scholarly journals Can the SCD test and terminal uridine nick-end labeling by flow cytometry technique (TUNEL/FCM) be used interchangeably to measure sperm DNA damage in routine laboratory practice?

2019 ◽  
Vol 29 (1) ◽  
Author(s):  
Cécile Grèze ◽  
Aline Guttmann ◽  
Hanae Pons-Rejraji ◽  
Marie-Paule Vasson ◽  
Jacqueline Lornage ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dna Damage ◽  
Gradient Centrifugation ◽  
Intraclass Correlation ◽  
Systematic Difference ◽  
Test Reliability ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Damage ◽  
Sperm Dna

Abstract Background Numerous tests have been proposed to evaluate sperm DNA integrity. To assess the sperm chromatin dispersion (SCD) test in an andrology laboratory, twenty-five men attending Clermont-Ferrand (France) University Hospital’s Center for Reproductive Medicine were recruited. Sperm DNA damage was measured in the same semen samples using the SCD test and the Terminal Uridine Nick-end Labeling by flow cytometry technique (TUNEL/FCM) after density gradient centrifugation. Results SCD test reliability between readings, readers or slides was clearly established with very high agreement between measurements (Intraclass correlation coefficient (ICC) at 0.97, 0.95 and 0.98 respectively). Despite very good agreement between the SCD test and TUNEL/FCM (ICC at 0.94), the SCD test tended to slightly but significantly underestimate DNA damage compared with TUNEL (p = 0.0127). This systematic difference between the two techniques was − 3.39 ± 1.45% (mean ± SE). Conclusions Andrology laboratories using the SCD test to measure sperm DNA damage need to know that it appears to give slightly underestimated measurements compared to TUNEL/FCM. However, this systematic underestimation is very small in amplitude. Both techniques give almost perfectly congruent results. Our study underlines the importance for each laboratory to validate its method to assess sperm DNA damage before implementing it in routine andrology lab practice.

scholarly journals Effect of Alcohol Consumption on the Sperm DNA Integrity: A Systematic Review

2017 ◽  
Vol 9 (13) ◽  
pp. 136
Author(s):  
Farah Hanan Fathihah Jaafar ◽  
Khairul Osman ◽  
Jaya Kumar ◽  
Siti Fatimah Ibrahim
Keyword(s):  
Dna Damage ◽  
Alcohol Consumption ◽  
Alcohol Withdrawal ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Damage ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Keywords Alcohol

There is no solid conclusion on the conventional sperm parameters in association with alcohol consumption, evaluation of sperm DNA integrity thus become a more reliable parameter. Hereby, this literature search was performed to summarize alcohol consumption on the sperm DNA integrity. A computerized database search was done through MEDLINE via Ovid (since 1946 until August 2017) and Cochrane was used. The following set of keywords: ‘alcohol consumption OR alcohol intake OR alcohol diet OR drinking alcohol OR ethanol diet’ AND ‘sperm DNA OR sperm chromatin OR sperm genome OR sperm histone OR sperm protamine’ were utilised. 24 articles were retrieved where only five studies conform to the inclusion criteria All studies demonstrated a negative effect of alcohol consumption on sperm DNA integrity, regardless of various range of alcohol doses and duration of alcohol consumption. Out of five studies reviewed, four studies were using a different approach to measure the sperm DNA damage. Hereby, this review identified a need to use a single approach of DNA damage test by having various method of alcohol administration and/or vice versa so that the extension of sperm DNA damage to alcohol consumption will have a better conclusion. On the same note, a few studies have reported the reversibility on conventional semen parameters, none has been done on the sperm DNA damage upon alcohol withdrawal. Therefore, the role of alcohol withdrawal on the reversibility of sperm DNA damage needs to be as well investigated further.

scholarly journals A cost for high levels of sperm competition in rodents: increased sperm DNA fragmentation

2016 ◽  
Vol 283 (1826) ◽  
pp. 20152708 ◽  
Author(s):  
Javier delBarco-Trillo ◽  
Olga García-Álvarez ◽  
Ana Josefa Soler ◽  
Maximiliano Tourmente ◽  
José Julián Garde ◽  
...  
Keyword(s):  
Dna Damage ◽  
Sperm Competition ◽  
Dna Fragmentation ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Sperm Dna Damage ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Physical And Chemical

Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage.

Effects of boar variability on comet-detected sperm-DNA damage following cryopreservation

2018 ◽  
Vol 58 (2) ◽  
pp. 252 ◽  
Author(s):  
L. Fraser ◽  
Ł. Zasiadczyk ◽  
C. S. Pareek
Keyword(s):  
Dna Damage ◽  
Membrane Integrity ◽  
Tail Length ◽  
Dna Integrity ◽  
Comet Tail ◽  
Sperm Dna Damage ◽  
Type Iv ◽  
Sperm Dna ◽  
Damage Type

Assessment of sperm-DNA integrity is a crucial issue in male fertility. In the present study, parameters derived from the image analysis of comets after single-cell gel electrophoresis were used to analyse the types of DNA damage of frozen–thawed boar spermatozoa. Semen, frozen in a cryoprotectant-free extender or in cryoprotectant-based extenders, was analysed for DNA fragmentation and with the following comet tail measures: percentage DNA in comet tail, comet tail length and olive tail moment. The percentages of sperm DNA damage in the comet tails were classified as Type 0 (no DNA damage), Type I (very low DNA damage), Type II (light DNA damage), Type III (medium DNA damage) and Type IV (heavy DNA damage). Sperm motility characteristics and membrane integrity were assessed in the pre-freeze and frozen–thawed semen samples. Assessment of sperm DNA fragmentation and comet tail measures showed marked inter-boar variability following cryopreservation. However, consistent differences among the boars, with respect to cryo-induced sperm DNA damage, were detected by the comet tail length and olive tail moment. Besides Type IV, all types of DNA damage were detected in the cryoprotectant-based extenders. It was found that the frequency of Type II and Type III of DNA damage of frozen–thawed spermatozoa was significantly greater in the cryoprotectant-based and cryoprotectant-free extenders respectively. Deterioration in the quality of the sperm DNA integrity was concomitant with a marked decline in sperm motility characteristics, reduced plasma membrane integrity and higher lipid peroxidation and aspartate aminotransferase activity after cryopreservation. It can be suggested that the comet-assay parameters, coupled with routine laboratory tests, are useful to improve the sperm evaluations of post-thaw quality of semen from individual boars and would offer more comprehensive information for a better understanding of the degree of cryo-induced sperm-DNA damage.

miR-424/322 is downregulated in the semen of patients with severe DNA damage and may regulate sperm DNA damage

2016 ◽  
Vol 28 (10) ◽  
pp. 1598 ◽  
Author(s):  
Kai Zhao ◽  
Yaoping Chen ◽  
Ruifeng Yang ◽  
Yang Bai ◽  
Cuiling Li ◽  
...  
Keyword(s):  
Dna Damage ◽  
Human Sperm ◽  
Vital Role ◽  
Dna Integrity ◽  
Dna Double Strand Breaks ◽  
Human Seminal Plasma ◽  
Sperm Dna Damage ◽  
Sperm Dna ◽  
Murine Homologue

Sperm DNA integrity is an essential factor for accurate transmission of genetic information. Human sperm DNA damage is a common cause of male infertility but the exact mechanism remains poorly understood. Considering the vital role of microRNA (miRNA) in multiple pathophysiological processes, we hypothesised that testicular miRNA is involved in sperm DNA damage during spermatogenesis. Infertile patients with high sperm DNA fragment index (DFI; n = 94) were selected from 1090 infertile men and a total of 18 testis-specific seminal miRNAs previously identified from human seminal plasma were chosen and tested. miR-29c and miR-424 were downregulated in men with high DFI. The inhibition of these two miRNAs in mice confirmed the role of miR-424 (murine homologue miR-322) in sperm DNA damage during spermatogenesis; by contrast, miR-29c exhibited a negative result. Thus, miR-424/322 is involved in sperm DNA damage. Furthermore, the dysregulation of this miRNA can induce DNA double-strand breaks during spermatogenesis.

scholarly journals Rapid rates of sperm DNA damage after activation in tench (Tinca tinca: Teleostei, Cyprinidae) measured using a sperm chromatin dispersion test

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Carmen López-Fernández ◽  
Matthew J G Gage ◽  
Francisca Arroyo ◽  
Altea Gosálbez ◽  
Ana M Larrán ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Dna Fragmentation ◽  
Living Organism ◽  
Sperm Chromatin ◽  
Tinca Tinca ◽  
External Fertilization ◽  
Sperm Dna Damage ◽  
Sperm Dna ◽  
Dispersion Test

Spermatozoal haplotypic DNA is prone to damage, leading to male fertility problems. So far, the assessment of sperm DNA breakage has been challenging because protamines render the nuclear chromatin highly compacted. Here, we report the application of a new test to quantify DNA fragmentation in spermatozoa of an externally fertilizing teleost fish. The sperm chromatin dispersion (SCD) test uses a species-specific lysing solution to generate controlled protein depletion that, followed by DNA-specific fluorescent labelling, allows an easy morphological discrimination between nuclei affected by DNA damage. Using tench (Tinca tinca) as our model, we first trialled the test against established, but more technically demanding, assays employing in situ nick translation (ISNT) and the comet assay. The SCD test showed high concordance with ISNT, comet assay measures and a chromatin-swelling test, confirming the application of this straightforward SCD technique to various aspects of reproductive biology. Second, we examined between-male variation in DNA damage, and measured changes through time following spermatozoal activation. Between-male variation in the basal levels of average DNA damage ranged from 0 to 20% of sperm showing damage, and all showed increases in DNA fragmentation through time (0–60 min). The rates of DNA damage increase are the fastest so far recorded in sperm for a living organism, and may relate to the external fertilization mode. Our findings have relevance for broodstock selection and optimizing IVF protocols routinely used in modern aquaculture.

scholarly journals Nuclear Integrity but Not Topology of Mouse Sperm Chromosome is Affected by Oxidative DNA Damage

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 501 ◽  
Author(s):  
Alexandre Champroux ◽  
Christelle Damon-Soubeyrand ◽  
Chantal Goubely ◽  
Stephanie Bravard ◽  
Joelle Henry-Berger ◽  
...  
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Chromosome 2 ◽  
Mouse Sperm ◽  
Sperm Dna Damage ◽  
Chromosome Architecture ◽  
Sperm Dna

Recent studies have revealed a well-defined higher order of chromosome architecture, named chromosome territories, in the human sperm nuclei. The purpose of this work was, first, to investigate the topology of a selected number of chromosomes in murine sperm; second, to evaluate whether sperm DNA damage has any consequence on chromosome architecture. Using fluorescence in situ hybridization, confocal microscopy, and 3D-reconstruction approaches we demonstrate that chromosome positioning in the mouse sperm nucleus is not random. Some chromosomes tend to occupy preferentially discrete positions, while others, such as chromosome 2 in the mouse sperm nucleus are less defined. Using a mouse transgenic model (Gpx5−/−) of sperm nuclear oxidation, we show that oxidative DNA damage does not disrupt chromosome organization. However, when looking at specific nuclear 3D-parameters, we observed that they were significantly affected in the transgenic sperm, compared to the wild-type. Mild reductive DNA challenge confirmed the fragility of the organization of the oxidized sperm nucleus, which may have unforeseen consequences during post-fertilization events. These data suggest that in addition to the sperm DNA fragmentation, which is already known to modify sperm nucleus organization, the more frequent and, to date, the less highly-regarded phenomenon of sperm DNA oxidation also affects sperm chromatin packaging.

scholarly journals Acridine Orange and Flow Cytometry: Which Is Better to Measure the Effect of Varicocele on Sperm DNA Integrity?

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Essam-Elden M. Mohammed ◽  
Eman Mosad ◽  
Asmaa M. Zahran ◽  
Diaa A. Hameed ◽  
Emad A. Taha ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acridine Orange ◽  
Pregnancy Outcome ◽  
Dna Fragmentation ◽  
Chromatin Condensation ◽  
Semen Parameters ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Sperm Dna Damage ◽  
Sperm Dna

We evaluated the effect of varicocelectomy on semen parameters and levels of sperm DNA damage in infertile men. A total of 75 infertile men with varicocele and 40 fertile men (controls) were included in this study. Semen analysis and sperm DNA damage expressed as the DNA fragmentation index using acridine orange staining and chromatin condensation test by flow cytometry were assessed before and 6 months after varicocelectomy. The patients were also followed up for 1 year for pregnancy outcome. Semen parameters were significantly lower in varicocele patients compared to controls (P<0.05). Mean percentages of sperm DNA fragmentation and sperm DNA chromatin condensation in patients were significantly higher than those in controls (P<0.05). After varicocelectomy, sperm DNA fragmentation improved significantly, whereas sperm chromatin condensation was not significantly changed. In 15 out of 75 varicocele patients, clinical pregnancy was diagnosed; those with positive pregnancy outcome had significant improvement in sperm count, progressive sperm motility, and sperm DNA fragmentation, but there was no significant difference in sperm DNA condensation compared to negative pregnancy outcome patients. We concluded from this study that acridine orange stain is more reliable method than flow cytometry in the evaluation of sperm DNA integrity after varicocelectomy.

scholarly journals Species-Specific Differences in Sperm Chromatin Decondensation Between Eutherian Mammals Underlie Distinct Lysis Requirements

2021 ◽  
Vol 9 ◽  
Author(s):  
Jordi Ribas-Maynou ◽  
Estela Garcia-Bonavila ◽  
Carlos O. Hidalgo ◽  
Jaime Catalán ◽  
Jordi Miró ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Condensation ◽  
Proteinase K ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Dna Breaks ◽  
Chromatin Decondensation ◽  
The Third ◽  
Core Length ◽  
Sperm Dna

Sperm present a highly particular DNA condensation that is acquired during their differentiation. Protamines are key elements for DNA condensation. However, whereas the presence of protamine 1 (P1) is conserved across mammalian species, that of protamine 2 (P2) has evolved differentially, existing only few species that use both protamines for sperm DNA condensation. In addition, altered P1/P2 ratios and alterations in the expression of P1 have previously been associated to infertility and DNA damage disorders. On the other hand, different methods evaluating DNA integrity, such as Sperm Chromatin Dispersion (SCD) and Comet tests, need a previous complete DNA decondensation to properly assess DNA breaks. Related with this, the present study aims to analyze the resilience of sperm DNA to decodensation in different eutherian mammals. Sperm samples from humans, horses, cattle, pigs and donkeys were used. Samples were embedded in low melting point agarose and treated with lysis solutions to induce DNA decondensation and formation of sperm haloes. The treatment consisted of three steps: (1) incubation in SDS + DTT for 30 min; (2) incubation in DTT + NaCl for 30 min; and (3) incubation in DTT + NaCl with or without proteinase K for a variable time of 0, 30, or 180 min. How incubation with the third lysis solution (with or without proteinase K) for 0, 30, and 180 min affected DNA decondensation was tested through analyzing core and halo diameters in 50 sperm per sample. Halo/core length ratio was used as an indicator of complete chromatin decondensation. While incubation time with the third lysis solution had no impact on halo/core length ratios in species having P1 and P2 (human, equine and donkey), DNA decondensation of pig and cattle sperm, which only present P1, significantly (P &lt; 0.05) increased following incubation with the third lysis solution for 180 min. In addition, the inclusion of proteinase K was found to accelerate DNA decondensation. In conclusion, longer incubations in lysis solution including proteinase K lead to higher DNA decondensation in porcine and bovine sperm. This suggests that tests intended to analyze DNA damage, such as halo or Comet assays, require complete chromatin deprotamination to achieve high sensitivity in the detection of DNA breaks.

P–103 Novel sperm preparation techniques compared with conventional preparation method

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
N Vahidi ◽  
F S Amjadi ◽  
F Kalat. sabz ◽  
Z Zandie ◽  
N Narimani
Keyword(s):  
Dna Damage ◽  
Zeta Potential ◽  
Dna Fragmentation ◽  
Sperm Parameters ◽  
Sperm Chromatin ◽  
P Value ◽  
Sperm Preparation ◽  
Sperm Dna Damage ◽  
Sperm Dna ◽  
Preparation Techniques

Abstract Study question Which sperm preparation technique separate the best quality sperm? Summary answer Microfluidic method improved the sperm parameters and decreased sperm DNA damage. What is known already About 40% of infertility issues are due to male factor. One of the known causes of male infertility is associated with low sperm parameters and high level DNA fragmentation. Sperm preparation techniques in ICSI procedures is used in order to obtain the best-quality sperm. Study design, size, duration The present study was designed to compare Microfluidic, Zeta potential, Magnetic Activated Cell Sorting (MACS) and Swim-up methods for sperm preparation and the effect of these methods on semen parameters and sperm DNA integrity in infertile men (n = 25) with a mean age of 38. Participants/materials, setting, methods In this study, each sample was divided into 4 groups, one part for preparing by Microfluidic method, one of them for preparing by Swim -up method, the other one was prepared by MACS and the last one was prepared by zeta potential. Then sperm count, viability, motility and morphology were assessed according to WHO 2010. DNA damage were assessed by Sperm DNA Fragmentation assay and sperm chromatin packaging assessed by CMA3 staining test Main results and the role of chance Sperm parameters including viability, motility, and morphology in the Microfluidic method were significantly improved and sperm DNA damage were significantly lower than three other methods (P-value &lt;0.05). The sperm parameters and sperm DNA damage after preparation by MACS and Zeta potential methods were not significantly different however in the Swim-up method sperm parameters were lower than three other methods (P-value &lt;0.05). Limitations, reasons for caution The fertilization and pregnancy rate of the resulting embryos are not available. Wider implications of the findings: Our results showed that Microfluidic can be an effective way to improve sperm quality of infertile male compared to conventional preparation methods. We also found instead of the MACS method, we can use the Zeta potential method according to their costs, for sperm preparation during ART cycle. Trial registration number *

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