Intra-individual variation of sperm DNA fragmentation in the Human ejaculate.

This retrospective study assessed the biological intra-individual variability of the percentage of sperm with DNA damage (SDF) observed in subsequent ejaculates of the same individual. Variation in SDF was analyzed using the Mean Signed Difference (MSD) statistic based on 131 individuals, comprising 333 ejaculates. Either two, three or four ejaculates were collected from each individual. With this cohort of individuals two main questions were addressed; (1) does the number of ejaculates analyzed influence the variability in the level of SDF associated with each individual? and (2) is the variability observed in SDF similar when individuals are ranked according to their level of SDF? Results showed that the variation observed in mean SDF was not different when 2, 3 or 4 ejaculates were analyzed; consequently, we suggest that the assessment of SDF based on two ejaculates is likely to be representative of the mean SDF expected for the individual. In parallel, it was determined that the variation in SDF increased as SDF increased; in individuals presenting with an SDF value of lower than 30% (potentially fertile), only 5% possessed levels of MSD that could be considered as variable as that presented by individuals presenting with a recurrent high SDF. Finally, we showed that a single assessment of SDF in individuals with medium SDF (20-30%) was less likely to be predictive of the SDF value in the next ejaculate, and therefore, less informative of the patient's SDF status.

Bacterial DNase activity as a putative inductor of sperm DNA fragmentation in infected bull frozen-thawed semen samples.

The aim of this study was to investigate the relationship between DNase activity associated with bacterial contamination of incubated bovine frozen-thawed spermatozoa and elevated sperm DNA fragmentation. Electrophoresis analysis of plasmid PBR322 incubated for 30 min at 37 °C with the supernatant of the diluent of frozen-thawed centrifuged bovine semen straws infected with bacteria showed clear evidence of DNase activity when compared to plasmid incubated in similarly prepared non-infected bovine diluent supernatant (Experiment 1). This DNase activity was subsequently found to be time dependent (0-60 min) and its activity prevented in the presence of EDTA (10 and 20 mM; Experiment 2). Semen straws infected (n = 10) and not infected (n = 10) with bacteria where incubated at 37 °C for up to 48h post-thaw. Semen infected with bacteria showed an exponential increase in bacterial growth and a corresponding increase in sperm DNA fragmentation. Non-infected semen samples showed no change in the incidence of sperm DNA fragmentation over the same period of incubation (Experiment 3). Our experiments reinforce the idea that exogenous DNases present in the semen should be considered as one of the primary contributing causes of sperm DNA fragmentation post ejaculation. In the case of the bull, post-thaw incubation of commercial straws contaminated with bacteria, resulted in increased levels of sperm DNA fragmentation, most likely associated with DNase activity (potentially restriction endonucleases) derived from the bacteria. Such adverse changes in sperm DNA fragmentation, as described here in vitro, may be also operative after insemination in the female reproductive tract (in vivo) and highlight the importance of implementing high levels of hygiene practice during semen processing, especially in light of future trends of bacterial resistance to the common antibiotics used in semen diluents.

The efficacy of novel centrifugation-free sperm selection (Io-Lix) on sperm parameters and ICSI reproductive outcomes.

RESEARCH QUESTION: What is the effect of a novel non-centrifugation method (Io-Lix) of sperm selection on sperm parameters and intracytoplasmic sperm injection (ICSI) reproductive outcomes? DESIGN: This pilot study elevated the capacity of the Io-Lix sperm selection protocol to improve sperm parameters (concentration, motility and sperm DNA fragmentation) of the neat ejaculate. Once established, the reproductive outcomes of Io-Lix selected spermatozoa were used for autologous and donor oocyte ICSI programmes and their efficacy compared with those using conventional swim-up. RESULTS: Io-Lix sperm selection resulted in lower sperm concentration yield (P < 0.001) and sperm DNA fragmentation (P < 0.001) but higher sperm motility (P < 0.001) when compared with spermatozoa in the neat ejaculate. When compared with swim-up sperm selection the Io-Lix protocol resulted in a 14.7% (P = 0.028) increase in pregnancy rate and 16.3% (P = 0.047) reduction in miscarriages in the autologous ICSI programme. A similar comparison of sperm selection procedures employed for a donor oocyte ICSI programme showed no difference in terms of their respective reproductive outcomes. CONCLUSIONS: The Io-Lix sperm selection protocol resulted in improved pregnancy rate and reduction in miscarriage when applied to autologous ICSI, which was attributed to a reduction in the proportion of spermatozoa with DNA damage post-selection. A similar finding was not apparent in the donor oocyte programme, which may be associated with the capacity of the donor oocyte to repair sperm DNA post-syngamy.

Sperm DNA damage in men with spinal cord injury: the relative incidence of single- and double-strand DNA breaks.

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

Reliability of the sperm chromatin dispersion assay to evaluate sperm deoxyribonucleic acid damage in men with infertility.

OBJECTIVE: To investigate the intraindividual agreement of the sperm chromatin dispersion (SCD) assay results to assess sperm DNA fragmentation (SDF) in men with infertility. DESIGN: Diagnostic test reliability study. SETTING: Andrology laboratories. PATIENT(S): A total of 219 men with infertility. INTERVENTION(S): Sperm DNA fragmentation assessment in two ejaculates of the same subjects within a 3-month interval, using the SCD assay performed and analyzed by the same observers under similar testing conditions. MAIN OUTCOME MEASURE(S): Cohen's κ statistics to assess the degree of agreement between the pairs of results after converting the nominal SCD values into categorical data, that is, normal (<20%), intermediate (21%-29%), and high (≥30%) SDF rates. We also assessed the pairs of results using reliability measures for numerical variables (intraclass correlation coefficient for consistency using the two-way mixed-effects model and Bland-Altman plots). RESULT(S): The degree of agreement in classifying patients according to normal and pathological SDF classes was overall substantial (κ = 0.632; 95% confidence interval [CI], 0.546-0.718). A total of 76.7% of individuals were classified under the same class using paired ejaculates. The agreement rate was highest (approximately 80%) in ejaculates initially classified as either normal or high and lowest (approximately 60%) among those with intermediate SDF levels. The frequency of intermediate SDF ejaculates downgraded to normal or upgrade to high SDF classes in the second test was similar (approximately 20%). The intraclass correlation coefficient was 0.856 (95% CI, 0.812-0.887), and the mean difference between the pairs of observations was 0.80% (95% CI, -0.72 to 2.23), indicating no systematic difference between paired observations. CONCLUSION(S): Our study indicates a substantial intraindividual agreement of paired SCD assay results to classify men with infertility into three SDF categories: normal, intermediate, and high. The reliability of the SCD assay was adequate and exceeded 0.80 using two ejaculates analyzed within a 3-month interval under similar conditions. Although this evidence overall supports a single SCD test for patient classification using predefined SDF thresholds, particularly when the first test shows normal or high SDF levels, one in four men will have discordant values in paired ejaculates. Clinicians should be judicious when using SDF test results in decision-making.

Optoelectronic manipulation of bio-droplets containing cells or macromolecules by active ferroelectric platforms.

Photovoltaic optoelectronic tweezers are a useful platform with many applications in optical manipulation and nanotechnology. They are based on electrical forces associated with the bulk photovoltaic effect presented by certain ferroelectric crystals, such as Fe doped lithium niobate. This manipulation technique has experienced huge developments in recent years, although its use in biology and biomedicine is still scarce. Recently, a novel strategy has been reported that extends the platform capabilities to the manipulation of polar droplets, such as water and aqueous bio-droplets, promising great potential for biological applications. In this work, we are taking this challenge, addressing the manipulation of cells and macromolecules contained inside the droplets by optoelectronic ferroelectric platforms. On the one hand, experiments of photoelectric induced migration of DNA and sperm droplets have been successfully developed and the corresponding droplet dynamics have been analyzed in depth. From this analysis, parameters of the biomaterial such as its concentration and its electrical charge have been evaluated, showing the sensing capabilities of the platform. In fact, the charge of sperm cells has been demonstrated to be negative, and the relative sperm concentration of the samples determined. On the other hand, experiments on the light-induced merging of two droplets have been carried out. Specifically, sperm droplets are mixed with droplets containing acridine orange, a convenient dye for visualization purposes. The spermatozoa become clearly visible in the final droplet through fluorescence imaging. The results point out the multiple possibilities of application of the optoelectronic ferroelectric platform in biology and biomedicine including the development of "lab on a chip" devices. Hence, these capabilities introduce these platforms as an efficient tool in biotechnology.

Free circulating DNA and DNase activity in the ejaculates of men with spinal cord injury.

STUDY DESIGN: Retrospective descriptive study. OBJECTIVES: To study the presence of cell-free DNA (cfDNA) and DNase activity in males with spinal cord injury (SCI) with elevated sperm DNA fragmentation. SETTING: Hospital in Toledo, Spain; University-based Genetics laboratory in Madrid, Spain. METHODS: Semen was collected from 15 males with spinal cord injury and elevated sperm DNA fragmentation (SDF). The presence and concentration of cfDNA was assessed using standard gel electrophoresis and microfluidic electrophoresis. DNase activity was evaluated in seminal plasma and under the presence of EDTA and EGTA to control the response of enzyme activity. cfDNA fragments were mapped on the sperm genome using FISH. All results were compared to 15 normozoospermic fertile donors. RESULTS: Standard gel electrophoresis revealed a cfDNA band of ~150 bp in all samples from males with SCI; this band was ocasionally accompanied by another band of ~90 bp. These bands were not observed in normozoospermic donors. Microfluidid electrophoresis only identified the equivalent band of 150 bp. No correlation was observed between the intensity of the two bands and the level of SDF in males with SCI. Although DNase activity was present in the seminal plasma of both normozoospermic donors and men with SCI it did not digest cfDNA. cfDNA fragments were found to be hybridized all over the sperm genome. CONCLUSIONS: SCI patients with elevated sperm DNA fragmentation showed a 150 bp DNA band of cfDNA in the seminal plasma, which appeared resistant to DNase activity.

Microencapsulation of human spermatozoa increases membrane stability and DNA longevity.

The microencapsulation of spermatozoa offers potential benefits for maintaining sperm survival in vitro. The technique has also resulted in the production of offspring in several domestic animal species, but as yet, it has not been successfully applied in human reproductive medicine. This study examined the effect of alginic acid microencapsulation on human sperm membrane integrity (viability) and sperm DNA fragmentation (SDF) following storage for 24 hr at 37°C. The cumulative sperm viability (Log-rank, Mantel-Cox; Chi-square = 114.95, p = .000) and cumulative sperm DNA fragmentation (Log-rank, Mantel-Cox; Chi-square = 187.86, p = .000) of encapsulated spermatozoa were substantially improved when compared to control spermatozoa. Significant differences in the dynamic behaviour of different individuals were only apparent for sperm viability in microencapsulated samples (p = .021) while no significant differences were observed in control spermatozoa (p = .245); the equivalent comparison for SDF showed no differences (control p = .320; microencapsulated p = .432). We present potential scenarios for the use of microencapsulated human spermatozoa in reproductive medicine.

Effect of Sperm Concentration and Storage Temperature on Goat Spermatozoa during Liquid Storage.

The use of cooled semen is relatively common in goats. There are a number of advantages of cooled semen doses, including easier handling of artificial insemination (AI) doses, transport, more AI doses per ejaculate, and higher fertility rates in comparison with frozen AI doses. However, cooled semen has a short shelf life. The objective of this study was to examine the effect of temperature and sperm concentration on the in vitro sperm quality during liquid storage for 48 h, including sperm motility and kinetics, response to oxidation, mitochondrial membrane potential (MMP) and DNA fragmentation in goats. Three experiments were performed. In the first, the effects of liquid preservation of semen at different temperatures (5 °C or 17 °C), durations (0, 24 and 48 h) and sperm concentrations (250 × 106 sperm/mL (1:2 dilution rate), 166.7 × 106 sperm/mL (1:3 dilution rate) or 50 × 106 sperm/mL (1:10 dilution rate)) on sperm motility and kinetics were studied. In the second experiment, the effect of temperature, sperm washing and concentration on sperm motility and DNA fragmentation was studied. Finally, the effect of sperm concentration and duration of storage at 5 °C on sperm motility, response to oxidative stress and MMP was examined. We found that refrigerated liquid storage of goat sperm impaired sperm quality, such as motility, MMP and response to oxidation, as storage time increased; however, sperm DNA fragmentation index was not significantly affected. Liquid storage at 5 °C preserved higher total motility than at 17 °C. Moreover, we observed that the reduction of sperm concentration below 500 × 106 sperm/mL did not seem to improve the quality of spermatozoa conserved in milk-based extender in the conditions tested.

Assessment of avian sperm DNA fragmentation using the sperm chromatin dispersion assay.

Herein we report a simple method for assessing avian sperm DNA fragmentation (SDF) using the sperm chromatin dispersion test (SCDt). The presence of sperm DNA damage was confirmed indirectly by correlating results of the SCDt determined in three bird species with results of a corresponding neutral comet assay (r=0.99; P<0.005). Frozen-thawed spermatozoa of each species were also incubated at 37°C for 5h and the within- and between-species variation of SDF, as an indicator of sperm DNA longevity, examined. The dynamic assessment of SDF using the SCDt revealed species and individual bird (rooster and turkey) differences in sperm DNA longevity.

Optoelectronic generation of bio-aqueous femto-droplets based on the bulk photovoltaic effect.

The generation and manipulation of small aqueous droplets is an important issue for nano- and biotechnology, particularly, when using microfluidic devices. The production of very small droplets has been frequently carried out by applying intense local electric fields to the fluid, which requires power supplies and metallic electrodes. This procedure complicates the device and reduces its versatility. In this work, we present a novel and flexible, to the best of our knowledge, electrodeless optoelectronic method for the production of tiny droplets of biologically friendly aqueous fluids. Our method takes advantage of the photoinduced electric fields generated by the bulk photovoltaic effect in iron-doped lithium niobate crystals. Two substrate configurations, presenting the polar ferroelectric axis either parallel or perpendicular to the active surface, have been successfully tested. In both crystal geometries, small droplets on the femtoliter scale have been obtained, although with a different spatial distributions correlated with the symmetry of the photovoltaic fields. The overall results demonstrate the effectiveness of the optoelectronic method to produce femtoliter droplets, both with pure water and with aqueous solutions containing biological material.

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

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

Multi-centre assessment of nitroblue tetrazolium reactivity in human semen as a potential marker of oxidative stress.

The nitroblue tetrazolium (NBT) reaction as a tracer of oxidative stress was examined in 707 ejaculates from seven clinics. Semen was initially surveyed by classifying the NBT reaction using a pre-established rank for the Oxisperm® test based on three colourimetric levels: L1, low (n = 141 [20%]); L2, medium (n = 538 [76%]) and L3, high (n = 28 [4%]). L3 was indicative of a high level of superoxide anions. Halosperm® chromatin dispersion assay was used to analyse samples of ejaculates 30 min after ejaculation; no difference was found in DNA fragmentation of L1 or L3; L3 category semen samples incubated for 24 h at 37oC showed a significantly faster rate (P < 0.001) of DNA damage than those in L1. The NBT reaction was further characterized in the ejaculates of 100 patients to determine the relative contribution of seminal plasma, spermatozoa, or both. Seminal plasma was the most significant fraction of •O2- localization, whereas sperm fractions generated detectable reactive oxygen species in only 32% of the ejaculates. Formazan precipitates were primarily associated with the sperm mid-piece and seminal leukocytes; however, not all spermatozoa stained positive to formazan and not all leukocytes presented with equivalent production of superoxide anions.

Two-Tailed Comet Assay (2T-Comet): Simultaneous Detection of DNA Single and Double Strand Breaks.

A modification of the original comet assay was developed for the simultaneous evaluation of DNA single strand breaks (SSBs) and double strand breaks (DSBs) in human spermatozoa. The two-dimensional perpendicular tail comet assay (2T-comet) combines non-denaturing and denaturant conditions to the same sperm nucleoid. In this case, the species-specific deproteinized sperm is first subjected to an electrophoretic field under non-denaturing conditions to mobilize isolated free discrete DNA fragments produced from DSBs; this is then followed by a second electrophoresis running perpendicular to the first one but under alkaline conditions to produce DNA denaturation, exposing SSBs on the same linear DNA chain or DNA fragments flanked by DSBs. This procedure results in a two dimensional comet tail emerging from the core where two types of original DNA affected molecule can be simultaneously discriminated. The 2T-comet is a fast, sensitive, and reliable procedure to distinguish between single and double strand DNA damage within the same cell. It is an innovative method for assessing sperm DNA integrity, which has important implications for human fertility and andrological pathology. This technique may be adapted to assess different DNA break types in other species and other cell types.

Reduced sperm DNA longevity is associated with an increased incidence of still born; evidence from a multi-ovulating sequential artificial insemination animal model.

PURPOSE: Using a rabbit model, we assessed the influence of sperm DNA longevity on female reproductive outcomes. METHODS: Semen was collected from 40 bucks, incubated at 38 °C for 24 h, and the rate of sperm DNA fragmentation (rSDF) was determined using the sperm chromatin dispersion assay. Males were allocated into high rSDF (>0.5 units of increase per hour) or low rSDF (<0.5 units of increase per hour) groups. High and low rSDF semen samples were sequentially artificially inseminated into the same doe to reduce female factor variability, and pregnancy outcomes were recorded. RESULTS: While there was no difference in SDFs between rSDF groups immediately after collection (T0), differences were significant after 2 h of incubation; SDFs determined at collection and rSDF behaved as independent characters (Pearson correlation = 0.099; P = 0.542). Following artificial insemination, the rate of stillborn pups was significantly higher in does inseminated by males with a high rSDF (14/21) compared to those with low rSDF (15/6); (contingency χ(2) 5.19; p = 0.022). The risk of stillborn when low rSDF rabbits were used for insemination was 0.16, but increased to 0.36 when high rSDF animals were used (odds ratio = 2.85; 95 % confidence interval = 1.4-2.7). CONCLUSION(S): Dynamic assessment of SDF coupled with natural multiple ovulation, high fecundity of the rabbit and control over female factor influence, provided a useful experimental model to demonstrate the adverse effect of reduced sperm DNA longevity on reproductive outcome.

DNA damage in spermatozoa from infertile men with varicocele evaluated by sperm chromatin dispersion and DBD-FISH.

PURPOSE: Evaluation of DNA integrity is an important test, possessing greater diagnostic and prognostic significance for couples requiring assisted reproduction. In this study, we evaluate the levels of DNA damage in infertile patients with varicocele with respect to fertile males by the sperm chromatin dispersion (SCD) test. The presence of DNA breaks in spermatozoa was confirmed by DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). METHODS: In this study, the frequency of sperm cells with fragmented DNA was studied in a group of 20 infertile patients with varicocele and compared with 20 fertile males. The spermatozoa were processed to classify different levels of DNA fragmentation using the Halosperm(®) kit, an improved SCD test, and DBD-FISH. RESULTS: Patients with varicocele showed 25.54 ± 28.17 % of spermatozoa with fragmented DNA, significantly higher than those of the group of fertile subjects (11.54 ± 3.88 %). The proportion of degraded cells in total sperm cells with fragmented DNA was sixfold higher in the case of patients with varicocele. The presence of DNA breaks in spermatozoa was confirmed by DBD-FISH. 5-bp Classical satellite-2 regions showed greater sensitivity to damage or "breakage" than alphoid satellite regions. CONCLUSIONS: Our finding preliminary demonstrated an increase of DNA fragmentation associated to severe sperm damage, in infertile patients with varicocele with respect to fertile males. 5-bp Classical satellite-2 regions showed greater sensitivity to damage or "breakage" than alphoid satellite regions.

Detection of DNA damage in cumulus cells using a chromatin dispersion assay.

DNA damage in cumulus cells (CCs) might be related with the developmental competence of the enclosed oocytes, however, conclusive studies are missing, partially due to the lack of a reliable, cheap, fast, and reproducible DNA damage test. We report the development of a chromatin dispersion test that allows for a fast evaluation of double strand DNA (ds-DNA) damage in CCs. The whole experiment was performed using CCs from 103 oocyte retrieval cycles evaluating the prototype D3-MAX ability (a chromatin dispersion based assay) to detect DNA breaks against in situ nick translation (ISNT) and a two tailed comet assay (TT-comet). Samples were collected from women younger than 35 years of age with a good response to stimulation. Pooled cumulus cells of MII oocytes were used. The chromatin dispersion assay results correlate with the double strand-DNA breaks values assessed by the TT-comet assay (Spearman Rho = 0.624; p = 0.003;), while the correlation was poor when compared to the single strand DNA (ss-DNA) breaks observed also with the TT-comet assay (Spearman Rho = -0.141; p = 0.554). ISNT showed a correspondence in the same cells between enzymatic incorporation of modified nucleotides and halos of chromatin dispersion. We conclude that D3-Max test detects mainly ds-DNA breaks in cumulus cells and is a reliable, fast, and easy reproducible assay suitable for routine clinical practices once the influence on oocyte quality has been established.

Diagnostic accuracy of sperm DNA degradation index (DDSi) as a potential noninvasive biomarker to identify men with varicocele-associated infertility.

PURPOSE: Varicocele is a frequent cause of impaired testicular function that has been associated with increased levels of sperm DNA fragmentation (SDF). Sperm with degraded DNA (DDS), as observed using the sperm chromatin dispersion (SCD) test, represent a subpopulation of spermatozoa with extensive DNA and nuclear protein damage. The aim of this work was to determine the usefulness of sperm DNA degradation index (DDSi) as a novel noninvasive biomarker to identify infertile men with varicocele. METHODS: A total of 593 semen samples obtained from men attending infertility clinics were analyzed for SDF and DDS with the SCD test. These samples were classified as: (1) fertile donors; (2) infertile patients with least two failed assisted reproduction cycles; (3) leukocytospermia; (4) Chlamydia trachomatis infection; (5) testicular cancer, and (6) infertile men with varicocele. The DDSi was obtained by determining the proportion of DDS in the whole sperm population presenting with fragmented DNA. The diagnostic accuracy of DDSi was evaluated by correlation coefficient and receiver operating characteristics analyses. RESULTS: A positive correlation (r ≥ 0.52) was observed between the SDF and the frequency of degraded sperm in all patient groups. The sperm DNA degradation index (DDSi) was at least twice as higher in infertile men with varicocele (mean: 0.54) compared with other clinical conditions and fertile donors (means ranging from 0.02 to 0.21; P < 0.0001). A DDSi ≥ 0.33 identified patients with varicocele with 94 % accuracy. CONCLUSION: Although DDS is not pathognomonic of varicocele, the DDSi is a useful noninvasive biomaker to identify infertile individuals with varicocele when examining sperm DNA damage during a routine semen analysis. This finding may alert practitioners and laboratories performing semen analysis that in the presence of an abnormal DDSi it is likely that a given patient has varicocele. It is therefore strongly recommended that such patients be referred to urologists in order to undergo a full andrological examination and be properly counseled.

Use of DBD-FISH for the study of cervical cancer progression.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a procedure to detect and quantify DNA breaks in single cells, either in the whole genome or within specific DNA sequences. This methodology combines microgel embedding of cells and DNA unwinding procedures with the power of FISH coupled to digital image analysis. Cells trapped within an agarose matrix are lysed and immersed in an alkaline unwinding solution that produces single-stranded DNA motifs beginning at the ends of internal DNA strand breaks. After neutralization, the microgel is dehydrated and the cells are incubated with fluorescently labeled DNA probes. The amount of hybridized probe at a target sequence correlates with the amount of single-stranded DNA generated during the unwinding step, which is in turn proportional to the degree of local DNA breakage. A general view of the technique is provided, emphasizing its versatility for evaluating the association between DNA damage and progressive stages of cervical neoplasia.

Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay.

Key ConceptsThe two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar-phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites "ALSs."DBD-FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to allow for the simultaneous evaluation of DSBs and SSBs in mammalian spermatozoa. Here we have compiled a retrospective overview of how the TT-comet assay has been used to investigate the structure and function of sperm DNA across a diverse range of mammalian species (eutheria, metatheria, and prototheria). When conducted as part of the TT-comet assay, we illustrate (a) how the alkaline comet single assay has been used to help understand the constitutive and transient changes in DNA structure associated with chromatin packing, (b) the capacity of the TT-comet to differentiate between the presence of SSBs and DSBs (c) and the possible implications of SSBs or DSBs for the assessment of infertility.