• Clinical and Experimental Reproductive Medicine
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• v.50 no.1
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• pp.44-52
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• 2023

Objective: The DNA integrity of spermatozoa that attach to fallopian tube (FT) cells is higher than spermatozoa that do not attach. FT epithelial cells can distinguish normal and abnormal sperm chromatin. This study investigated the effects of sperm with a high-DNA fragmentation index (DFI) from men with unexplained repeated implantation failure (RIF) on the Toll-like receptor (TLR) signaling pathway in human FT cells in vitro. Methods: Ten men with a RIF history and high-DFI and 10 healthy donors with low-DFI comprised the high-DFI (>30%) and control (<30%) groups, respectively. After fresh semen preparation, sperm were co-cultured with a human FT epithelial cell line (OE-E6/E7) for 24 hours. RNA was extracted from the cell line and the human innate and adaptive immune responses were tested using an RT2 profiler polymerase chain reaction (PCR) array. Results: The PCR array data showed significantly higher TLR-1, TLR-2, TLR-3, TLR-6, interleukin 1α (IL-1α), IL-1β, IL-6, IL-12, interferon α (IFN-α), IFN-β, tumor necrosis factor α (TNF-α), CXCL8, GM-CSF, G-CSF, CD14, ELK1, IRAK1, IRAK2, IRAK4, IRF1, IRF3, LY96, MAP2K3, MAP2K4, MAP3K7, MAP4K4, MAPK8, MAPK8IP3, MYD88, NFKB1, NFKB2, REL, TIRAP, and TRAF6 expression in the high-DFI group than in the control group. These factors are all involved in the TLR-MyD88 signaling pathway. Conclusion: The MyD88-dependent pathway through TLR-1, TLR-2, and TLR-6 activation may be one of the main inflammatory pathways activated by high-DFI sperm from men with RIF. Following activation of this pathway, epithelial cells produce inflammatory cytokines, resulting in neutrophil infiltration, activation, phagocytosis, neutrophil extracellular trap formation, and apoptosis.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.9
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• pp.1411-1420
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• 2020

Objective: The aim of study was to investigate the effects of season and single layer centrifugation (SLC) before cryopreservation on post-thaw bull sperm quality in Thailand. Methods: Semen was collected from 6 bulls (Bos indicus) in summer, rainy season and winter 2014 through 2016. Semen characteristics, sperm morphology, sperm kinematics, viability, chromatin structure and mitochondrial membrane were evaluated. Meteorological data were available from the local meteorological station; Results: Season had an effect on semen characteristics in the raw ejaculate, with higher proportions of normal spermatozoa and lower abnormalities in winter than in the other two seasons. Sperm kinematics, viability, DNA fragmentation index, and mitochondrial membrane potential were not different between seasons. Sperm samples selected by SLC had greater normal morphology and a lower proportion with bent tails than controls and higher values of progressive motility (PRO), beat cross frequency, linearity, straightness, wobble (WOB), and lower values of slow motility, velocity average path (VAP), velocity curved line, and amplitude of lateral head displacement than controls. In addition, SLC-selection had a favorable effect on PRO, VAP, and WOB that differed among seasons. Conclusion: Our results suggested that these bulls were well adapted to their location, with season having an effect on sperm morphology. Moreover, SLC could be used prior to cryopreservation, regardless of season, to enhance normal morphology and kinematics of bull sperm samples without adversely affecting other parameters of sperm quality. However, there was considerable variation among bulls in DNA fragmentation index, mitochondrial membrane potential and sperm viability. In addition, SLC had a positive effect on sperm morphology and sperm kinematics, which could be expected to influence fertility.

• Clinical and Experimental Reproductive Medicine
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• v.48 no.1
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• pp.61-68
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• 2021

Objective: This study was conducted to investigate the relationship of semen parameters in samples used for intracytoplasmic sperm injection (ICSI) with fertilization and pregnancy rates in infertile couples. Methods: In this prospective study of Infertile couples with male factor infertility that had undergone ICSI, fractions of the same semen samples obtained for microinjection (to ensure the best predictability) were evaluated to determine the semen parameters and sperm DNA fragmentation index (DFI) on the day of oocyte recovery. Results: In total, 120 couples completed the study and were subdivided into fertilized (n=87) and non-fertilized couples (n=33). The fertilized couples were further classified into pregnant (n=48) and non-pregnant (n=39) couples. Compared to non-fertilized and non-pregnant couples, fertilized and pregnant couples showed statistically significantly higher sperm viability and percentage of normal sperm morphology, as well as significantly lower sperm DFI values. A receiver operating characteristic curve analysis of data from the 120 ICSI cycles showed that sperm viability, normal sperm morphology percentages, and sperm DFI were significant prognostic indicators of fertilization at cutoff values of 40%, 7%, and 46%, respectively. A sperm DFI of 46% showed sensitivity and specificity of 95% and 90%, respectively, for predicting fertilization, and no clinical pregnancies occurred in couples with a sperm DFI above 46%. Conclusion: Semen parameters from the ICSI day sample, especially sperm viability, normal morphology, and DFI, had an impact on fertilization and pregnancy outcomes in ICSI cycles.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.4
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• pp.277-283
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• 2020

Objective: The sperm DNA fragmentation index (DFI) guides the clinician's choice of an appropriate assisted reproductive technology (ART) procedure. The DFI can be determined using commercially available methodologies, including sperm chromatin dispersion (SCD) kits and sperm chromatin structure assay (SCSA). Currently, when DFI is evaluated using SCD kits, the result is analyzed in reference to the SCSA-derived threshold for the choice of an ART procedure. In this study, we compared DFI values obtained using SCSA with those obtained using SCD and determined whether the difference affects the choice of ART procedure. Methods: We compared SCSA to two SCD kits, CANfrag (n=36) and Halosperm (n=31), to assess the DFI values obtained, the correlations between tests, the technical repeatability, and the impact of DFI on the choice of ART. Results: We obtained higher median DFI values using SCD kits than when using SCSA, and this difference was significant for the CANfrag kit (p<0.001). The SCD kits had significantly higher coefficients of variation than SCSA (p<0.001). In vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) would be chosen for a significantly higher proportion of patients if a decision were made based on DFI derived from SCD rather than DFI determined using SCSA (p=0.003). Conclusion: Our results indicate that SCD kit-specific thresholds should be established in order to avoid the unnecessary use of IVF/ICSI based on sperm DNA damage for the management of infertility. Appropriate measures should be taken to mitigate the increased variability inherent to the methods used in these tests.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.1
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• pp.53-62
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• 2023

Objective: The aim of this study was to investigate the relationships of serum folate (vitamin B9), cobalamin (vitamin B12) levels and diet with semen parameters (semen standard parameters [SSP] and DNA fragmentation index [DFI]) in infertile men. Methods: Sperm samples were assessed for SSP and DFI (using the sperm chromatin dispersion test). Serum vitamin concentrations were measured with an immuno-electrochemiluminescence assay, and men completed a semi-quantitative food frequency questionnaire (FFQ). Results: Serum folate levels were positively correlated with sperm progressive motility and DFI. A comparison of SSP between two groups of patients according to serum folate concentration (B9 <4.840 ng/mL and B9 ≥4.840 ng/mL) showed significantly higher sperm concentration and sperm progressive motility in the latter group. However, there was no difference between these groups regarding DFI. Interestingly, serum folate levels were significantly higher in patients with a high DFI (using the cut-offs of 30% or 18%). FFQ data showed that the consumption of fruits and egg yolk correlated positively with sperm concentration and sperm motility, respectively. Conclusion: Serum folate levels showed significant associations with sperm concentration and sperm progressive motility. However, the positive association of serum folate with DFI raises the need for careful prescription of folate supplements.

• 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.

• Journal of Nutrition and Health
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• v.40 no.2
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• pp.138-146
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• 2007

Food irradiation has been steadily increased in many countries concomitantly with increasing international trades. Harmful contaminants naturally occurred from foods which contain high levels of unsaturated fatty acids that are easily oxidized can affect the human anti-oxidation system through the generation of free radicals. Moreover, previous studies proved that ${\gamma}$-irradiation may cause production of free radicals in food. We investigated the effect of ${\gamma}$-irradiated soybeans in relation to oxidative stress in mice. Oxidative index of mice was evaluated by TBARS, DNA fragmentation in various organs such as blood lymphocytes, liver and kidney. Forty male ICR mice were equally divided into 4 groups and fed control diet or ${\gamma}$-irradiated diet containing 50% soybeans (5, 10, and 20 kGy, respectively) for 8 weeks. Peroxide values of the irradiated diets were higher than that of the non-irradiated one and increased according to the storage period. There was no significant difference in weight gain as well as in TBARS value in plasma and kidney of all groups. Liver TBARS value of the group fed with irradiated diet at 20 kGy increased significantly compared with the control group (p < 0.05). DNA oxidative damage as measured by alkaline comet assay showed that % tail DNA in the blood lymphocytes of 5 kGy and 10 kGy groups increased significantly over the control group (p < 0.05). Also, tail moments of 5 kGy and 10 kGy groups were higher than that of the control group. Ultrastructural examination shows myeline figures and swollen mitochondria in parietal and intestinal epithelial cells of the group fed with irradiated diet. Therefore, considering unsaturated fatty acid content, consumption of soybeans ${\gamma}$-irradiated with over 20 kGy or repeatedly may decrease the body's antioxidant mechanism.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.1
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• pp.10-17
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• 2011

Objective: To explore potential relationships between sperm DNA integrity and both semen parameters and clinical outcomes. Methods: Semen analysis of 498 samples was performed according to the 2010 criteria of the World Health Organization. The sperm DNA fragmentation Index (DFI) of the semen samples was assessed using a neutral comet assay. Results: Sperm DFI showed a significant correlation with semen parameters, including the patient's age, sperm viability, motility, morphology, and number of leukocytes (p<0.05). The sperm DFI values for asthenozoospermic (15.2%), oligoteratozoospermic (18.3%), asthenoteratozoospermic (17.5%), and oligoasthenoteratozoospermic semen samples (21.3%) were significantly higher than that observed in normozoospermic semen samples (10.5%, p<0.05). A sperm DFI value of 14% was used as a threshold of sperm DFI in assessing whether DNA was highly damaged. In 114 IVF-ET cycles, the fertilization rate of the sperm DFI <14% group (70 cycles, 61.7%) was significantly higher than that observed for the ${\geq}14%$ group (44 cycles, 55.3%), but there was no difference in the other clinical outcomes between the two groups. In the ${\geq}14%$ group, the pregnancy rates of the ICSI cycles (40.0%) and half-ICSI (44.0%) were higher than conventional IVF cycles (30.7%), but the difference was not statistically significant. Conclusion: Along with the conventional semen analysis, the sperm DFI assessed using the comet assay was shown to improve the quality of the semen evaluation. To evaluate the precise effect of ICSI on pregnancy rates in the patients who demonstrate high sperm DFI values, further study is necessary.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.3
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• pp.101-105
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• 2015

Objective: Several publications have established a relationship between sperm DNA damage and male factor infertility, based on data from America, Europe, and Asia. This study aimed to compare the extent of sperm DNA damage in sperm samples from Nigerian men with unexplained infertility and in sperm samples from a fertile group composed of sperm donors who had successfully impregnated a female partner naturally or through assisted conception. Methods: A total of 404 men underwent male fertility evaluation at Androcare Laboratories and Cryobank participated in this study. Semen analysis and a sperm chromatin structure assay (SCSA) were performed on all subjects. Results: The men in the unexplained infertility group were slightly older than the men in the fertile sperm group ($36{\pm}10$ years vs. $32{\pm}6$ years, p=0.051). No significant difference was observed between the two groups in semen analysis parameters ($p{\geq}0.05$). Men in the unexplained infertility group with normal semen parameters had a significantly higher DNA fragmentation index (DFI) than men in the fertile sperm group ($27.5%{\pm}7.0%$ vs. $14.1%{\pm}5.3%$, p<0.05). In the unexplained infertility group, 63% of the men had a DFI greater than 20%, compared to 4% in the fertile sperm group. In the unexplained infertility group, 15.2% of the subjects had a DFI greater than 30%, compared to 1% in the fertile sperm group. Conclusion: Our study showed that the SCSA may be a more reliable predictor of fertility potential than traditional semen analysis in cases of unexplained infertility.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.16
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• pp.6967-6972
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• 2015

Background: Cigarette smoking and tobacco chewing are common modes of consuming tobacco all over the world. Parents need to be aware that germ cell integrity is vital for birth of healthy offspring as biological parenting begins much before birth of a child and even before conception. The present study was conducted to determine the etiology of non-familial sporadic heritable retinoblastoma (NFSHRb), by evaluating oxidative sperm DNA damage in fathers due to use of tobacco (smoking and chewing). Materials and Methods: We recruited 145 fathers of NFSHRb children and 53 fathers of healthy children (controls) in the study. Tobacco history was obtained by personal interview. Seminal reactive oxygen species (ROS) in semen, sperm DNA fragmentation index (DFI) and 8 hydroxy 2' deoxyguanosine (8-OHdG) levels in sperm were evaluated. The RB1 gene was screened in genomic blood DNA of parents of children with NFSHRb and controls. Odds ratios (ORs) derived from conditional logistic regression models. Results: There was significant difference in the levels of ROS (p<0.05), DFI (p<0.05) and 8-OHdG (p<0.05) between tobacco users and non-users. The OR of NFSHRb for smokers was 7.29 (95%CI 2.9-34.5, p<0.01), for tobacco chewers 4.75 (2.07-10.9, p<0.05) and for both 9.11 (3.79-39.2; p<0.01). Conclusions: This study emphasizes the adverse effect of tobacco on the paternal genome and how accumulation of oxidative damage in sperm DNA may contribute to the etiology of NFSHRb. In an ongoing parallel study in our laboratory, 11 of fathers who smoked underwent. Meditation and yoga interventions, showed significant decline in levels of highly mutagenic oxidised DNA adducts after 6 months. Thus our lifestyle and social habits impact sperm DNA integrity and simple interventions like yoga and meditation are therapeutic for oxidative damage to sperm DNA.