Journal of Assisted Reproduction and Genetics

, Volume 29, Issue 4, pp 305–311 | Cite as

Release of superoxide dismutase-1 by day 3 embryos of varying quality and implantation potential

  • Catherine M. H. Combelles
  • Emily A. Holick
  • Catherine Racowsky



To determine if the antioxidant superoxide dismutase-1 (SOD1 or Cu,Zn-SOD) is released by cultured human cleavage-stage embryos and to assess any link between SOD1 and implantation potential.


Women (n = 91; ≤40 years old) undergoing IVF treatment with transfer of one or two 8-cell embryos that resulted in 0 or 100% implantation were included. Following individual embryo culture, spent medium samples (n = 122) were collected and levels of SOD1 protein were measured by an enzyme-linked immunosorbent assay. SOD1 detection and concentration in embryo spent medium were analyzed in relation to embryo fragmentation and symmetry scores, and implantation (viable fetus at >12 weeks).


Cleavage-stage embryos release SOD1 protein into the spent culture medium. Neither detection nor concentration of SOD1 was related to implantation. There was a positive relationship between increased embryo fragmentation scores and SOD1 release, with no apparent association with symmetry. In non-pregnant cycles, the release of SOD1 decreased with increasing maternal age.


While SOD1 does not predict implantation potential of select good-quality embryos, our data support the need to evaluate the biological significance of released SOD1 by embryos of varying quality and from patients of varying age.


Embryo culture medium Secreted protein Superoxide dismutase-1 Cu,Zn-superoxide dismutase Pregnancy outcome 


  1. 1.
    Cummins JM, Breen TM, Harrison KL, Shaw JM, Wilson LM, Hennessey JF. A formula for scoring human embryo growth rates in in vitro fertilization: its value in predicting pregnancy and in comparison with visual estimates of embryo quality. J In Vitro Fert Embryo Transf. 1986;3:284–95.CrossRefPubMedGoogle Scholar
  2. 2.
    Steer CV, Mills CL, Tan SL, Campbell S, Edwards RG. The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme. Hum Reprod. 1992;7:117–9.PubMedGoogle Scholar
  3. 3.
    Giorgetti C, Terriou P, Auquier P, Hans E, Spach JL, Salzmann J, Roulier R. Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers. Hum Reprod. 1995;10:2427–31.PubMedGoogle Scholar
  4. 4.
    Holte J, Berglund L, Milton K, Garello C, Gennarelli G, Revelli A, Bergh T. Construction of an evidence-based integrated morphology cleavage embryo score for implantation potential of embryos scored and transferred on day 2 after oocyte retrieval. Hum Reprod. 2007;22:548–57.CrossRefPubMedGoogle Scholar
  5. 5.
    Racowsky C, Ohno-Machado L, Kim J, Biggers JD. Is there an advantage in scoring early embryos on more than one day? Hum Reprod. 2009;24:2104–13.CrossRefPubMedGoogle Scholar
  6. 6.
    Guerif F, Le Gouge A, Giraudeau B, Poindron J, Bidault R, Gasnier O, Royere D. Limited value of morphological assessment at days 1 and 2 to predict blastocyst development potential: a prospective study based on 4042 embryos. Hum Reprod. 2007;22:1973–81.CrossRefPubMedGoogle Scholar
  7. 7.
    Katz-Jaffe MG, McReynolds S, Gardner DK, Schoolcraft WB. The role of proteomics in defining the human embryonic secretome. Mol Hum Reprod. 2009;15:271–7.CrossRefPubMedGoogle Scholar
  8. 8.
    Seli E, Robert C, Sirard MA. OMICS in assisted reproduction: possibilities and pitfalls. Mol Hum Reprod. 2010;16:513–30.CrossRefPubMedGoogle Scholar
  9. 9.
    Warner CM, Lampton PW, Newmark JA, Cohen J. Symposium: innovative techniques in human embryo viability assessment. Soluble human leukocyte antigen-G and pregnancy success. Reprod Biomed Online. 2008;17:470–85.CrossRefPubMedGoogle Scholar
  10. 10.
    Nieder GL, Weitlauf HM, Suda-Hartman M. Synthesis and secretion of stage-specific proteins by peri-implantation mouse embryos. Biol Reprod. 1987;36:687–99.CrossRefPubMedGoogle Scholar
  11. 11.
    Fridovich I. Oxygen toxicity: a radical explanation. J Exp Biol. 1998;201:1203–9.PubMedGoogle Scholar
  12. 12.
    Mondola P, Annella T, Santillo M, Santangelo F. Evidence for secretion of cytosolic CuZn superoxide dismutase by Hep G2 cells and human fibroblasts. Int J Biochem Cell Biol. 1996;28:677–81.CrossRefPubMedGoogle Scholar
  13. 13.
    Mondola P, Annella T, Seru R, Santangelo F, Iossa S, Gioielli A, Santillo M. Secretion and increase of intracellular CuZn superoxide dismutase content in human neuroblastoma SK-N-BE cells subjected to oxidative stress. Brain Res Bull. 1998;45:517–20.CrossRefPubMedGoogle Scholar
  14. 14.
    Cimini V, Ruggiero G, Buonomo T, Seru R, Sciorio S, Zanzi C, Santangelo F, Mondola P. CuZn-superoxide dismutase in human thymus: immunocytochemical localisation and secretion in thymus-derived epithelial and fibroblast cell lines. Histochem Cell Biol. 2002;118:163–9.PubMedGoogle Scholar
  15. 15.
    Scott R, Seli E, Miller K, Sakkas D, Scott K, Burns DH. Noninvasive metabolomic profiling of human embryo culture media using Raman spectroscopy predicts embryonic reproductive potential: a prospective blinded pilot study. Fertil Steril. 2008;90:77–83.CrossRefPubMedGoogle Scholar
  16. 16.
    Seli E, Sakkas D, Scott R, Kwok SC, Rosendahl SM, Burns DH. Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril. 2007;88:1350–7.CrossRefPubMedGoogle Scholar
  17. 17.
    Paszkowski T, Clarke RN. Antioxidative capacity of preimplantation embryo culture medium declines following the incubation of poor quality embryos. Hum Reprod. 1996;11:2493–5.PubMedGoogle Scholar
  18. 18.
    Bedaiwy M, Agarwal A, Said TM, Goldberg JM, Sharma RK, Worley S, Falcone T. Role of total antioxidant capacity in the differential growth of human embryos in vitro. Fertil Steril. 2006;86:304–9.CrossRefPubMedGoogle Scholar
  19. 19.
    Bedaiwy MA, Falcone T, Mohamed MS, Aleem AA, Sharma RK, Worley SE, Thornton J, Agarwal A. Differential growth of human embryos in vitro: role of reactive oxygen species. Fertil Steril. 2004;82:593–600.CrossRefPubMedGoogle Scholar
  20. 20.
    Bedaiwy MA, Mahfouz RZ, Goldberg JM, Sharma R, Falcone T. Abdel Hafez MF, Agarwal A: Relationship of reactive oxygen species levels in day 3 culture media to the outcome of in vitro fertilization/intracytoplasmic sperm injection cycles. Fertil Steril. 2010;94:2037–42.CrossRefPubMedGoogle Scholar
  21. 21.
    Wiener-Megnazi Z, Shiloh H, Avraham L, Lahav-Baratz S, Koifman M, Reznick AZ, Auslender R, Dirnfeld M. Oxidative parameters of embryo culture media may predict treatment outcome in in vitro fertilization: a novel applicable tool for improving embryo selection. Fertil Steril. 2011;95:979–84.CrossRefPubMedGoogle Scholar
  22. 22.
    Reichman DE, Politch J, Ginsburg ES, Racowsky C. Extended in vitro maturation of immature oocytes from stimulated cycles: an analysis of fertilization potential, embryo development, and reproductive outcomes. J Assist Reprod Genet. 2010;27:347–56.CrossRefPubMedGoogle Scholar
  23. 23.
    Racowsky C, Combelles CM, Nureddin A, Pan Y, Finn A, Miles L, Gale S, O'Leary T, Jackson KV. Day 3 and day 5 morphological predictors of embryo viability. Reprod Biomed Online. 2003;6:323–31.CrossRefPubMedGoogle Scholar
  24. 24.
    Harvey MB, Arcellana-Panlilio MY, Zhang X, Schultz GA, Watson AJ. Expression of genes encoding antioxidant enzymes in preimplantation mouse and cow embryos and primary bovine oviduct cultures employed for embryo coculture. Biol Reprod. 1995;53:532–40.CrossRefPubMedGoogle Scholar
  25. 25.
    Wrenzycki C, De Sousa P, Overstrom EW, Duby RT, Herrmann D, Watson AJ, Niemann H, O'Callaghan D, Boland MP. Effects of superovulated heifer diet type and quantity on relative mRNA abundances and pyruvate metabolism in recovered embryos. J Reprod Fertil. 2000;118:69–78.CrossRefPubMedGoogle Scholar
  26. 26.
    Lequarre AS, Feugang JM, Malhomme O, Donnay I, Massip A, Dessy F, Van Langendonckt A. Expression of Cu/Zn and Mn superoxide dismutases during bovine embryo development: influence of in vitro culture. Mol Reprod Dev. 2001;58:45–53.CrossRefPubMedGoogle Scholar
  27. 27.
    Santillo M, Secondo A, Seru R, Damiano S, Garbi C, Taverna E, Rosa P, Giovedi S, Benfenati F, Mondola P. Evidence of calcium- and SNARE-dependent release of CuZn superoxide dismutase from rat pituitary GH3 cells and synaptosomes in response to depolarization. J Neurochem. 2007;102:679–85.CrossRefPubMedGoogle Scholar
  28. 28.
    Turner BJ, Atkin JD, Farg MA, Zang DW, Rembach A, Lopes EC, Patch JD, Hill AF, Cheema SS. Impaired extracellular secretion of mutant superoxide dismutase 1 associates with neurotoxicity in familial amyotrophic lateral sclerosis. J Neurosci. 2005;25:108–17.CrossRefPubMedGoogle Scholar
  29. 29.
    Gomes C, Keller S, Altevogt P, Costa J. Evidence for secretion of Cu, Zn superoxide dismutase via exosomes from a cell model of amyotrophic lateral sclerosis. Neurosci Lett. 2007;428:43–6.CrossRefPubMedGoogle Scholar
  30. 30.
    Mondola P, Ruggiero G, Seru R, Damiano S, Grimaldi S, Garbi C, Monda M, Greco D, Santillo M. The Cu, Zn superoxide dismutase in neuroblastoma SK-N-BE cells is exported by a microvesicles dependent pathway. Brain Res Mol Brain Res. 2003;110:45–51.CrossRefPubMedGoogle Scholar
  31. 31.
    Goto Y, Noda Y, Mori T, Nakano M. Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radic Biol Med. 1993;15:69–75.CrossRefPubMedGoogle Scholar
  32. 32.
    Johnson MH, Nasr-Esfahani MH. Radical solutions and cultural problems: could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro? Bioessays. 1994;16:31–8.CrossRefPubMedGoogle Scholar
  33. 33.
    Orsi NM, Leese HJ. Protection against reactive oxygen species during mouse preimplantation embryo development: role of EDTA, oxygen tension, catalase, superoxide dismutase and pyruvate. Mol Reprod Dev. 2001;59:44–53.CrossRefPubMedGoogle Scholar
  34. 34.
    Betts DH, Madan P. Permanent embryo arrest: molecular and cellular concepts. Mol Hum Reprod. 2008;14:445–53.CrossRefPubMedGoogle Scholar
  35. 35.
    Bain NT, Madan P, Betts DH. The early embryo response to intracellular reactive oxygen species is developmentally regulated. Reprod Fertil Dev. 2011;23:561–75.CrossRefPubMedGoogle Scholar
  36. 36.
    Yang HW, Hwang KJ, Kwon HC, Kim HS, Choi KW, Oh KS. Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos. Hum Reprod. 1998;13:998–1002.CrossRefPubMedGoogle Scholar
  37. 37.
    Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med. 2010;48:749–62.CrossRefPubMedGoogle Scholar
  38. 38.
    Jurisicova A, Varmuza S, Casper RF. Programmed cell death and human embryo fragmentation. Mol Hum Reprod. 1996;2:93–8.CrossRefPubMedGoogle Scholar
  39. 39.
    Hardy K. Apoptosis in the human embryo. Rev Reprod. 1999;4:125–34.CrossRefPubMedGoogle Scholar
  40. 40.
    Kimura N, Tsunoda S, Iuchi Y, Abe H, Totsukawa K, Fujii J. Intrinsic oxidative stress causes either 2-cell arrest or cell death depending on developmental stage of the embryos from SOD1-deficient mice. Mol Hum Reprod. 2010;16:441–51.CrossRefPubMedGoogle Scholar
  41. 41.
    Tarin JJ, Gomez-Piquer V, Pertusa JF, Hermenegildo C, Cano A. Association of female aging with decreased parthenogenetic activation, raised MPF, and MAPKs activities and reduced levels of glutathione S-transferases activity and thiols in mouse oocytes. Mol Reprod Dev. 2004;69:402–10.CrossRefPubMedGoogle Scholar
  42. 42.
    Matos L, Stevenson D, Gomes F, Silva-Carvalho JL, Almeida H. Superoxide dismutase expression in human cumulus oophorus cells. Mol Hum Reprod. 2009;15:411–9.CrossRefPubMedGoogle Scholar
  43. 43.
    Tatone C, Carbone MC, Falone S, Aimola P, Giardinelli A, Caserta D, Marci R, Pandolfi A, Ragnelli AM, Amicarelli F. Age-dependent changes in the expression of superoxide dismutases and catalase are associated with ultrastructural modifications in human granulosa cells. Mol Hum Reprod. 2006;12:655–60.CrossRefPubMedGoogle Scholar
  44. 44.
    Carbone MC, Tatone C. Delle Monache S, Marci R, Caserta D, Colonna R, Amicarelli F: Antioxidant enzymatic defences in human follicular fluid: characterization and age-dependent changes. Mol Hum Reprod. 2003;9:639–43.CrossRefPubMedGoogle Scholar
  45. 45.
    Tarin JJ. Potential effects of age-associated oxidative stress on mammalian oocytes/embryos. Mol Hum Reprod. 1996;2:717–24.CrossRefPubMedGoogle Scholar
  46. 46.
    Tatone C, Amicarelli F, Carbone MC, Monteleone P, Caserta D, Marci R, Artini PG, Piomboni P, Focarelli R. Cellular and molecular aspects of ovarian follicle ageing. Hum Reprod Update. 2008;14:131–42.CrossRefPubMedGoogle Scholar
  47. 47.
    Li J, Foote RH, Simkin M. Development of rabbit zygotes cultured in protein-free medium with catalase, taurine, or superoxide dismutase. Biol Reprod. 1993;49:33–7.CrossRefPubMedGoogle Scholar
  48. 48.
    Nonogaki T, Noda Y, Narimoto K, Umaoka Y, Mori T. Protection from oxidative stress by thioredoxin and superoxide dismutase of mouse embryos fertilized in vitro. Hum Reprod. 1991;6:1305–10.PubMedGoogle Scholar
  49. 49.
    Nonogaki T, Noda Y, Narimoto K, Umaoka Y, Mori T. Effects of superoxide dismutase on mouse in vitro fertilization and embryo culture system. J Assist Reprod Genet. 1992;9:274–80.CrossRefPubMedGoogle Scholar
  50. 50.
    Ali AA, Bilodeau JF, Sirard MA. Antioxidant requirements for bovine oocytes varies during in vitro maturation, fertilization and development. Theriogenology. 2003;59:939–49.CrossRefPubMedGoogle Scholar
  51. 51.
    Liu Z, Foote RH. Development of bovine embryos in KSOM with added superoxide dismutase and taurine and with five and twenty percent O2. Biol Reprod. 1995;53:786–90.CrossRefPubMedGoogle Scholar
  52. 52.
    Luvoni GC, Keskintepe L, Brackett BG. Improvement in bovine embryo production in vitro by glutathione-containing culture media. Mol Reprod Dev. 1996;43:437–43.CrossRefPubMedGoogle Scholar
  53. 53.
    Thomas M, Jain S, Kumar GP, Laloraya M. A programmed oxyradical burst causes hatching of mouse blastocysts. J Cell Sci. 1997;110(Pt 14):1597–602.PubMedGoogle Scholar
  54. 54.
    Nasr-Esfahani MH, Aitken JR, Johnson MH. Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development. 1990;109:501–7.PubMedGoogle Scholar
  55. 55.
    Dalvit GC, Cetica PD, Pintos LN, Beconi MT. Reactive oxygen species in bovine embryo in vitro production. Biocell. 2005;29:209–12.PubMedGoogle Scholar
  56. 56.
    Lapointe J, Bilodeau JF. Antioxidant defenses are modulated in the cow oviduct during the estrous cycle. Biol Reprod. 2003;68:1157–64.CrossRefPubMedGoogle Scholar
  57. 57.
    El Mouatassim S, Guerin P, Menezo Y. Mammalian oviduct and protection against free oxygen radicals: expression of genes encoding antioxidant enzymes in human and mouse. Eur J Obstet Gynecol Reprod Biol. 2000;89:1–6.CrossRefPubMedGoogle Scholar
  58. 58.
    Guerin P, Menezo Y. Review: role of tubal environment in preimplantation embryogenesis: application to co-culture assays. Zygote. 2011;19:47–54.CrossRefPubMedGoogle Scholar
  59. 59.
    Leyens G, Knoops B, Donnay I. Expression of peroxiredoxins in bovine oocytes and embryos produced in vitro. Mol Reprod Dev. 2004;69:243–51.CrossRefPubMedGoogle Scholar
  60. 60.
    Harvey AJ, Kind KL, Thompson JG. REDOX regulation of early embryo development. Reproduction. 2002;123:479–86.CrossRefPubMedGoogle Scholar
  61. 61.
    Dumollard R, Carroll J, Duchen MR, Campbell K, Swann K. Mitochondrial function and redox state in mammalian embryos. Semin Cell Dev Biol. 2009;20:346–53.CrossRefPubMedGoogle Scholar
  62. 62.
    Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature. 1988;332:459–61.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Catherine M. H. Combelles
    • 1
  • Emily A. Holick
    • 1
  • Catherine Racowsky
    • 2
  1. 1.Biology DepartmentMiddlebury CollegeMiddleburyUSA
  2. 2.Department of Obstetrics and Gynecology, Brigham and Women’s HospitalHarvard Medical SchoolBostonUSA

Personalised recommendations