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An analysis of 2 decades (2003–2022) of seismicity recorded by the Spanish and Portuguese seismic networks along the West Iberian passive margin has resulted in a better understanding of the distribution of moderate seismic activity in this intraplate submarine area. The study provides a precise trend of specific alignments inferred from the density maps of seismicity, giving an accurate depiction of event distribution along two wide stripes that extend for 700 km through the ocean floor in the WNW–ESE direction. These bands are parallel to the Africa–Eurasia plate boundary but are distinctly separated from its related seismicity by approximately 300 and 700 km, respectively. This is a sufficient distance to be considered intraplate activity. When trying to relate this seismicity to structural and geophysical features, a conclusive picture does not emerge. The earthquakes occur indiscriminately across thinned continental, hyperextended, and exhumed mantle rift domains. They fade out in proximity to undisputed oceanic crust, but some events extend beyond. The hypocentral depths signal a considerable number of events nucleating in the upper mantle. The focal mechanisms, although scarce, are predominantly strike-slip. Considering these observations, hypotheses ranging from subduction initiation and development of strained corridors to local structures of the margin are discussed in order to explain this relatively anomalous seismicity. However, some of them lack convincing arguments, while others are too vague. None of them are flawless, suggesting that several factors may be at play. Despite being one of the most probed passive margins in the world, the present geodynamic status of the West Iberian Margin manifested in its modern seismicity remains unknown. Interpreting these data within a global tectonic plate framework, together with the potential addition of seafloor seismometers, may provide the key to understanding this activity along one of the most archetypical margins of the Atlantic Ocean.

The cross-correlation of ambient noise records registered by seismic networks has proven to be a valuable tool to obtain new insights into the crustal structure at different scales. Based on 2 to 14 s period Rayleigh and Love dispersion data extracted from the seismic ambient noise recorded by 20 three-component broadband stations belonging to two different temporary experiments, we present the first (i) upper-crustal (1–12 km) high-resolution shear wave velocity and (ii) radial anisotropy variation models of the continental crust in NW Iberia. The area of study represents one of the best-exposed cross sections along the Variscan orogen of western Europe, showing the transition between the external eastern zones towards the internal areas in the west. Both the 2-D maps and an E–W transect reveal a close correspondence with the main geological domains of the Variscan orogen. The foreland fold-and-thrust belt of the orogen, the Cantabrian Zone, is revealed by a zone of relatively low shear wave velocities (2.3–3.0 km s−1), while the internal zones generally display higher homogeneous velocities (> 3.1 km s−1). The boundary between the two zones is clearly delineated in the models, depicting the arcuate shape of the orogenic belt. The velocity patterns also reveal variations of the elastic properties of the upper crust that can be linked to major Variscan structures, such as the basal detachment of the Cantabrian Zone, the stack of nappes involving pre-Variscan basement, or sedimentary features such as the presence of thick syn-orogenic siliciclastic wedges. Overall, the radial anisotropy magnitude varies between −5 % and 15 % and increases with depth. The depth pattern suggests that the alignment of cracks is the main source of anisotropy at < 8 km depths, although the intrinsic anisotropy seems to be significant in the West Asturian–Leonese Zone, the low-grade slate belt adjacent to the Cantabrian Zone. At depths > 8 km, widespread high and positive radial anisotropies are observed, which we attribute to the presence of subhorizontal alignments of grains and minerals in relation to the pre- or syn-orogenic deformation associated with the Variscan orogenesis.

This study was designed to develop a semiautomatic computer assisted methodology to evaluate the membrane integrity of ram spermatozoa using a commercial kit based on acridine orange/propidium iodide (AO/PI) labelling and ImageJ software. The study was divided into two experiments. In the first trial, the new computer-assisted method was validated by mixing fresh semen samples with different volumes of freeze killed spermatozoa to determine proportions of damaged spermatozoa in the final samples. The proportion of damaged spermatozoa in each sample determined by the automated procedure where highly correlated (R2=0.97, p

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a recently developed technique that allows cell-by-cell detection and quantification of DNA breakage in the whole genome or within specific DNA sequences. The present investigation was conducted to adapt the methodology of DBD-FISH to the visualization and evaluation of DNA damage in buccal epithelial cells. DBD-FISH revealed that DNA damage increased significantly according to H2O2 concentration (r2=0.91). In conclusion, the DBD-FISH technique is easy to apply in buccal cells and provides prompt results that are easy to interpret. Future studies are needed to investigate the potential applicability of a buccal cell DBD-FISH model to human biomonitoring and nutritional work.

Whole comparative genomic hybridization (W-CGH) is a new technique that reveals cryptic differences in highly repetitive DNA sequences, when different genomes are compared using metaphase or interphase chromosomes. W-CGH provides a quick approach to identify differential expansion of these DNA sequences at the single-chromosome level in the whole genome. In this study, we have determined the frequency of constitutive chromatin polymorphisms in the centromeric regions of human chromosomes using a whole-genome in situ cross-hybridization method to compare the whole genome of five different unrelated individuals. Results showed that the pericentromeric constitutive heterochromatin of chromosome 6 exhibited a high incidence of polymorphisms in repetitive DNA families located in pericentromeric regions. The constitutive heterochromatin of chromosomes 5 and 9 was also identified as highly polymorphic. Although further studies are necessary to corroborate and assess the overall incidence of these polymorphisms in human populations, the use of W-CGH could be pertinent and of clinical relevance to assess rapidly, from a chromosomal viewpoint, genome similarities and differences in closely related genomes such as those of relatives, or in more specific situations such as bone marrow transplantation where chimerism is produced in the recipient.

This study investigated possible variations in the chromatin structure of koilocytes resulting from human papillomavirus (HPV) infection. Alkaline-labile sites (ALS) were detected with the DNA breakage detection–fluorescence in situ hybridization (DBD-FISH) technique using a whole human genome DNA probe obtained from individuals without koilocytosis. The variable levels of ALS present were measured quantitatively using image analysis after whole-genome DNA hybridization. A significant increase in the number of ALS was observed in koilocytes compared with normal cells. We demonstrated that the presence of ALS could be an indicator of chromatin change in koilocytes caused by HPV infection.

The genome of stallion (Spanish breed) and donkey (Spanish endemic Zamorano-Leonés) were compared using whole comparative genomic in situ hybridization (W-CGH) technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.

The genome of stallion (Spanish breed) and donkey (Spanish endemic Zamorano-Leonés) were compared using whole comparative genomic in situ hybridization (W-CGH) technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.

The NW sector of the Iberian Peninsula has been classically considered as a seismically stable area, with only dispersed and low-magnitude activity. Around the village of Becerreá (Lugo) a significant concentration of earthquakes is observed. Since the beginning of instrumental record in 1979, seven seismic crisis have been reported in that zone, including 14 events of magnitude gt>4 and a larger event of magnitude 5.1mbLg (VII EMS) in 1997. Even if this seismicity is to be related to the general compressive regime between Africa and Eurasia, the seismotectonic characterization of the area is still controversial, as the different hypotheses considered are based on insufficiently accurate data. New insights to the problem are discussed here, coming from the deployment of a seismic portable network between 1999 and 2002, which has provided new accurate seismic data. The corresponding events are distributed around a subvertical axis, reaching a depth of 12km, and epicentres tend to concentrate beneath the intersection of the main Becerreá fault and secondary, ENE/WSW oriented faults. This intersection structure is thus interpreted as being responsible for the relatively high level of seismic activity over the area.

Introduction: In post-COVID survivors, transforming growth factor-beta-1 (TGF-β1) might mediate fibroblast activation, resulting in persistent fibrosis., Methods: In this study, 82 survivors of COVID-19-associated ARDS were examined at 6- and 24-months post-ICU discharge. At 6-months, quantitative CT analysis of lung attenuation was performed and active TGF-β1 was measured in blood and exhaled breath condensate (EBC)., Results: At 6-months of ICU-discharge, patients with reduced DmCO/alveolar volume ratio exhibited higher plasma and EBC levels of active TGF-β1. Plasma TGF-β1 levels were elevated in dyspneic survivors and directly related to the high-attenuation lung volume. In vitro, plasma and EBC from survivors induced profibrotic changes in human primary fibroblasts in a TGF-β receptor-dependent manner. Finally, at 6-months, plasma and EBC active TGF-β1 levels discriminated patients who, 24-months post-ICU-discharge, developed gas exchange impairment., Discussion: TGF-β1 pathway plays a pivotal role in the early-phase fibrotic abnormalities in COVID-19-induced ARDS survivors, with significant implications for long-term functional impairment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Alfaro, Casitas, Díaz-García, García-Tovar, Galera, Torres-Vargas, Fernández-Velilla, López-Fernández, Añón, Quintana-Díaz, García-Río and Cubillos-Zapata.)

This study examines the association between nurses' perception of caring and attitudes toward caring for dying patients in end-of-life care units. We used a descriptive cross-sectional design with convenience sampling. Data were collected from May to June 2021 through self-reported questionnaires from 303 Spanish nurses (mean age = 48 years, 78.9% female). Participants completed the Caring Dimension Inventory (CDI-25) and the Frommelt Attitudes Toward Care of the Dying scale. Demographic and training information were collected. Data were analyzed using Stata 16.0. Attitudes toward Care of the Dying Patient were significantly higher in nurses with palliative care training (β = 2.829, p = 0.018) and those who scored higher on the psychosocial (β = 0.789, p = 0.0001) and technical dimensions (β = 0.487, p = 0.011) of the CDI-25. Multivariate analysis identified the psychosocial dimension of the CDI-25 scale (β = 0.783, p = 0.0001) and palliative care training (β = 2.686, p = 0.017) as the only significant variables associated with Attitudes toward Care of the Dying Patient. Overall, nurses exhibited a positive attitude toward caring for dying patients. Our results could potentially help nursing directors identify those with the necessary qualities and training for optimal end-of-life care and to further plan training courses to provide optimal care in end-of-life situations.

Background: Understanding the enduring respiratory consequences of severe COVID-19 is crucial for comprehensive patient care. This study aims to evaluate the impact of post-COVID conditions on respiratory sequelae of severe acute respiratory distress syndrome (ARDS)., Methods: We examined 88 survivors of COVID-19-associated severe ARDS six months post-intensive care unit (ICU) discharge. Assessments included clinical and functional evaluation as well as plasma biomarkers of endothelial dysfunction, inflammation, and viral response. Additionally, an in vitro model using human umbilical vein endothelial cells (HUVECs) explored the direct impact of post-COVID plasma on endothelial function., Results: Post-COVID patients with impaired gas exchange demonstrated persistent endothelial inflammation marked by elevated ICAM-1, IL-8, CCL-2, and ET-1 plasma levels. Concurrently, systemic inflammation, evidenced by NLRP3 overexpression and elevated levels of IL-6, sCD40-L, and C-reactive protein, was associated with endothelial dysfunction biomarkers and increased in post-COVID patients with impaired gas exchange. T-cell activation, reflected in CD69 expression, and persistently elevated levels of interferon-β (IFN-β) further contributed to sustained inflammation. The in vitro model confirmed that patient plasma, with altered levels of sCD40-L and IFN-β proteins, has the capacity to alter endothelial function., Conclusions: Six months post-ICU discharge, survivors of COVID-19-associated ARDS exhibited sustained elevation in endothelial dysfunction biomarkers, correlating with the severity of impaired gas exchange. NLRP3 inflammasome activity and persistent T-cell activation indicate on going inflammation contributing to persistent endothelial dysfunction, potentially intensified by sustained viral immune response., (© 2024. The Author(s).)

Introduction: Although higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Interestingly, adenosinergic signaling has emerged as a powerful immune checkpoint driving tumor development and progression., Methods: Here, we explored the expression of the adenosinergic ecto-enzymes CD39 and CD73 in T-lymphocytes of OSA patients without any evidence of cancer, as well as their soluble forms in plasma (sCD39 and sCD73), along with adenosine. In addition, we explored the role of intermittent hypoxia (IH) in this context by in vitro models., Results: Our results showed that CD39 is upregulated while CD73 is downregulated in OSA T-cells' membrane. Moreover, our findings suggest that IH, through HIF-1, mediates the upregulation of both CD39 and CD73; and that CD73 downregulation could be mediated by a higher release of sCD73 by OSA T-lymphocytes. Importantly, we found that both sCD39 and sCD73 are upregulated in OSA plasma, suggesting T-lymphocytes as a potential source for plasmatic sCD73. Finally, our data propose the alterations in CD39/CD73 axis could underlie the upsurge of adenosine levels in the plasma of OSA patients., Conclusion: Our study reveals a hypoxia-mediated alteration of the CD39/CD73 axis in OSA patients, which could trigger ADO upregulation, thus potentially contributing to the immune suppressive environment and ultimately facilitating tumor development and progression. Therefore, our data highlights the need for new longitudinal studies evaluating CD39 and/or CD73 as potential cancer-risk prognostic biomarkers in OSA patients., (Copyright © 2024 SEPAR. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Hyperinflammation, hypercoagulation and endothelial injury are major findings in acute and post-COVID-19. The SARS-CoV-2 S protein has been detected as an isolated element in human tissues reservoirs and is the main product of mRNA COVID-19 vaccines. We investigated whether the S protein alone triggers pro-inflammatory and pro-coagulant responses in primary cultures of two cell types deeply affected by SARS-CoV-2, such are monocytes and endothelial cells., Methods: In human umbilical vein endothelial cells (HUVEC) and monocytes, the components of NF-κB and the NLRP3 inflammasome system, as well as coagulation regulators, were assessed by qRT-PCR, Western blot, flow cytometry, or indirect immunofluorescence., Results: S protein activated NF-κB, promoted pro-inflammatory cytokines release, and triggered the priming and activation of the NLRP3 inflammasome system resulting in mature IL-1β formation in both cell types. This was paralleled by enhanced production of coagulation factors such as von Willebrand factor (vWF), factor VIII or tissue factor, that was mediated, at least in part, by IL-1β. Additionally, S protein failed to enhance ADAMTS-13 levels to counteract the pro-coagulant activity of vWF multimers. Monocytes and HUVEC barely expressed angiotensin-converting enzyme-2. Pharmacological approaches and gene silencing showed that TLR4 receptors mediated the effects of S protein in monocytes, but not in HUVEC., Conclusion: S protein behaves both as a pro-inflammatory and pro-coagulant stimulus in human monocytes and endothelial cells. Interfering with the receptors or signaling pathways evoked by the S protein may help preventing immune and vascular complications driven by such an isolated viral element. Video Abstract., (© 2024. The Author(s).)

Introduction: Although higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Recently, the adhesion receptor P-selectin glycoprotein-1 (PSGL 1) has been identified as a novel immune checkpoint, which are recognized major hallmarks in several types of cancer and have revolutionized cancer therapy., Methods: The expression of PSGL-1 and its ligands VISTA and SIGLEC-5 was assessed in the leucocytes of OSA patients and control subjects exploring the role of intermittent hypoxia (IH) using in vitro models. In addition, PSGL-1 impact on T-cells function was evaluated by ex vivo models., Results: Data showed PSGL-1 expression is upregulated in the T-lymphocytes from patients with severe OSA, indicating a relevant role of hypoxemia mediated by intermittent hypoxia. Besides, results suggest an inhibitory role of PSGL-1 on T-cell proliferation capacity. Finally, the expression of SIGLEC-5 but not VISTA was increased in monocytes from OSA patients, suggesting a regulatory role of intermittent hypoxia., Discussion: In conclusion, PSGL-1 might constitute an additional immune checkpoint leading to T-cell dysfunction in OSA patients, contributing to the disruption of immune surveillance, which might provide biological plausibility to the higher incidence and aggressiveness of several tumors in these patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Díaz-García, García-Sánchez, Alfaro, López-Fernández, Mañas, Cano-Pumarega, López-Collazo, García-Río and Cubillos-Zapata.)

Obstructive sleep apnea (OSA) patients are at special risk of suffering atherosclerosis, leading to major cardiovascular diseases. Notably, the transforming growth factor (TGF-β) plays a crucial role in the development and progression of atherosclerosis. In this context, the central regulator of TGF-β pathway, SMAD4 (small mother against decapentaplegic homolog 4), has been previously reported to be augmented in OSA patients, which levels were even higher in patients with concomitant cardiometabolic diseases. Here, we analyzed soluble and intracellular SMAD4 levels in plasma and monocytes from OSA patients and non-apneic subjects, with or without early subclinical atherosclerosis (eSA). In addition, we used in vitro and ex vivo models to explore the mechanisms underlying SMAD4 upregulation and release. Our study confirmed elevated sSMAD4 levels in OSA patients and identified that its levels were even higher in those OSA patients with eSA. Moreover, we demonstrated that SMAD4 is overexpressed in OSA monocytes and that intermittent hypoxia contributes to SMAD4 upregulation and release in a process mediated by NLRP3. In conclusion, this study highlights the potential role of sSMAD4 as a biomarker for atherosclerosis risk in OSA patients and provides new insights into the mechanisms underlying its upregulation and release to the extracellular space.

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., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflicts of interest., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

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.

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 × 10 6 sperm/mL (1:2 dilution rate), 166.7 × 10 6 sperm/mL (1:3 dilution rate) or 50 × 10 6 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 × 10 6 sperm/mL did not seem to improve the quality of spermatozoa conserved in milk-based extender in the conditions tested.

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 × 10 6 /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 × 10 6 /mL was approximately 3.3 times higher when compared to samples containing 25 × 10 6 /mL and 3.9 higher in comparison with samples adjusted to 12 × 10 6 /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 × 10 6 /mL in order to avoid a higher susceptibility of the sperm DNA molecule towards fragmentation.

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.

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.

This study compared the potential of assessing sperm DNA fragmentation (SDF) from neat semen and the subsequent swim-up (SU) procedure to predict pregnancy when conducting ICSI of fertile donor oocytes. Infertile females (n=81) were transferred embryos resulting from intracytoplasmic sperm injection (ICSI) of their partner's spermatozoa and proven donor oocytes. This model normalized the impact of female factor in putative sperm DNA repair. Semen was blindly assessed for SDF using Halosperm immediately following ejaculation (NS) and after swim-up at the time of ICSI fertilisation. There was a decrease in SDF values of the ejaculated semen sample following the swim-up protocol (P=0.000). Interestingly, pregnancy could be equally predicted from SDF values derived from either neat or swim-up semen samples. Receiver operator curves and the derived Youden's indices determined SDF cutoff values for NS and SU of 24.8% and 17.5%, respectively. Prediction of pregnancy from NS SDF had a sensitivity of 75% and a specificity of 69%, whereas for SU SDF was 78% and 73%, respectively. While increased levels of SDF negatively impact reproductive outcome, we have shown that a reduction in SDF following sperm selection using ICSI with proven donor oocytes is not mandatory for achieving pregnancy. This suggests that a certain level of DNA damage that is not detectable using current technologies could be impacting on the relative success of assisted reproductive technology (ART) procedures. Consequently, we propose a modification of the so called 'iceberg model' as a possible rationale for understanding the role of SDF in reproductive outcome.

We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a recently developed technique that allows cell-by-cell detection and quantification of DNA breakage in the whole genome or within specific DNA sequences. The present investigation was conducted to adapt the methodology of DBD-FISH to the visualization and evaluation of DNA damage in buccal epithelial cells. DBD-FISH revealed that DNA damage increased significantly according to H2O2 concentration (r2=0.91). In conclusion, the DBD-FISH technique is easy to apply in buccal cells and provides prompt results that are easy to interpret. Future studies are needed to investigate the potential applicability of a buccal cell DBD-FISH model to human biomonitoring and nutritional work.

This experiment documented the incidence and variability of sperm characteristics found in freshly collected and ex vivo-manipulated semen samples from a population of disease-free captive koalas with a special emphasis on the dynamic aspects of DNA fragmentation. These changes were analyzed in light of the putative negative effect of iatrogenic damage after chilled storage and cryopreservation with respect to different semen extender compositions to maximize sperm longevity. Sperm DNA fragmentation (SDF) dynamics (SDF assessment after a varying period of time) was investigated with the sperm chromatin dispersion assay after either dilution in tris-citrate media and chilled preservation at 4°C for upward of 16 days or cryopreservation in either glycerol or dimethylacetamide (DMA) tris-citrate-based cryoprotectant media; corresponding data on progressive sperm motility, plasma membrane integrity, and the proportion of koala sperm with relaxed chromatin were also recorded. SDF analysis of the captive koala population revealed a low mean (± SEM) basal level of only 6.7% ± 0.9%. The percentage of progressive sperm motility, percentage of intact sperm plasma membranes, and the percentage of relaxed chromatin did not correlate significantly with that of basal SDF. Moreover, despite the absence of cysteine residues in marsupial protamines, koala spermatozoa showed remarkable stability in terms of their DNA integrity after the incubation of either fresh, chilled, or frozen-thawed semen samples; observations of progressive motility (P < .05) and plasma membrane integrity (P < .05) revealed that chilled koala spermatozoa declined after 4 days, whereas the incidence of relaxed chromatin increased after 8 days. Although koala SDF increased significantly (P < .05) with the period of chilled storage, these values remained less than 16% after 16 days storage and subsequent incubation at 35°C for a further 48 hours. Survivorship of prefreeze sperm DNA damage was not different when compared with sperm frozen in DMA or between sperm frozen in DMA or glycerol; however, spermatozoa frozen in glycerol showed a higher (P = .042) rate of DNA fragmentation than prefreeze spermatozoa. This result differed from that of observations of progressive motility, plasma membrane integrity, and relaxed chromatin, which were all adversely affected (P < .05) after cryopreservation in either glycerol or DMA; however, the postthaw characteristics of sperm cryopreserved in either glycerol or DMA were not different. After thawing, koala sperm chromatin tended to decondense; however, the incidence of sperm DNA fragmentation was not correlated with the incidence of sperm chromatin relaxation after glycerol (R = .2) or DMA (R = -.04) cryopreservation.

This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic injury model appears to explain post-thaw changes in koala SDF, the mechanisms resulting in relaxed chromatin require further study. A lack of correlation between the percentage of sperm with relaxed chromatin and SDF suggests that the timing of these pathologies are asynchronous. We propose an integrative model of cryo-induced osmotic injury that involves a combination of structural damage (rupture of membrane) and oxidative stress that first leads to the reduction of MMP and the relaxation of chromatin, which is then ultimately followed by an increase in DNA fragmentation.

Some methods for determining sperm DNA fragmentation, such as the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion test (SCD), provide additional information about particular subgroups of spermatozoa with specific irregularities. Thus, SCSA recognizes a specific sperm subpopulation, the high-DNA stainability sperm subpopulation (HDS), and SCD recognizes the so-called DNA-degraded sperm (DDS) subpopulation. Although some studies associate the presence of these subpopulations with specific aspects related to infertility, the relationship between both sperm subpopulations and their preponderance in specific clinical groups of infertile males has not been extensively investigated. In this study, HDS and DDS subpopulations were determined in a total of 37 human males: 8 males with proven fertility, 9 infertile males with asthenoteratozoospermia, 10 carriers of chromosomal reorganizations, and 10 infertile males with clinical varicocele. Results showed a significant increase of the DDS subpopulation (P < .001) in both the varicocele patient (16.85 ± 7.24) and carrier of rearranged genome (11.6 ± 5.23) groups, but not in patients with asthenoteratozoospermia (3.88 ± 1.55) or fertile donors (2.62 ± 1.68). No statistical differences were detected for the HDS subpopulation (P = .542), but the highest values were found in the varicocele and rearranged-genome groups. However, no correlation between the HDS and DDS subpopulations were found (r = 0.196; P = .244), suggesting that both represent a different class of sperm subpopulation in the ejaculate. A significant increase in HDS, and especially DDS, can be associated with the presence of varicocele or the rearrangement of chromosomes. Specific diagnostic tests to confirm the diagnosis must be performed in patients with increased DDS and HDS values.

Objective: To evaluate the relationship between duration of sexual abstinence and sperm selection on sperm DNA fragmentation (SDF)., Design: Prospective study based on normozoospermic individuals., Setting: Fertility and IVF unit and university unit for research., Patient(s): Two cohorts of normozoospermic individuals: 21 men (aged 25-35 years) attending a clinic and with clearly adverse female factors; and a group of 12 selected donors (aged 20-25 years)., Intervention(s): SDF assessment using the sperm chromatin the dispersión test (Halosperm) in two cohorts of normozoospermic men., Main Outcome Measure(s): SDF assessment after 24 hours of abstinence with recurrent ejaculation (one every 24 hours) using neat sperm samples; and SDF assessment before and after sperm selection with abstinence of 3 hours., Result(s): Lower baseline levels of SDF were observed after shorter periods of abstinence between ejaculations (24 hours and 3 hours) than those recommended. This effect is much more marked after quick repetitive ejaculation (3 hours of abstinence) and sperm selection., Conclusion(s): The present results challenge the role of abstinence in current male infertility treatments and suggest that SDF can be efficiently reduced by a biological practice consisting of short-term recurrent ejaculation coupled with effective sperm selection., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains.

Whole comparative genomic hybridization (W-CGH) is a new technique that reveals cryptic differences in highly repetitive DNA sequences, when different genomes are compared using metaphase or interphase chromosomes. W-CGH provides a quick approach to identify differential expansion of these DNA sequences at the single-chromosome level in the whole genome. In this study, we have determined the frequency of constitutive chromatin polymorphisms in the centromeric regions of human chromosomes using a whole-genome in situ cross-hybridization method to compare the whole genome of five different unrelated individuals. Results showed that the pericentromeric constitutive heterochromatin of chromosome 6 exhibited a high incidence of polymorphisms in repetitive DNA families located in pericentromeric regions. The constitutive heterochromatin of chromosomes 5 and 9 was also identified as highly polymorphic. Although further studies are necessary to corroborate and assess the overall incidence of these polymorphisms in human populations, the use of W-CGH could be pertinent and of clinical relevance to assess rapidly, from a chromosomal viewpoint, genome similarities and differences in closely related genomes such as those of relatives, or in more specific situations such as bone marrow transplantation where chimerism is produced in the recipient.

The genome of stallion (Spanish breed) and donkey (Spanish endemic Zamorano-Leonés) were compared using whole comparative genomic in situ hybridization (W-CGH) technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.