• 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.

• Clinical and Experimental Reproductive Medicine
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• v.51 no.2
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• pp.135-141
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• 2024

Objective: Recently, oral antioxidants in combined forms have been used to treat men with idiopathic infertility. This study aimed to evaluate the effects of treatment with vitamin C, vitamin E, selenium, zinc, arginine, L-carnitine, and coenzyme Q10 on sperm quality parameters, DNA integrity, reproductive hormones, and pregnancy rates in men with infertility and idiopathic oligoasthenoteratozoospermia (OAT). Methods: A prospective study was conducted on 420 men with infertility and idiopathic OAT who took an oral supplement of antioxidant SP-Power tablets twice daily for 6 months. Semen quality, reproductive hormones, and the DNA fragmentation index (DFI) were evaluated at baseline and at 3 and 6 months after supplementation, using the World Health Organization 2021 guidelines. Results: No significant difference was observed in volume or the percentage of typical morphology during treatment. A significant improvement in sperm concentration was observed after supplementation (8.67±1.41, 12.17±1.91, and 19.01±0.86 at baseline, 3, and 6 months respectively, p<0.01). The total motility, progressive motility, and total motile sperm count also increased significantly (p<0.01), whereas the DFI decreased after 6 months. There was an increase in normal FSH levels and testosterone levels after 6 months of supplementation of antioxidant SP-Power but these differences were not statistically significant (p=not significant and p=0.06, respectively). Conclusion: Supplementation with SP-Power tablets improved sperm quality parameters, sperm DFI, some reproductive hormones, and pregnancy rates in men with infertility and idiopathic OAT, which could be attributed to the supplement's synergistic antioxidant action. Further studies are needed to determine the effects of supplementation on oxidative stress markers.

• Biomedical Science Letters
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• v.7 no.4
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• pp.181-190
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• 2001

This study was designed to investigate the effects of reactive oxygen species (ROS) on DNA stability in human spermatozoa. To verify human spermatozoa were incubated with xanthine-xanthine oxidase (X 100$\mu$M-XO 50 mlU ~ 400 mIU), $H_2O_2$ (125 $\mu$M ~ 1 mM), sodium nitroprusside (SNP 0.1 $\mu$M ~ 100 $\mu$M) or lymphocyte. Otherwise, spermatozoa were incubated under low $O_2$ (5%) condition. Damage of sperm DNA was analyzed by single cell electrophoresis (Comet assay) and flow cytometry after acridine orange staining. In the presence of ROS, there was increase in DNA damage. The rate of DNA single strand breakage (9.0$\pm$1.0% ~ 46.0$\pm$4.6%) and DNA fragmentation (7.51$\pm$1.0% ~ 29.5$\pm$4.6%) were similar regardless of the kinds of ROS and exposure time. DNA damage in the lower $O_2$ condition (5%) was lower than ambient $O_2$ condition (20%). Taken together, it suggested that sperm DNA might be damaged by ROS. In the presence of ROS, increase in DNA damage and chromatin instability was obvious in spite of short exposure. Although present study reconfirmed that sperm incubation in the low concentration of ROS have the benefit m the induction of capacitation and Ah, the increase in DNA damage by ROS and possible genetic problem should be considered before the human trials.

• Journal of Veterinary Clinics
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• v.30 no.6
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• pp.442-448
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• 2013

The aim of the present study was to develop glycerol-free TRIS extender using glucose for dog sperm cryopreservation. We determined the appropriate concentration of glucose in glycerol-free TRIS and the exposure time in glycerol-free TRIS containing 0.3 M glucose at $4^{\circ}C$. Ejaculates of six dog sperm were cooled in glycerol-free TRIS through $4^{\circ}C$ for 100 min, cooled at $4^{\circ}C$ in TRIS with different glucose concentrations 0 M, 0.04 M, 0.1 M, 0.2 M and 0.3 M, respectively for 30 min followed by cryopreservation. After thawing at $37^{\circ}C$ for 25 sec, membrane and acrosome integrities of dog sperm were evaluated. In addition, the effect of exposure time (10, 30, 50 and 70 min) of sperm to glycerol-free TRIS containing 0.3 M glucose at $4^{\circ}C$ on progressive motility, viability, and DNA integrity following sperm cryopreservation was studied. Membrane integrity and acrosome integrity were assessed by 6-carboxyfluoresceindiacetate (6-CFDA)/propidium iodide (PI) fluorescent staining and Pisum sativum agglutinin conjugated to fluorescein isothiocyanate, respectively. DNA integrity was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling, using flow cytometry. Sperm frozen in glycerol-free TRIS supplemented with 0.2 M or 0.3 M glucose have an intact plasma membrane (CFDA+/PI-) after cryopreservation than sperm frozen in the extenders with lower glucose concentrations (p<0.05). Acrosome integrity was significantly higher in the 0.3 M group than less than 0.1 M groups (p<0.05). The sperm DNA fragmentation index did not differ according to exposure time, although progressive motility was significantly higher in the 50 min exposure group than the other groups (p<0.05). These results indicate that cryopreservation of dog sperm is feasible and yields more motile sperm following freezing and thawing in glycerol-free TRIS containing 0.3 M glucose with the exposure time for 50 min at $4^{\circ}C$.

• Clinical and Experimental Reproductive Medicine
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• v.51 no.3
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• pp.213-224
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• 2024

Objective: Some age-related testicular changes, such as Sertoli cell vacuolization and blood-testis barrier breakdown, reduce total sperm production and male fertility. Therefore, this study investigated the effect of vitamin E on restoring testicular function in aged mice. Sperm cryo-resistance was also assessed. Methods: Twenty-eight 48-week-old male Naval Medical Research Institute mice were divided into four groups for a daily gavage of vitamin E: the control group received distilled water, while the three treatment groups were administered 100, 200, and 400 mg/kg, respectively, for 4 weeks. Subsequently, semen analyses, DNA fragmentation index (DFI), and protamine deficiency tests were conducted. Testicular histology, tissue antioxidant enzyme activity, and gene expression levels were also assessed. Results: The two higher dosages of vitamin E were associated with a higher sperm count, greater progressive motility, and improved sperm morphology (p<0.05). These benefits were also evident after sperm freezing (p<0.05). Although chromatin abnormalities increased following vitrification, the treatment groups showed better outcomes (p<0.05). The tubular diameter, epithelium height, and luminal diameters remained unchanged with age. The tissue antioxidant capacity was greater in the groups receiving the high doses of vitamin E. Additionally, significant increases in inhibitor of DNA binding protein-4 (Id4) and GDNF family receptor alpha-1 (Gfra1) expression were observed in the higher vitamin E dosage groups, and promyelocytic leukemia zinc finger protein (Plzf) expression was notably present in the 400 mg/kg treatment group compared to the control group (p<0.05). Conclusion: Antioxidant supplementation might enhance reproductive outcomes in aging males. The observed effects included improved sperm cryo-resistance, which is advantageous for future applications such as sperm freezing or fertility preservation.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.1
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• pp.49-56
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• 2022

Objective: Oxidative stress and sperm DNA fragmentation (SDF) have been linked to idiopathic male infertility (IMI). Various antioxidants have been tried to improve semen parameters and fertility potential in IMI patients, but with inconsistent results. The study aimed to compare the effects of coenzyme Q10 (CoQ10) and Centrum multivitamins on semen parameters, seminal antioxidant capacity, and SDF in infertile men with idiopathic oligoasthenospermia (OA). Methods: This prospective controlled clinical study involved 130 patients with idiopathic OA and 58 fertile controls. The patients were divided randomly into two groups: the first group received CoQ10 (200 mg/day orally) and the second group received Centrum multivitamins (1 tablet/day) for 3 months. Semen parameters, CoQ10 levels, reactive oxygen species (ROS), total antioxidant capacity (TAC), catalase, SDF, and serum hormone levels (follicle-stimulating hormone, luteinizing hormone, testosterone, and prolactin) were compared at baseline and after 3 months. Results: Both CoQ10 and Centrum improved sperm concentration and motility, but the improvement was greater with Centrum therapy (p<0.05). Similarly, both therapies improved antioxidant capacity, but TAC and catalase improvement was greater (p<0.01 and p<0.001 respectively) with CoQ10, whereas ROS (p<0.01) and SDF (p<0.001) improvements were greater with Centrum administration. Centrum therapy was associated with reduced serum testosterone (p<0.05). Conclusion: In conclusion, both CoQ10 and Centrum were effective in improving semen parameters, antioxidant capacity, and SDF, but the improvement was greater with Centrum than with CoQ10. Therefore, Centrum-as a source of combined antioxidants-may provide more effective results than individual antioxidants such as CoQ10 in the treatment of infertile men with idiopathic OA.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.197-204
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• 2019

This study was investigated to test whether the zygote recognized the topoisomerase II beta (TOP2B) mediated DNA fragmentation in epididymal spermatozoa or the nuclease degradation in vas deferens spermatozoa by testing for the presence of gammaH2AX (γH2AX). The γH2AX is phosphorylation of histone protein H2AX on serine 139 occurs at sites flanking DNA double-stranded breaks (DSBs). The presence of γH2AX in the pronuclei of mouse zygotes which were injected with DNA broke epididymal spermatozoa was tested by immunohistochemistry at 5 and 9 h post fertilization, respectively. Paternal pronuclei that arose from epididymal spermatozoa treated with divalent cations did not stain for γH2AX at 5 h. On the other hand, in embryos injected with vas deferences spermatozoa that had been treated with divalent cations, γH2AX was only present in paternal pronuclei, and not the maternal pronuclei at 5 h. Interestingly, both pronuclei stained positively for γH2AX for all treatments and controls at 9 h after sperm injection. In conclusion, the embryos recognize DNA that is damaged by nuclease, but not by TOP2B because H2AX in phosphorylated in paternal pronuclei resulting from spermatozoa treated with fragmented DNA from vas deferens spermatozoa treated with divalent cations, but not from epididymal spermatozoa treated the same way.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.9
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• pp.4517-4525
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• 2016

Background: Sperm DNA damage is underlying aetiology of poor implantation and pregnancy rates but also affects health of offspring and may also result in denovo mutations in germ line and post fertilization. This may result in complex diseases, polygenic disorders and childhood cancers. Childhood cancer like retinoblastoma (RB) is more prevalent in developing countries and the incidence of RB has increased more than three fold in India in the last decade. Recent studies have documented increased incidence of cancers in children born to fathers who consume alcohol in excess and tobacco or who were conceived by assisted conception. The aetiology of childhood cancer and increased disease burden in these children is lin ked to oxidative stress (OS) and oxidative DNA damage( ODD) in sperm of their fathers. Though several antioxidants are in use to combat oxidative stress, the effect of majority of these formulations on DNA is not known. Yoga and meditation cause significant decline in OS and ODD and aid in regulating OS levels such that reactive oxygen speues meditated signal transduction, gene expression and several other physiological functions are not disrupted. Thus, this study aimed to analyze sperm ODD as a possible etiological factor in childhood cancer and role of simple life style interventions like yoga and meditation in significantly decreasing seminal oxidative stress and oxidative DNA damage and thereby decreasing incidence of childhood cancers. Materials and Methods: A total of 131 fathers of children with RB (non-familial sporadic heritable) and 50 controls (fathers of healthy children) were recruited at a tertiary center in India. Sperm parameters as per WHO 2010 guidelines and reactive oxygen species (ROS), DNA fragmentation index (DFI), 8-hydroxy-2'-deoxy guanosine (8-OHdG) and telomere length were estimated at day 0, and after 3 and 6 months of intervention. We also examined the compliance with yoga and meditation practice and smoking status at each follow-up. Results: The seminal mean ROS levels (p<0.05), sperm DFI (p<0.001), 8-OHdG (p<0.01) levels were significantly higher in fathers of children with RB, as compared to controls and the relative mean telomere length in the sperm was shorter. Levels of ROS were significantly reduced in tobacco users (p<0.05) as well as in alcoholics (p<0.05) after intervention. DFI reduced significantly (p<0.05) after 6 months of yoga and meditation practice in all groups. The levels of oxidative DNA damage marker 8-OHdG were reduced significantly after 3 months (p<0.05) and 6 months (p<0.05) of practice. Conclusions: Our results suggest that OS and ODD DNA may contribute to the development of childhood cancer. This may be due to accumulation of oxidized mutagenic base 8OHdG, and elevated MDA levels which results in MDA dimers which are also mutagenic, aberrant methylation pattern, altered gene expression which affect cell proliferation and survival through activation of transcription factors. Increased mt DNA mutations and aberrant repair of mt and nuclear DNA due to highly truncatred DNA repair mechanisms all contribute to sperm genome hypermutability and persistant oxidative DNA damage. Oxidative stress is also associated with genome wide hypomethylation, telomere shortening and mitochondrial dysfunction leading to genome hypermutability and instability. To the best of our knowledge, this is the first study to report decline in OS and ODD and improvement in sperm DNA integrity following adoption of meditation and yoga based life style modification.This may reduce disease burden in next generation and reduce incidence of childhood cancers.

• Animal Bioscience
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• v.34 no.2
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• pp.198-204
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• 2021

Objective: The aim of this study was to ascertain the effects of adding green tea extract (GTE) to skim milk-egg yolk (SM-EY) extender on both the quality of post-thawed bull semen and the pregnancy rates of the recipient cows. Methods: Twelve ejaculates from four Simmental bulls, aged 3 to 5 years and weighing 900 to 950 kg, were diluted SM-EY extender, added with 0, 0.05, 0.1, and 0.15 mg GTE/100 mL extender and then frozen. After four weeks storage in liquid nitrogen, the sperm were thawed and evaluated for viability, motility, intact plasma membrane (IPM), and DNA fragmentation. Meanwhile, the estrus cycles of 48 recipient cows were synchronized by intramuscular administration of a single injection of 5 mg prostaglandin F2α. Estrus cows were divided into four equal groups and inseminated artificially 18 to 20 h after the onset of estrus by using semen from each extender group. Pregnancy was diagnosed by measuring serum progesterone levels at 21 days, followed by transrectal palpation 90 days after insemination. Results: The findings revealed that adding 0.1 mg of GTE/100 mL extender produced the highest percentages of sperm viability (70.67%±1.75%), motility (69.17%±1.47%), and IPM (69.23%±1.21%) and the lowest percentage of DNA fragmentation (3.00%±0.50%). The pregnancy diagnosis revealed that all cows (36/36) inseminated using frozen semen in GTE addition extender were pregnant (pregnancy rate 100%), whereas the pregnancy rate of the control group was 83.33% (10/12). Conclusion: It may be concluded that 0.1 mg GTE/100 mL extender yields the best quality of spermatozoa and that all variants doses of GTE in extender produce a higher pregnancy rate among recipient cows.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.247-252
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• 2019

We observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. According to the results from each freezing extender, the sperm membrane integrity (HOST: Hypoosmotic Swelling Test) analysis in TCGGD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Glycerol 3%, Dimethylsulpoxide 3.5 M) is 59.8 ± 0.7, TCGSD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Sucrose 0.1 M, Dimethylsulpoxide 3.5 M) is 59.3 ± 0.5 were significantly higher (p < 0.05) among the experimental groups. And MMPs analysis result, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of active MMP-2 was the highest in sperms frozen in TCGSD and TCGD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Dimethylsulpoxide 3.5 M), Meanwhile, sperms from the TCGGD and TCGED (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Ethylene glycol 3%, Dimethylsulpoxide 3.5 M) group showed lower level of active MMP-2 expression. Together, these results indicate that adding glycerol or sucrose to the sperm freezing buffer would not only suppress MMPs expression but also minimize DNA fragmentation, providing a mean to improve the success rate in the in vitro manipulation of rabbit sperms. Therefore, these results suggest that TCGGD or TCGSD extender method for freezing-thawing of rabbit sperm increased the viability after thawing.