Evaluation of the sperm parameters, oxidative stress, and histopathological effects of vitamin B12 in preventing Helicobacter pylori-induced testicular toxicity: An experimental study


Background: Helicobacter pylori (H. pylori) causes sterility by affecting the reproductive system. vitamin B12 improves sperm quantity and function.

Objective: Vitamin B12 protection against H. pylori adverse effects was investigated.

Materials and Methods: 40 C57 male mice (6 wk) were randomly assigned to 4 equal groups (n = 10) including, group 1 (control without any intervention), group 2 (H), 3 (HP), and 4 (HB) received 1×109 colony forming unit (CFU) of H. pylori, 1×109 CFU of H. pylori+phosphate buffered saline, 1×109 CFU of H. pylori+50 μg/kg vitamin B12 intraperitoneally, respectively. In the induction groups, the H. pylori was orogasterically injected 3 times with 1 cc phosphate buffered saline throughout the day. Then testicular metrics, sperm motility, viability, quantity, and shape, plasma levels of malondialdehyde (MDA), superoxide dismutase, glutathione peroxidase, and total antioxidant capacity were measured. Also, testicular-tissue changes were examined using Johnson scores, tubular differentiation index, and spermatogenesis index. Vitamin B12, homocysteine, and testosterone serum levels were examined.

 Results: The results showed a significantly lower Johnson score, tubular differentiation index, and spermatogenesis index, and serum level of testosterone and homocysteine as well as a higher MDA level in the H and HP groups than the HB group (p < 0.05). In contrast, the highest superoxide dismutase and glutathione peroxidase enzymes activity and total antioxidant capacity as well as the lowest serum level of MDA were found in the HB group compared to other groups (p < 0.05).

Conclusion: Vitamin B12 increased antioxidant enzyme activity, enhanced sperm parameters, and decreased injury to testicular tissue. It can be used as a potent antioxidant in reducing testicular damage induced by H. pylori.

Key words: Helicobacter pylori, Reproductive medicine, Oxidative stress.


[1] Wang S, Zhang K, Yao Y, Li J, Deng S. Bacterial infections affect male fertility: A focus on the oxidative stress-autophagy axis. Front Cell Dev Biol 2021; 9: 727812.

[2] Oghbaei H, Rastgar Rezaei Y, Nikanfar S, Zarezadeh R, Sadegi M, Latifi Z, et al. Effects of bacteria on male fertility: Spermatogenesis and sperm function. Life Sci 2020; 256: 117891.

[3] Biggs SN, Kennedy J, Lewis SL, Hearps S, O’Bryan MK, McLachlan R, et al. Lifestyle and environmental risk factors for unexplained male infertility: Study protocol for Australian Male Infertility Exposure (AMIE), a case-control study. Reprod Health 2023; 20: 32.

[4] Moridi A, Roozbeh N, Yaghoobi H, Soltani S, Dashti S, Shahrahmani N, et al. Etiology and risk factors associated with infertility. Int J Women’s Health Reprod Sci 2019; 7: 353–346.

[5] Marchiani S, Baccani I, Tamburrino L, Mattiuz G, Nicolò S, Bonaiuto C, et al. Effects of common gram-negative pathogens causing male genitourinary-tract infections on human sperm functions. Sci Rep 2021; 11: 19177.

[6] Eini F, Azizi Kutenaei M, Zareei F, Sharifian Dastjerdi Z, Hosseinzadeh Shirzeyli M, Salehi E. Effect of bacterial infection on sperm quality and DNA fragmentation in subfertile men with Leukocytospermia. BMC Mol Cell Biol 2021; 22: 42.

[7] Moretti E, Figura N, Campagna MS, Gonnelli S, Iacoponi F, Collodel G. Sperm parameters and semen levels of inflammatory cytokines in Helicobacter pylori-infected men. Urology 2015; 86: 47–41.

[8] Kamarehei F, Taheri M, Mohammadi Y, Shokoohizadeh L, Alikhani MY. Prevalence of Helicobacter pylori virulence genes among Iranian infected patients: A systematic review and meta-analysis. Rev Res Med Microbiol 2020; 31: 200–191.

[9] Cho J, Prashar A, Jones NL, Moss SF. Helicobacter pylori infection. Gastroenterol Clin North Am 2021; 50: 261–282.

[10] Taylor NS, Fox JG. Animal models of Helicobacter-induced disease: Methods to successfully infect the mouse. Methods Mol Biol 2012; 921: 131–142.

[11] Shubina OS, Dudenkova NA. The effect of lead on the process of spermatogenesis in sex glands of male albino rats. Vet World 2016; 9: 1129–1134.

[12] Salehinezhad F, Eshraghi H, Kadivar A, Shirian S, Asghari A, Aali E, et al. Amelioration effects of vitamin E, melatonin, L-carnitine, and atorvastatin, on destructive effects of busulfan in the testes of male rats: A gene expression evaluation. Kafkas Univ Vet Fak Derg 2019; 25: 709–716.

[13] Gholami-Ahangaran M, Karimi-Dehkordi M, Akbari Javar A, Haj Salehi M, Ostadpoor M. A systematic review on the effect of Ginger (Zingiber officinale) on improvement of biological and fertility indices of sperm in laboratory animals, poultry and humans. Vet Med Sci 2021; 7: 1959–1969.

[14] Ghajari G, Nabiuni M, Amini E. The association between testicular toxicity induced by Li2Co3 and protective effect of Ganoderma lucidum: Alteration of Bax & c-Kit genes expression. Tissue Cell 2021; 72: 101552.

[15] Nabi A, Entezari F, Miresmaeili SM, Vahidi S, Lorian K, Anbari F, et al. Evaluation of sperm parameters and DNA integrity following different incubation times in PVP medium. Urol J 2022; 19: 232–237.

[16] Marchetti F, Aardema M, Beevers C, Yauk CL, Young R, Williams A. Simulation of mouse and rat spermatogenesis to inform genotoxicity testing using OECD test guideline 488. Mutat Res Genet Toxicol Environ Mutagen 2018; 832–833: 19–28.

[17] Demir M, Ozdem S. Evaluation of total antioxidant capacity in human seminal plasma. Int J Med Biochem 2022; 5: 54–59.

[18] Menevse E, Dursunoglu D, Cetin N, Korucu EN, Erbayram FZ. Evaluation of sialic acid, malondialdehyde and glutathione levels in infertile male. Rev Int Androl 2022; 20: 266–273.

[19] Ghajari Gh, Moosavi R. Evaluation of the effects of diazinon toxin on some reproductive parameters in male rats. Pers Med J 2022; 7: 30–35.

[20] Faria C, Zakout R, Araújo M. Helicobacter pylori and autoimmune diseases. Biomed Pharmacother 2013; 67: 347–349.

[21] Mohammadi N, Shirian S, Gorji A, Roshanpajouh M, Ahmadi E, Nazari H, et al. The potential protective effect of melatonin and N-acetylcysteine alone and in combination on opioid-induced testicular dysfunction and degeneration in rat. Reprod Toxicol 2023; 120: 108453.

[22] Zangoie R, Eshraghi R, Shirian S, Nazari H, Aali E. Melatonin synergistically enhances protective effect of atorvastatin against busulfan-induced spermatogenesis injuries in a rat model. Comparat Clin Pathol 2020; 29: 161–166.

[23] Elsaed WM, Bedeer RF, Eladl MA. Ameliorative effect of vitamin B12 on seminiferous epithelium of cimetidine-treated rats: A histopathological, immunohistochemical and ultrastructural study. Anat Cell Biol 2018; 51: 52–61.

[24] El-Kady HM. The plausibility of Helicobacter pylori and CagA strains related infertility among males in Alexandria, Egypt. J High Inst Public Health 2019; 49: .63–56

[25] Kohli A, Kumar R. Role of antioxidants of natural herbs in management of male infertility. J Res Appl Sci Biotechnol 2023; 2: 55–80.

[26] Adelakun SA, Ukwenya VO, Akintunde OW. Vitamin B12 ameliorate tramadol-induced oxidative stress, endocrine imbalance, apoptosis and NO/iNOS/NF-?B expression in Sprague Dawley rats through regulatory mechanism in the pituitary-gonadal axis. Tissue Cell 2022; 74: 101697.

[27] Banihani SA. Vitamin B12 and semen quality. Biomolecules 2017; 7: 42.

[28] Beltrame FL, Sasso-Cerri E. Vitamin B12-induced spermatogenesis recovery in cimetidine-treated rats: Effect on the spermatogonia number and sperm concentration. Asian J Androl 2017; 19: 567–572.

[29] Froese DS, Fowler B, Baumgartner MR. Vitamin B12, folate, and the methionine remethylation cycle-biochemistry, pathways, and regulation. J Inherit Metab Dis 2019; 42: 673–685.

[30] Rehman T, Shabbir MA, Inam-Ur-Raheem M, Manzoor MF, Ahmad N, Liu ZW, et al. Cysteine and homocysteine as biomarker of various diseases. Food Sci Nutr 2020; 8: .4707–4696

[31] Asadi N, Bahmani M, Kheradmand A, Rafieian-Kopaei M. The impact of oxidative stress on testicular function and the role of antioxidants in improving it: A review. J Clin Diagn Res 2017; 11: IE01-IE05.

[32] Ritchie C, Ko EY. Oxidative stress in the pathophysiology of male infertility. Andrologia 2021; 53: e13581.

[33] Asghari A, Akbari G, Meghdadi A, Mortazavi P. Protective effect of metformin on testicular ischemia/ reperfusion injury in rats. Acta Cir Bras 2016; 31: 411–416.