The association between iron, calcium, and oxidative stress in seminal plasma and sperm quality

  • Oumaima Ammar
  • Zohra Houas
  • Meriem Mehdi
Research Article


The present study aimed to determine the level of iron and calcium in the seminal plasma of men with different fertility potentials and to examine its relationship with oxidative stress. Seventy-nine sub-fertile patients with asthenoteratozoospermia (AT), n 27; teratoleucozoospermia (TL), n 20; teratozoospermia (Terato), n 32; and 29 healthy donors were included. The ability of spermatozoa to produce reactive oxygen species (ROS) was evaluated by using nitroblue tetrazolium (NBT) staining. The lipid peroxidation end product, malondialdehyde (MDA), and the trace element levels (iron and calcium) were measured spectrophotometrically. Iron and calcium concentrations in seminal plasma of the patient groups were significantly more elevated than the normal group. Nevertheless, both calcium and iron showed strong negative correlations with the total sperm motility and normal sperm morphology, but only iron was positively and significantly associated with multiple anomalies index and seminal leucocyte concentration. On the other hand, the rates of MDA and ROS production in semen were significantly higher in the three abnormal groups than in controls. These two oxidative stress biomarkers were significantly associated with the percentage of atypical forms in semen. However, only semen ROS level was significantly associated with the decreased sperm motility and the sperm leucocytes concentration. Meanwhile, there are positive correlations between seminal iron and calcium content and the studied oxidative stress biomarkers. Oxidative stress and trace element excess are implicated in low sperm quality. Iron and calcium might be the mediators of the effects of oxidative damage and induces lipid peroxidation.


Male infertility Iron Calcium Oxidative stress Sperm 



The authors thank all couples, doctors, and researchers for their cooperation. We also thank our research team in the reproductive biology and human cytogenetic laboratory for their support.


This work was supported by funds allocated to the Research Unit of Histology and Genetic UR12ES10 by the Ministère Tunisien de l’Enseingement Supérieur et de la Recherché Scientifique.

Compliance with ethical standards

Informed consent

This protocol was approved by the local ethics committees le comité d’éthique médicale et de recherche de l’hopital universitaire Fattouma Bourguiba Monastir. All patients and controls had previously given informed consent for the study.

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Agarwal A, Sekhon LH (2010) The role of antioxidant therapy in the treatment of male infertility. Hum Fertil 13:217–225. CrossRefGoogle Scholar
  2. Agarwal A, Ikemoto I, Loughlin KR (1994) Relationship of sperm parameters with levels of reactive oxygen species in semen specimens. J Urol 152:107–110CrossRefGoogle Scholar
  3. Agarwal A, Tvrda E, Sharma R (2014) Relationship amongst teratozoospermia, seminal oxidative stress and male infertility. Reprod Biol Endocrinol 12:2–9. CrossRefGoogle Scholar
  4. Aitken RJ, West KM (1990) Analysis of the relationship between reactive oxygen species production and leucocyte infiltration in fractions of human semen separated on Percoll gradients. Int J Androl 13(6):433–451. CrossRefGoogle Scholar
  5. Aitken RJ, Buckingham D, West K, Wu FC, Zikopoulos K, Richardson DW (1992) Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors. J Reprod Fertil 94:451–462. CrossRefGoogle Scholar
  6. Aitken RJ, Harkiss D, Buckingham D (1993a) Relationship between iron-catalysed lipid peroxidation potential and human sperm function. J Reprod Fertil 98(1):257–265. CrossRefGoogle Scholar
  7. Aitken RJ, Harkiss D, Buckingham DW (1993b) Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev 35(3):302–315. CrossRefGoogle Scholar
  8. Ammar O, Tesnim A, Zohra H et al (2018a) Investigation on the origin of sperm morphological defects: oxidative attacks, chromatin immaturity, and DNA fragmentation. Environ Sci Pollut Res 25:1–12. Google Scholar
  9. Ammar O, Haouas Z, Hamouda B, Hamdi H, Hellara I, Jlali A, Cheikh HB, Mehdi M (2018b) Relationship between sperm DNA damage with sperm parameters, oxidative markers in teratozoospermic men. Eur J Obstet Gynecol Reprod Biol 233:70–75. CrossRefGoogle Scholar
  10. Arumugam K (1994) Endometriosis: endometriosis and infertility: raised iron concentration in the peritoneal fluid and its effect on the acrosome reaction. Hum Reprod 9(6):1153–1157. CrossRefGoogle Scholar
  11. Atig F, Raffa M, Ben AH et al (2011) Altered antioxidant status and increased lipid per-oxidation in seminal plasma of Tunisian infertile men. Int J Biol Sci 8:139–149. CrossRefGoogle Scholar
  12. Atig F, Kerkeni A, Saad A, Ajina M (2017) Effects of reduced seminal enzymatic antioxidants on sperm DNA fragmentation and semen quality of Tunisian infertile men. J Assist Reprod Genet 34:373–381. CrossRefGoogle Scholar
  13. Auger J, Eustash F (2000) Standardisation de la classification morphologique des spermatozoïdes humains selon la méthode de David modifiée. Andrologie 10:358–373CrossRefGoogle Scholar
  14. Aydemir B, Kiziler AR, Onaran I, Alici B, Ozkara H, Akyolcu MC (2006) Impact of Cu and Fe concentrations on oxidative damage in male infertility. Biol Trace Elem Res 112:193–204. CrossRefGoogle Scholar
  15. Ayinde OC, Ogunnowo S, Ogedegbe RA (2012) Influence of Vitamin C and Vitamin E on testicular zinc content and testicular toxicity in lead exposed albino rats. BMC Pharmacol Toxicol 14:13–17. Google Scholar
  16. Aziz N, Saleh RA, Sharma RK, Lewis-Jones I, Esfandiari N, Thomas AJ Jr, Agarwal A (2004) Novel association between sperm reactive oxygen species production, sperm morphological defects, and the sperm deformity index. Fertil Steril 81:349–354. CrossRefGoogle Scholar
  17. Barroso G, Morshedi M, Oehninger S (2000) Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 5(1):9–19. Google Scholar
  18. Benoff S, Jacob A, Hurley IR (2000) Male infertility and environmental exposure to lead and cadmium. Hum Reprod Update 6(2):107–121CrossRefGoogle Scholar
  19. Birben E, Murat U, Md S et al (2012) Oxidative stress and antioxidant defense. WAO 5(1):9–19. Google Scholar
  20. Chandra A, Goswami H, Sengupta P (2012a) Dietary calcium induced cytological and biochemical changes in thyroid. Environ Toxicol Pharmacol 34:454–465. CrossRefGoogle Scholar
  21. Chandra AK, Sengupta P, Goswami H, Sarkar M (2012b) Excessive dietary calcium in the disruption of structural and functional status of adult male reproductive system in rat with possible mechanism. Mol Cell Biochem 364:181–119. CrossRefGoogle Scholar
  22. Esfandiari N, Sharma RK, Saleh RA, Thomas AJ Jr, Agarwal A (2003) Utility of the nitroblue tetrazolium reduction test for assessment of reactive oxygen species production by seminal leukocytes and spermatozoa. J Androl 24(6):862–870. CrossRefGoogle Scholar
  23. Fraczek M, Szkutnik D, Sanocka D, Kurpisz M (2001) Peroxidation components of sperm lipid membranes in male infertility. 72(2):73–79Google Scholar
  24. Gil-Guzman E, Ollero M, Lopez M et al (2001) Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation. Hum Reprod 16:1922–1930. CrossRefGoogle Scholar
  25. Halliwell B (2007) Biochemistry of oxidative stress. Biochem Soc Trans 35(5):1147–1150. CrossRefGoogle Scholar
  26. Halliwell B, Cross CE (1994) Oxygen-derived species: their relation to human disease and environmental stress. Environ Health Perspect 102(10):5–12. Google Scholar
  27. Hong CY, Chiang BN, Turner P (1984) Calcium ion is the key regulator of human sperm function. Lancet. 2(8417–8418):1449–1451. CrossRefGoogle Scholar
  28. Huang YL, Tseng WC, Lin TH (2001) In vitro effects of metal ions (Fe2+, Mn2+, Pb2+) on sperm motility and lipid peroxidation in human semen. J Toxicol Environ Health 62:259–267. CrossRefGoogle Scholar
  29. Jana K, Samanta PK (2006) Evaluation of single intratesticular injection of calcium chloride for nonsurgical sterilization in adultalbino rats. Contraception 73:289–300. CrossRefGoogle Scholar
  30. Keshtgar S, Fanaei H, Bahmanpour S, Azad F, Ghannadi A, Kazeroni M (2012) In vitro effects of tocopherol on teratozoospermic semen samples. Andrologia 1:721–727. CrossRefGoogle Scholar
  31. Kovalski N, de Lamirande E, Gagnon C (1992) Reactive oxygen species generated by human neutrophils inhibit sperm motility, protective effect of seminal plasma and scavengers. Fertil Steril 58(4):809–816. CrossRefGoogle Scholar
  32. Machado RB, Bernardes CR, De Souza IM et al (2013) Is lipid profile determination necessary in women wishing to use oral contraceptives? Contraception 87:801–805. CrossRefGoogle Scholar
  33. Makker K, Agarwal A, Sharma R (2009) Oxidative stress & male infertility. Indian J Med Res 14(8):470–485 129:357 367Google Scholar
  34. Marzec-Wróblewska U, Kamiński P, Łakota P, Szymański M, Wasilow K, Ludwikowski G, Kuligowska-Prusińska M, Odrowąż-Sypniewska G, Stuczyński T, Michałkiewicz J (2011) Zinc and iron concentration and SOD activity in human semen and seminal plasma. Biol Trace Elem Res 143:167–177. CrossRefGoogle Scholar
  35. Massányi P, Trandzik J, Nad P et al (2004) Semen concentration of trace elements in stallions and relation to the spermatozoa quality. Trace Elem Elect 40(5):1097–1105. Google Scholar
  36. Morton BE, Sagadraca R, Fraser C (1978) Sperm motility within the mammalian epididymis: species variation and correlation with free calcium levels in epididymal plasma. Fertil Steril 29:695–698. CrossRefGoogle Scholar
  37. Muratori M, Tamburrino L, Marchiani S et al (2015) Investigation on the origin of sperm DNA fragmentation: role of apoptosis, immaturity and oxidative stress. Mol Med 21:109–122. CrossRefGoogle Scholar
  38. Nishida S, Yamano S, Aono T (1996) Exposure of human sperm to low calcium medium enhances fertilizing ability. Arch Androl 36:145–153CrossRefGoogle Scholar
  39. Ochsendorf FR, Thiele J, Fuchs J, Schüttau H, Freisleben HJ, Buslau M, Milbradt R (1994) Chemiluminescence in semen of infertile men. Andrologia 26(5):289–293. CrossRefGoogle Scholar
  40. Pasqualotto FF, Sharma RK, Nelson DR, Thomas AJ Jr, Agarwal A (2000a) Relationship between oxidative stress, semen characteristics, and clinical diagnosis in men undergoing infertility investigation. Fertil Steril 73(3):459–464. CrossRefGoogle Scholar
  41. Pasqualotto FF, Sharma RK, Potts JM, Nelson DR, Thomas AJ Jr, Agarwal A (2000b) Seminal oxidative stress in patients with chronic prostatitis. Urology. 55:881–885CrossRefGoogle Scholar
  42. Perera D, Pizzey A, Campbell A, Katz M, Porter J, Petrou M, Irvine DS, Chatterjee R (2002) Sperm DNA damage in potentially fertile homozygous beta-thalassaemia patients with iron overload. Hum Reprod 17:1820–1825. CrossRefGoogle Scholar
  43. Prien SD, Lox CD, Messer RH, DeLeon FD (1990) Seminal concentrations of total and ionized calcium from men with normal and decreased motility. Fertil Steril 54(1):171–172CrossRefGoogle Scholar
  44. Ragan HA, Mast TJ (1990) Cadmium inhalation and male reproductive toxicity. Rev Environ Contam Toxicol 114:1–22. Google Scholar
  45. Rao B, Soufir JC, Martin M, David G (1989) Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility. Gamete Res 24(2):127–134. CrossRefGoogle Scholar
  46. Rengan AK, Agarwal A, van der Linde M, du Plessis SS (2012) An investigation of excess residual cytoplasm in human spermatozoa and its distinction from the cytoplasmic droplet. Reprod Biol Endocrinol 10:92. CrossRefGoogle Scholar
  47. Schmid TE, Grant PG, Marchetti F, Weldon RH, Eskenazi B, Wyrobek AJ (2013) Elemental composition of human semen is associated with motility and genomic sperm defects among older men. Hum Reprod 28(1):274–282. CrossRefGoogle Scholar
  48. Slivkova J, Popelkova M, Massanyi P, Toporcerova S, Stawarz R, Formicki G, Lukac N, Putała A, Guzik M (2009) Concentration of trace elements in human semen and relation to spermatozoa quality. J Environ Sci Health A Tox Hazard Subst Environ Eng 15:441–448. Google Scholar
  49. Suleiman SA, Eamin Ali M, Zaki ZMS et al (1996) Lipid peroxidation and human sperm motility: protective role of vitamin. J Androl 17(5):530–537. Google Scholar
  50. Tunc O, Thompson J, Tremellen K (2010) Development of the NBT assay as a marker of sperm oxidative stress. Int J Androl 33(1):13–21. CrossRefGoogle Scholar
  51. Tvrda E, Peer R, Sikka SC, Agarwal A (2015) Iron and copper in male reproduction: a double-edged sword. J Assist Reprod Genet 32(1):3–16. CrossRefGoogle Scholar
  52. Venkatesh S, Riyaz AM, Shamsi MB, Kumar R, Gupta NP, Mittal S, Malhotra N, Sharma RK, Agarwal A, Dada R (2009) Clinical significance of reactive oxygen species in semen of infertile Indian men. Andrologia 41(4):251–256. CrossRefGoogle Scholar
  53. Vernet P, Aitken RJ, Drevet JR (2004) Antioxidant strategies in the epididymis. Mol Cell Endocrinol 216:31–39. CrossRefGoogle Scholar
  54. Wellejus A, Poulsen HE, Loft S (2000) Iron-induced oxidative DNA damage in rat sperm cells in vivo and in vitro. Free Radic Res 32(1):75–83. CrossRefGoogle Scholar
  55. Whittington K, Ford WCL (1998) The effect of incubation periods under 95% oxygen on the stimulated acrosome reaction and motility of human spermatozoa. Mol Hum Reprod 4(11):1053–1057. CrossRefGoogle Scholar
  56. Whittington K, Ford WC (1999) Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions. Int J Androl 22(4):229–235Google Scholar
  57. Wong WY, Flik G, Groenen PMW, Swinkels DW, Thomas CMG, Copius-Peereboom JHJ, Merkus HMWM, Steegers-Theunissen RPM (2001) The impact of calcium, magnesium, zinc, and copper in blood and seminal plasma on semen parameters in men. Reprod Toxicol 15(2):131–136. CrossRefGoogle Scholar
  58. World health organisation (2010) World Health Organization laboratory manual for the examination and processing of human semen. WHO PressGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Histology Embryology and Cytogenetic (UR 12 ES 10), Faculty of MedicineUniversity of MonastirMonastirTunisia
  2. 2.Laboratory of Cytogenetics and Reproductive Biology, Center of Maternity and Neonatology, MonastirFattouma Bourguiba University Teaching HospitalMonastirTunisia

Personalised recommendations