Skip to main content
SpringerLink
Log in

In Vitro Studies of Antioxidants for Male Reproductive Health

  • Chapter
  • First Online:
Male Infertility

  • 1999 Accesses

Abstract

The detrimental effects of oxidants on spermatozoa were suggested close to 70 years ago with the demonstration that oxygen is sperm toxic. Later studies confirmed the susceptibility of spermatozoa to oxidative stress and the fact that human spermatozoa and semen leukocytes can generate reactive oxygen species (ROS). These observations have led to studies on the role of antioxidants in protecting spermatozoa from oxidative stress in vitro. The purpose of this chapter is to discuss the rationale for antioxidant therapy in male infertility and evaluate the data on the efficacy of in vitro antioxidant preparations on sperm function. A review of the literature demonstrates a beneficial effect of in vitro antioxidants in protecting spermatozoa from exogenous oxidants and cryopreservation (and subsequent thawing). However, the protective effect of in vitro antioxidants on sperm preparations subjected to endogenous ROS and gentle sperm processing has not been established.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Agarwal A, Saleh RA. Role of oxidants in male infertility: rationale, significance, and treatment. Urol Clin North Am. 2002;29:817–27.

    Article  PubMed  Google Scholar 

  2. Agarwal A, Sharma RK, Nallella KP, Thomas Jr AJ, Alvarez JG, Sikka SC. Reactive oxygen species as an independent marker of male factor infertility. Fertil Steril. 2006;86:878–85.

    Article  PubMed  CAS  Google Scholar 

  3. Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil Steril. 1992;57:409–16.

    PubMed  CAS  Google Scholar 

  4. Zini A, de Lamirande E, Gagnon C. Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase-and catalase-like activities in seminal plasma and spermatozoa. Int J Androl. 1993;16:183–8.

    Article  PubMed  CAS  Google Scholar 

  5. Aitken RJ, Irvine DS, Wu FC. Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol. 1991;164:542–51.

    PubMed  CAS  Google Scholar 

  6. Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients. Fertil Steril. 1997;68:519–24.

    Article  PubMed  CAS  Google Scholar 

  7. Shen HM, Chia SE, Ong CN. Evaluation of oxidative DNA damage in human sperm and its association with male infertility. J Androl. 1999;20:718–23.

    PubMed  CAS  Google Scholar 

  8. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod. 2000;15:1338–44.

    Article  PubMed  CAS  Google Scholar 

  9. Gomez E, Buckingham DW, Brindle J, Lanzafame F, Irvine DS, Aitken RJ. Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. J Androl. 1996;17:276–87.

    PubMed  CAS  Google Scholar 

  10. Muratori M, Piomboni P, Baldi E, Filimberti E, Pecchioli P, Moretti E, et al. Functional and ultrastructural features of DNA-fragmented human sperm. J Androl. 2000;21:903–12.

    PubMed  CAS  Google Scholar 

  11. de Lamirande E, Jiang H, Zini A, Kodama H, Gagnon C. Reactive oxygen species and sperm physiology. Rev Reprod. 1997;2:48–54.

    Article  PubMed  Google Scholar 

  12. Aitken RJ, West K, Buckingham D. Leukocytic infiltration into the human ejaculate and its association with semen quality, oxidative stress, and sperm function. J Androl. 1994;15:343–52.

    PubMed  CAS  Google Scholar 

  13. Aitken RJ, Paterson M, Fisher H, Buckingham DW, van Duin M. Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J Cell Sci. 1995;108(Pt 5):2017–25.

    PubMed  CAS  Google Scholar 

  14. de Lamirande E, Gagnon C. Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum Reprod. 1995;10 Suppl 1:15–21.

    PubMed  CAS  Google Scholar 

  15. Griveau JF, Le Lannou D. Reactive oxygen species and human spermatozoa: physiology and pathology. Int J Androl. 1997;20:61–9.

    Article  PubMed  CAS  Google Scholar 

  16. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod. 1998;59:1037–46.

    Article  PubMed  CAS  Google Scholar 

  17. Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl. 1987;8:338–48.

    PubMed  CAS  Google Scholar 

  18. de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility. J Androl. 1992;13:379–86.

    PubMed  Google Scholar 

  19. de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J Androl. 1992;13:368–78.

    PubMed  Google Scholar 

  20. Yang MH, Schaich KM. Factors affecting DNA damage caused by lipid hydroperoxides and aldehydes. Free Radic Biol Med. 1996;20:225–36.

    Article  PubMed  CAS  Google Scholar 

  21. Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil. 1987;81:459–69.

    Article  PubMed  CAS  Google Scholar 

  22. Lewis SE, Aitken RJ. DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res. 2005;322:33–41.

    Article  PubMed  CAS  Google Scholar 

  23. Jeulin C, Soufir JC, Weber P, Laval-Martin D, Calvayrac R. Catalase activity in human spermatozoa and seminal plasma. Gamete Res. 1989;24:185–96.

    Article  PubMed  CAS  Google Scholar 

  24. Gagnon C, Iwasaki A, De Lamirande E, Kovalski N. Reactive oxygen species and human spermatozoa. Ann N Y Acad Sci. 1991;637:436–44.

    Article  PubMed  CAS  Google Scholar 

  25. Jow WW, Schlegel PN, Cichon Z, Phillips D, Goldstein M, Bardin CW. Identification and localization of copper-zinc superoxide dismutase gene expression in rat testicular development. J Androl. 1993;14:439–47.

    PubMed  CAS  Google Scholar 

  26. Zini A, Schlegel PN. Expression of glutathione peroxidases in the adult male rat reproductive tract. Fertil Steril. 1997;68:689–95.

    Article  PubMed  CAS  Google Scholar 

  27. Zini A, Schlegel PN. Catalase mRNA expression in the male rat reproductive tract. J Androl. 1996;17:473–80.

    PubMed  CAS  Google Scholar 

  28. Zini A, Schlegel PN. Identification and characterization of antioxidant enzyme mRNAs in the rat epididymis. Int J Androl. 1997;20:86–91.

    Article  PubMed  CAS  Google Scholar 

  29. Holmes RP, Goodman HO, Shihabi ZK, Jarow JP. The taurine and hypotaurine content of human semen. J Androl. 1992;13:289–92.

    PubMed  CAS  Google Scholar 

  30. Lewis SE, Boyle PM, McKinney KA, Young IS, Thompson W. Total antioxidant capacity of seminal plasma is different in fertile and infertile men. Fertil Steril. 1995;64:868–70.

    PubMed  CAS  Google Scholar 

  31. Sanocka D, Miesel R, Jedrzejczak P, Kurpisz MK. Oxidative stress and male infertility. J Androl. 1996;17:449–54.

    PubMed  CAS  Google Scholar 

  32. Smith R, Vantman D, Ponce J, Escobar J, Lissi E. Total antioxidant capacity of human seminal plasma. Hum Reprod. 1996;11:1655–60.

    Article  PubMed  CAS  Google Scholar 

  33. Aitken RJ, Buckingham DW, Carreras A, Irvine DS. Superoxide dismutase in human sperm suspensions: relationship with cellular composition, oxidative stress, and sperm function. Free Radic Biol Med. 1996;21:495–504.

    Article  PubMed  CAS  Google Scholar 

  34. Appasamy M, Muttukrishna S, Pizzey AR, Ozturk O, Groome NP, Serhal P, et al. Relationship between male reproductive hormones, sperm DNA damage and markers of oxidative stress in infertility. Reprod Biomed Online. 2007;14:159–65.

    Article  PubMed  CAS  Google Scholar 

  35. Fraga CG, Motchnik PA, Shigenaga MK, Helbock HJ, Jacob RA, Ames BN. Ascorbic acid protects against endogenous oxidative DNA damage in human sperm. Proc Natl Acad Sci USA. 1991;88:11003–6.

    Article  PubMed  CAS  Google Scholar 

  36. Song GJ, Norkus EP, Lewis V. Relationship between seminal ascorbic acid and sperm DNA integrity in infertile men. Int J Androl. 2006;29:569–75.

    Article  PubMed  CAS  Google Scholar 

  37. Verit FF, Verit A, Kocyigit A, Ciftci H, Celik H, Koksal M. No increase in sperm DNA damage and seminal oxidative stress in patients with idiopathic infertility. Arch Gynecol Obstet. 2006;274:339–44.

    Article  PubMed  CAS  Google Scholar 

  38. Silver EW, Eskenazi B, Evenson DP, Block G, Young S, Wyrobek AJ. Effect of antioxidant intake on sperm chromatin stability in healthy nonsmoking men. J Androl. 2005;26:550–6.

    Article  PubMed  CAS  Google Scholar 

  39. Fraga CG, Motchnik PA, Wyrobek AJ, Rempel DM, Ames BN. Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat Res. 1996;351:199–203.

    Article  PubMed  Google Scholar 

  40. Hampl JS, Taylor CA, Johnston CS. Vitamin C deficiency and depletion in the United States: the Third National Health and Nutrition Examination Survey, 1988 to 1994. Am J Public Health. 2004;94:870–5.

    Article  PubMed  Google Scholar 

  41. Jacob RA. Assessment of human vitamin C status. J Nutr. 1990;120 Suppl 11:1480–5.

    PubMed  CAS  Google Scholar 

  42. Ryle PR, Thomson AD. Nutrition and vitamins in alcoholism. Contemp Issues Clin Biochem. 1984;1:188–224.

    PubMed  CAS  Google Scholar 

  43. Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl. 1988;9:367–76.

    PubMed  CAS  Google Scholar 

  44. Potts RJ, Notarianni LJ, Jefferies TM. Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation. Mutat Res. 2000;447:249–56.

    Article  PubMed  CAS  Google Scholar 

  45. Twigg J, Irvine DS, Houston P, Fulton N, Michael L, Aitken RJ. Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol Hum Reprod. 1998;4:439–45.

    Article  PubMed  CAS  Google Scholar 

  46. Aitken RJ. Founders’ Lecture. Human spermatozoa: fruits of creation, seeds of doubt. Reprod Fertil Dev. 2004;16:655–64.

    Article  PubMed  Google Scholar 

  47. Said TM, Agarwal A, Sharma RK, Thomas Jr AJ, Sikka SC. Impact of sperm morphology on DNA damage caused by oxidative stress induced by beta-nicotinamide adenine dinucleotide phosphate. Fertil Steril. 2005;83:95–103.

    Article  PubMed  CAS  Google Scholar 

  48. Cho C, Willis WD, Goulding EH, Jung-Ha H, Choi YC, Hecht NB, et al. Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nat Genet. 2001;28:82–6.

    PubMed  CAS  Google Scholar 

  49. De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, Hedges A, et al. DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2′-deoxyguanosine, a marker of oxidative stress. Biol Reprod. 2009;81:517–24.

    Article  PubMed  Google Scholar 

  50. Libman J, Gabriel MS, Sairam MR, Zini A. Catalase can protect spermatozoa of FSH receptor knock-out mice against oxidant-induced DNA damage in vitro. Int J Androl. 2010;33:818–22.

    Article  PubMed  CAS  Google Scholar 

  51. Aitken RJ, Buckingham DW, Brindle J, Gomez E, Baker HW, Irvine DS. Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma. Hum Reprod. 1995;10:2061–71.

    PubMed  CAS  Google Scholar 

  52. Aitken RJ, Buckingham D, Harkiss D. Use of a xanthine oxidase free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J Reprod Fertil. 1993;97:441–50.

    Article  PubMed  CAS  Google Scholar 

  53. Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod. 1998;13:896–900.

    Article  PubMed  CAS  Google Scholar 

  54. Russo A, Troncoso N, Sanchez F, Garbarino JA, Vanella A. Propolis protects human spermatozoa from DNA damage caused by benzo[a]pyrene and exogenous reactive oxygen species. Life Sci. 2006;78:1401–6.

    Article  PubMed  CAS  Google Scholar 

  55. Sierens J, Hartley JA, Campbell MJ, Leathem AJ, Woodside JV. In vitro isoflavone supplementation reduces hydrogen peroxide-induced DNA damage in sperm. Teratog Carcinog Mutagen. 2002;22:227–34.

    Article  PubMed  CAS  Google Scholar 

  56. Zini A, Sigman M. Are tests of sperm DNA damage clinically useful? Pros and cons. J Androl. 2009;30:219–29.

    Article  PubMed  CAS  Google Scholar 

  57. Aitken RJ, Fisher HM, Fulton N, Gomez E, Knox W, Lewis B, et al. Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine. Mol Reprod Dev. 1997;47:468–82.

    Article  PubMed  CAS  Google Scholar 

  58. Twigg J, Fulton N, Gomez E, Irvine DS, Aitken RJ. Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum Reprod. 1998;13:1429–36.

    Article  PubMed  CAS  Google Scholar 

  59. Anderson D, Schmid TE, Baumgartner A, Cemeli-Carratala E, Brinkworth MH, Wood JM. Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay). Mutat Res. 2003;544:173–8.

    Article  PubMed  CAS  Google Scholar 

  60. Cemeli E, Schmid TE, Anderson D. Modulation by flavonoids of DNA damage induced by estrogen-like compounds. Environ Mol Mutagen. 2004;44:420–6.

    Article  PubMed  CAS  Google Scholar 

  61. Dobrzynska MM, Baumgartner A, Anderson D. Antioxidants modulate thyroid hormone- and noradrenaline-induced DNA damage in human sperm. Mutagenesis. 2004;19:325–30.

    Article  PubMed  CAS  Google Scholar 

  62. Griveau JF, Le Lannou D. Effects of antioxidants on human sperm preparation techniques. Int J Androl. 1994;17:225–31.

    Article  PubMed  CAS  Google Scholar 

  63. Oeda T, Henkel R, Ohmori H, Schill WB. Scavenging effect of N-acetyl-l-cysteine against reactive oxygen species in human semen: a possible therapeutic modality for male factor infertility? Andrologia. 1997;29:125–31.

    Article  PubMed  CAS  Google Scholar 

  64. Verma A, Kanwar KC. Effect of vitamin E on human sperm motility and lipid peroxidation in vitro. Asian J Androl. 1999;1:151–4.

    PubMed  CAS  Google Scholar 

  65. Zheng RL, Zhang H. Effects of ferulic acid on fertile and asthenozoospermic infertile human sperm motility, viability, lipid peroxidation, and cyclic nucleotides. Free Radic Biol Med. 1997;22:581–6.

    Article  PubMed  CAS  Google Scholar 

  66. Calamera JC, Fernandez PJ, Buffone MG, Acosta AA, Doncel GF. Effects of long-term in vitro incubation of human spermatozoa: functional parameters and catalase effect. Andrologia. 2001;33:79–86.

    Article  PubMed  CAS  Google Scholar 

  67. Chi HJ, Kim JH, Ryu CS, Lee JY, Park JS, Chung DY, et al. Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Hum Reprod. 2008;23:1023–8.

    Article  PubMed  CAS  Google Scholar 

  68. Donnelly ET, McClure N, Lewis SE. Glutathione and hypotaurine in vitro: effects on human sperm motility, DNA integrity and production of reactive oxygen species. Mutagenesis. 2000;15:61–8.

    Article  PubMed  CAS  Google Scholar 

  69. Donnelly ET, McClure N, Lewis SE. The effect of ascorbate and alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa. Mutagenesis. 1999;14:505–12.

    Article  PubMed  CAS  Google Scholar 

  70. Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W. The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity. Hum Reprod. 1998;13:1240–7.

    Article  PubMed  CAS  Google Scholar 

  71. Esteves SC, Sharma RK, Thomas Jr AJ, Agarwal A. Cryopreservation of human spermatozoa with pentoxifylline improves the post-thaw agonist-induced acrosome reaction rate. Hum Reprod. 1998;13:3384–9.

    Article  PubMed  CAS  Google Scholar 

  72. Brennan AP, Holden CA. Pentoxifylline-supplemented cryoprotectant improves human sperm motility after cryopreservation. Hum Reprod. 1995;10:2308–12.

    PubMed  CAS  Google Scholar 

  73. Bell M, Wang R, Hellstrom WJ, Sikka SC. Effect of cryoprotective additives and cryopreservation protocol on sperm membrane lipid peroxidation and recovery of motile human sperm. J Androl. 1993;14:472–8.

    PubMed  CAS  Google Scholar 

  74. Wang R, Sikka SC, Veeraragavan K, Bell M, Hellstrom WJ. Platelet activating factor and pentoxifylline as human sperm cryoprotectants. Fertil Steril. 1993;60:711–5.

    PubMed  CAS  Google Scholar 

  75. Check DJ, Kiefer D, Katsoff D, Check JH. Effect of pentoxifylline added to freezing media on subsequent post-thaw hypoosmotic swelling test and other semen parameters. Arch Androl. 1995;35:161–3.

    Article  PubMed  CAS  Google Scholar 

  76. Park NC, Park HJ, Lee KM, Shin DG. Free radical scavenger effect of rebamipide in sperm processing and cryopreservation. Asian J Androl. 2003;5:195–201.

    PubMed  CAS  Google Scholar 

  77. Askari HA, Check JH, Peymer N, Bollendorf A. Effect of natural antioxidants tocopherol and ascorbic acids in maintenance of sperm activity during freeze-thaw process. Arch Androl. 1994;33:11–5.

    Article  PubMed  CAS  Google Scholar 

  78. Branco CS, Garcez ME, Pasqualotto FF, Erdtman B, Salvador M. Resveratrol and ascorbic acid prevent DNA damage induced by cryopreservation in human semen. Cryobiology. 2010;60:235–7.

    Article  PubMed  CAS  Google Scholar 

  79. Li Z, Lin Q, Liu R, Xiao W, Liu W. Protective effects of ascorbate and catalase on human spermatozoa during cryopreservation. J Androl. 2010;31:437–44.

    Article  PubMed  CAS  Google Scholar 

  80. Martinez-Soto JC, de Dioshourcade J, Gutierrez-Adan A, Landeras JL, Gadea J. Effect of genistein supplementation of thawing medium on characteristics of frozen human spermatozoa. Asian J Androl. 2010;12:431–41.

    Article  PubMed  CAS  Google Scholar 

  81. Thomson LK, Fleming SD, Aitken RJ, De Iuliis GN, Zieschang JA, Clark AM. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum Reprod. 2009;24:2061–70.

    Article  PubMed  CAS  Google Scholar 

  82. Taylor K, Roberts P, Sanders K, Burton P. Effect of antioxidant supplementation of cryopreservation medium on post-thaw integrity of human spermatozoa. Reprod Biomed Online. 2009;18:184–9.

    Article  PubMed  Google Scholar 

  83. Nallella KP, Sharma RK, Allamaneni SS, Aziz N, Agarwal A. Cryopreservation of human spermatozoa: comparison of two cryopreservation methods and three cryoprotectants. Fertil Steril. 2004;82:913–8.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Surgery, St. Mary’s Hospital, 3830 Lacombe Avenue, Room B270, Montreal, QC, Canada, H3T 1M5

    Armand Zini MD

  2. Division of Urology, Department of Surgery, Royal Victoria Hospital, McGill University, Montreal, QC, Canada, H3A 1A1

    Maria San Gabriel PhD

Authors
  1. Armand Zini MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria San Gabriel PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Armand Zini MD .

Editor information

Editors and Affiliations

  1. University of Florida, Director, Robotic Surgery, Winter Haven Hospital &, 200 Avenue F. N.E., Winter Haven, 33881, Florida, USA

    Sijo J. Parekattil

  2. Center for Reproductive Medicine, Department of Urology, Cleveland Clinic, Euclid Avenue 9500, Cleveland, 44195, Ohio, USA

    Ashok Agarwal

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this chapter

Cite this chapter

Zini, A., Gabriel, M.S. (2012). In Vitro Studies of Antioxidants for Male Reproductive Health. In: Parekattil, S., Agarwal, A. (eds) Male Infertility. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-3335-4_38

Download citation

  • DOI: https://doi.org/10.1007/978-1-4614-3335-4_38

  • Published:

  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4614-3334-7

  • Online ISBN: 978-1-4614-3335-4

  • eBook Packages: MedicineMedicine (R0)

Publish with us

Policies and ethics

Search

Navigation