• Clinical and Experimental Reproductive Medicine
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• v.37 no.3
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• pp.245-251
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• 2010

Objective: Human sperm nucleus DNA damage may negatively affect pregnancy outcome, and the spermatozoa of infertile men have more DNA damage than that of fertile men. The aim of this study was to evaluate the effect of microsurgical varicocelectomy on human sperm nucleus DNA integrity. Methods: We reviewed the medical records of 18 subfertile male patients who underwent microsurgical varicocelectomy at our hospital from April 2006 to April 2007. Varicocele was diagnosed by physical examination and Doppler ultrasound. Standard semen analysis was performed in 18 patients before and 4 months after microsurgical varicoceletcomy using a computer assisted semen analyzer. Sperm nucleus DNA integrity was assessed by a single-cell gel electrophoresis (comet assay). Results: No recurrence of varicocele was observed after 4 months later. The DNA fragmentation index improved after varicocelectomy compared with pre-operatively (19.3 versus 13.7%, respectively, p<0.05). Semen analysis parameters (total count, concentration, motile sperm, viability, strict morphology) increased after varicocelectomy, but the difference did not reach statistical significance. Conclusion: Our data suggest that microsurgical varicocelectomy can improve semen analysis parameters and human sperm nucleus DNA integrity in infertile men with varicocele.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.3
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• pp.939-944
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• 2016

Background: Cell-free DNA circulating in blood is a candidate biomarker for malignant tumors. Unlike uniformly truncated DNA released from apoptotic non diseased cells, DNA released from necrotic cancer cells varies in size. Objectives: To measure the DNA integrity index in serum and the absolute DNA concentration to assess their clinical utility as potential serum biomarkers for colorectal carcinoma (CRC) compared to CEA and CA19-9. Materials and Methods: Fifty patients with CRC, 10 with benign colonic polyps and 20 healthy sex and age matched volunteers, were investigated by real time PCR of ALU repeats (ALU q-PCR) using two sets of primers (115 and 247 bp) amplifying different lengths of DNA fragments. The DNA integrity index was calculated as the ratio of q-PCR results of ALU 247/ALU 115bp. Results: Serum DNA integrity was statistically significantly higher in CRC patients compared to the benign and control groups (p<0.001). ROC curves for differentiating CRC patients from normal controls and benign groups had areas under curves of 0.90 and 0.85 respectively. Conclusions: The DNA integrity index is superior to the absolute DNA concentration as a potential serum biomarker for screening and diagnosis of CRC. It may also serve as an indicator for monitoring the progression of CRC patients. Combining CEA and CA19-9 with either of the genetic markers studied is better than either of them alone.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.265-271
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• 2013

Cryopreservation and in vitro fertilization (IVF) protocols are important in genetic studies and applications to transgenic animals. Various studies about boar sperm cryopreservation have been studied for a long time. Those were about the use of extenders, the choice of sugars, the cooling and warming rates. The factors that influence the boar sperm are the dramatic changes in temperatures, osmotic and toxic stresses, and reactive oxygen species (ROS) generation. Among these factors, ROS generation is the main damage to DNA which is a principal genetic material and the most important for the practical applications. So we wondered whether ROS generation could be reduced. In previous study, monothioglycerol (MTG) was essential for the culture of embryo stem cells. Therefore we added MTG in the freezing extender based on lactose-egg yolk (LEY) with trehalose. For the assessment of the frozen-thawed sperm, we focused onmotility, membrane integrity and DNA damage. First, we used a computer-aided sperm analysis system for overall conditions of sperm such as motility and viability. Then we performed the sperm chromatin structure assay for DNA integrity and hypo-osmotic swelling test for membrane integrity. And our result showed the existence of MTG in the freezing extender caused less damage to DNA and higher motility in frozen-thawed boar sperm. Also we checked a relative antioxidant activity of MTG in modified Modena B extender. We concluded that this reagent can activate sperm mitochondria at MTG $0.2{\mu}M$, contribute to sperm motility and DNA integrity but there was no significant difference on membrane integrity. Also antioxidant activity of MTG in modified Modena B extender was proved.

• Journal of Preventive Medicine and Public Health
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• v.45 no.4
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• pp.235-243
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• 2012

Objectives: Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples. Methods: Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments. Results: The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study. Conclusions: Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.

• BMB Reports
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• v.56 no.2
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• pp.108-113
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• 2023

Cohesin is a ring-shaped protein complex that comprises the SMC1, SMC3, and α-kleisin proteins, STAG1/2/3 subunits, and auxiliary factors. Cohesin participates in chromatin remodeling, chromosome segregation, DNA replication, and gene expression regulation during the cell cycle. Mitosis-specific α-kleisin factor RAD21 and meiosis-specific α-kleisin factor REC8 are expressed in embryonic stem cells (ESCs) to maintain pluripotency. Here, we demonstrated that RAD21 and REC8 were involved in maintaining genomic stability and modulating chromatin modification in murine ESCs. When the kleisin subunits were depleted, DNA repair genes were downregulated, thereby reducing cell viability and causing replication protein A (RPA) accumulation. This finding suggested that the repair of exposed single-stranded DNA was inefficient. Furthermore, the depletion of kleisin subunits induced DNA hypermethylation by upregulating DNA methylation proteins. Thus, we proposed that the cohesin complex plays two distinct roles in chromatin remodeling and genomic integrity to ensure the maintenance of pluripotency in ESCs.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.35-43
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• 2009

Objectives: Seminal concentration of tumor necrosis factor-alpha (TNF-${\alpha}$) relevant to sperm nuclear DNA integrity has not been studied. The present study aimed to evaluate seminal concentration of TNF-${\alpha}$ in correlation with sperm parameters and nuclear DNA integrity in asymptomatic healthy donors. Methods: Semen samples were obtained by masturbation from forty-five healthy donors. Results: Sperm quality was assessed by computer-assisted semen analysis and nuclear DNA integrity measured by the TUNEL assay in raw semen. TNF-${\alpha}$ concentrations were measured by ELISA in frozen-thawed seminal plasmas. Sperm DNA fragmentation rates were ranged between 1.9% and 53.0% (mean${\pm}$SD, 12.4${\pm}$9.6%). Univariate analysis revealed that DNA fragmentation rate was not associated with sperm concentration or motility but had a correlation with linearity negatively (r=-0.325, p=0.03) and age positively (r=0.484, p=0.001). The mean seminal concentration of TNF-${\alpha}$ was 4.9 pg/mL with a range from 1.1 to 22.6 pg/mL. The TNF-${\alpha}$ concentration had no correlation with clinically relevant parameters of sperm quality or nuclear DNA fragmentation rate. Conclusion: Our results indicate that sperm nuclear DNA fragmentation may be not associated with seminal TNF-${\alpha}$ level or sperm quality in asymptomatic healthy donors.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.1
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• pp.17-24
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• 2018

Objective: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. Methods: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values < 0.05 were considered to indicate statistical significance. Results: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. Conclusion: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.

• Clinical and Experimental Reproductive Medicine
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• v.51 no.1
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• pp.42-47
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• 2024

Objective: This study evaluated the effects of temperature and storage time on the quality and DNA integrity of freeze-dried sperm from individuals with normozoospermia. Methods: Normal sperm samples from 15 men aged 24 to 40 years were studied. Each sample was divided into six groups: fresh, freezing (frozen in liquid nitrogen), freeze-dried then preserved at room temperature for 1 month (FD-1m-RT), freeze-dried then preserved at room temperature for 2 months (FD-2m-RT), freeze-dried then preserved at 4 ℃ for 1 month (FD-1m-4 ℃), and freeze-dried then preserved at 4 ℃ for 2 months (FD-2m-4 ℃). The morphology, progressive motility, vitality, and DNA integrity of the sperm were evaluated in all groups. Results: In all freeze-dried groups, sperm cells were immotile after rehydration. The freeze-dried groups also showed significantly less sperm vitality than the fresh and frozen groups. Significantly more morphological sperm abnormalities were found in the freeze-dried groups, but freeze-drying did not lead to a significantly higher DNA fragmentation index (DFI). The DFI was significantly higher in the FD-2m-RT group than in the other freeze-dried groups. Conclusion: The freeze-drying method preserved the integrity of sperm DNA. The temperature and duration of storage were also identified as factors that influenced the DFI. Accordingly, more research is needed on ways to improve sperm quality in the freeze-drying process.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.3
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• pp.335-340
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• 2006

A large number of toxicological substances and pharmacological and physical agents can cause reproductive intervention at the cellular and molecular level. The present study was designed to assess the effect of mercury ($HgCl_2$) at 50 to $550{\mu}M$ concentration ranges, in vitro, on the sperm membrane and DNA integrity, viability, and acrosomal status of normal bull spermatozoa. The samples were processed for sperm analyses using semen-diluting fluid (PBS, pH 7.2). We recorded a sharp increase in the lipid peroxidation/LPO rate; the highest was at $550{\mu}M$ mercury concentration, indicating a deleterious effect of mercury on the sperm membrane intactness. There was also a strong negative correlation between LPO rate and % viable spermatozoa (R = 0.987, p<0.001). Data obtained from a comet assay technique revealed that mercury is capable of inducing DNA breaks in the sperm nuclei. Interestingly, 92% of DNA breaks were double-stranded. The correlation between LPO rate and % DNA breaks was 0.984. Performing the gelatin test indicates that mercury is able to alter the integrity of acrosomal membranes showing an abnormal acrosome reaction. In this regard, a strong link was found between LPO rate and % halos (R = 0.990, p<0.001). Collectively, mercury proved to be a potent oxidant in the category of environmental factors affecting bull spermatozoa. Hence, considering the wide spread use of mercury and its compounds, these metals should be regarded with more concern.

• Journal of Life Science
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• v.33 no.7
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• pp.586-592
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• 2023

Oxidative stress is a critical factor affecting the quality and viability of sperm during boar semen storage. Oxidative stress is also a significant concern during the process of freezing semen. The process of semen storage involves exposing the sperm to various stressors, including temperature changes, cryoprotectants, and extended periods of incubation. In addition, oxidative stress can lead to the production of reactive oxygen species (ROS) within the sperm, resulting in oxidative damage to cellular components, such as lipids, proteins, and DNA. Striking a balance between ROS production and the antioxidant defense system is crucial for maintaining sperm viability and functionality during semen storage. Moreover, the prolonged storage of boar semen leads to an increase in ROS levels, which can impair sperm motility, membrane integrity, and DNA integrity. ROS-induced lipid peroxidation affects the fluidity and stability of sperm membranes, leading to decreased sperm motility. Moreover, oxidative damage to the DNA can result in DNA fragmentation, compromising the genetic integrity of the sperm. In conclusion, oxidative stress is a significant challenge in maintaining sperm quality during boar semen storage. Understanding the mechanisms underlying oxidative stress and their impacts on sperm function is crucial for developing effective strategies to minimize oxidative damage and improve sperm storage outcomes.