline in chromatin quality between 20 and 31 h when stored at 5 °C; however, spermatozoa from fertile stallions did not change during the time intervals analyzed. These data suggest that sperm DNA from some subfertile stallions may decline at a greater rate than spermatozoa from fertile stallions when exposed to similar storage conditions. [Copyright &y& Elsevier]

Objective To determine consequences of lymphoma treatments on sperm characteristics and sperm DNA, and to evaluate predictors of sperm recovery. Design Multicenter prospective longitudinal study of patients analyzed before treatment and after 3, 6, 12, and 24 months. Setting University hospitals. Patient(s) Seventy-five Hodgkin lymphoma and non-Hodgkin lymphoma patients and a control group of 257 fertile men. Intervention(s) Semen analyses, and sperm DNA and chromatin assessments. Main Outcome Measure(s) Comparisons of sperm characteristics before and after treatment. Result(s) Patients already had altered sperm characteristics before lymphoma treatment, with no identified risk factor. Sperm count, total sperm count, motility, and vitality decreased after treatment, with lowest values at 3 and 6 months. Twelve months after treatment, mean sperm count recovered to pretreatment values after doxorubicin, bleomycin, vinblastine, darcarbacine (ABVD) or ABVD + radiotherapy, but not after doxorubicin, cyclophosphamide, vincristine, prednisone (CHOP) or mechlorethamine, oncovin, procarbazine, prednisone (MOPP) chemotherapies. It was noteworthy that 7% of patients remained azoospermic at 24 months. After 24 months, Kaplan-Meier estimates showed that more than 90% of patients will recover normal sperm count after ABVD or ABVD + radiotherapy vs. 61% for CHOP chemotherapies. In multivariate analyses including diagnosis and treatment protocol, only pretreatment total sperm count was related to recovery. Compared with a control group, lymphoma patients had higher sperm chromatin alterations and DNA fragmentation before any treatment. After treatment, DNA fragmentation assessed by TUNEL assay and sperm chromatin structure assay decreased from 3 and 6 months, respectively, while remaining higher than in the control group during follow-up. Conclusion(s) Lymphoma patients had altered sperm DNA and chromatin before treatment. Lymphoma treatment had damaging effects on spermatogenesis. These data on both the recovery period according to treatment modalities and the pre- and post-treatment chromatin status of sperm are useful tools for counseling patients wishing to conceive. [ABSTRACT FROM AUTHOR]

Objective: To determine the consequences of adjuvant testicular germ cell tumor treatment (TGCT) on sperm characteristics and sperm DNA, and to evaluate the predictors of sperm recovery. Design: Multicenter prospective longitudinal study of patients analyzed before treatment and after 3, 6, 12, and 24 months. Setting: University hospitals. Patient(s): One hundred twenty-nine volunteer TGCT patients and a control group of 257 fertile men. Intervention(s): Routine semen analyses, sperm DNA, and chromatin assessments. Main Outcome Measure(s): Comparisons of mean sperm characteristics before and after treatment, with sperm recovery analyzed by the Kaplan-Meier method. Result(s): The quantitative and qualitative sperm characteristics decreased after treatment, with lowest values at 3 and 6 months and with variations according to treatment type. The mean total sperm count recovered to pretreatment values at 12 months after treatment after two or fewer bleomycin, etoposide, and cisplatin (BEP) cycles, but not after radiotherapy or more than two BEP cycles. Only the treatment modalities and pretreatment sperm production were related to recovery of the World Health Organization reference sperm values. An increased proportion of patients had elevated high sperm DNA stainability at 6 months after radiotherapy. Conclusion(s): Adjuvant treatments for testicular germ cell tumor have drastic effects on spermatogenesis and sperm chromatin quality. These new data on both the recovery period according to treatment modalities and the post-treatment chromatin status of sperm are useful tools for counseling patients wishing to conceive. [ABSTRACT FROM AUTHOR]

The characteristics of Halosperm® make this kit a reasonable alternative to allow basic and clinical research on sperm DNA fragmentation in any basic laboratory around the world. [Copyright &y& Elsevier]