Skip to main content
Springer Nature Link
Log in

Composition of protein supplements used for human embryo culture

  • Embryo Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

To determine the composition of commercially available protein supplements for embryo culture media and test if differences in protein supplement composition are biologically relevant in a murine model.

Methods

Amino acid, organic acid, ion and metal content were determined for 6 protein supplements: recombinant human albumin (AlbIX), human serum albumin (HSA and Buminate), and three complex protein supplements (SSS, SPS, LGPS). To determine if differences in the composition of these supplements are biologically relevant, mouse one-cell embryos were collected and cultured for 120 hours in each protein supplement in Global media at 5 and 20 % oxygen in an EmbryoScope time-lapse incubator. The compositions of six protein supplements were analyzed for concentrations of 39 individual amino acids, organic acids, ions and elements. Blastocyst development and cell cycle timings were calculated at 96-hours of culture and the experiments were repeated in triplicate. Blastocyst gene expression was analyzed.

Results

Recombinant albumin had the fewest undefined components , the lowest concentration of elements detected, and resulted in high blastocyst development in both 5 and 20 % oxygen. Buminate, LGPS and SPS had high levels of transition metals whereas SSS had high concentrations of amino acids. Pre-compaction mouse embryo development was delayed relative to embryos in AlbIX for all supplements and blastocyst formation was reduced in Buminate, SPS and SSS.

Conclusions

The composition of protein supplements are variable, consisting of previously undescribed components. High concentrations of pro-oxidant transition metals were most notable. Blastocyst development was protein dependent and showed an interaction with oxygen concentration and pro-oxidant supplements.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

References

  1. Kane MT. Variability in different lots of commercial bovine serum albumin affects cell multiplication and hatching of rabbit blastocysts in culture. J Reprod Fertil. 1983;69:555–8.

    Article  CAS  PubMed  Google Scholar 

  2. McKiernan SH, Bavister BD. Different lots of bovine serum albumin inhibit or stimulate in vitro development of hamster embryos. In Vitro Cell Dev Biol. 1992;28A:154–6.

    Article  CAS  PubMed  Google Scholar 

  3. Meintjes M. Media composition: macromolecules and embryo growth. Methods Mol Biol. 2012;912:107–27.

    CAS  PubMed  Google Scholar 

  4. Leonard PH, Charlesworth MC, Benson L, Walker DL, Fredrickson JR, Morbeck DE. Variability in protein quality used for embryo culture: embryotoxicity of the stabilizer octanoic acid. Fertil Steril. 2013;100:544–9.

    Article  CAS  PubMed  Google Scholar 

  5. Yu MW, Finlayson JS. Quantitative determination of the stabilizers octanoic acid and N-acetyl-DL-tryptophan in human albumin products. J Pharm Sci. 1984;73:82–6.

    Article  CAS  PubMed  Google Scholar 

  6. Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update. 1995;1:91–148.

    Article  CAS  PubMed  Google Scholar 

  7. Blake D, Svalander P, Jin M, Silversand C, Hamberger L. Protein supplementation of human IVF culture media. J Assist Reprod Genet. 2002;19:137–43.

    Article  PubMed Central  PubMed  Google Scholar 

  8. Batt PA, Gardner DK, Cameron AW. Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro. Reprod Fertil Dev. 1991;3:601–7.

    Article  CAS  PubMed  Google Scholar 

  9. Caro CM, Trounson A. The effect of protein on preimplantation mouse embryo development in vitro. J In Vitro Fert Embryo Transf. 1984;1:183–7.

    Article  CAS  PubMed  Google Scholar 

  10. Leveille MC, Carnegie J, Tanphaichitr N. Effects of human sera and human serum albumin on mouse embryo culture. J Assist Reprod Genet. 1992;9:45–52.

    Article  CAS  PubMed  Google Scholar 

  11. Menezo Y, Testart J, Perrone D. Serum is not necessary in human in vitro fertilization, early embryo culture, and transfer. Fertil Steril. 1984;42:750–5.

    CAS  PubMed  Google Scholar 

  12. Adler A, Reing AM, Bedford JM, Alikani M, Cohen J. Plasmanate as a medium supplement for in vitro fertilization. J Assist Reprod Genet. 1993;10:67–71.

    Article  CAS  PubMed  Google Scholar 

  13. Weathersbee PS, Pool TB, Ord T. Synthetic serum substitute (SSS): a globulin-enriched protein supplement for human embryo culture. J Assist Reprod Genet. 1995;12:354–60.

    Article  CAS  PubMed  Google Scholar 

  14. Meintjes M, Chantilis SJ, Ward DC, Douglas JD, Rodriguez AJ, Guerami AR, et al. A randomized controlled study of human serum albumin and serum substitute supplement as protein supplements for IVF culture and the effect on live birth rates. Hum Reprod. 2009;24:782–9.

    Article  CAS  PubMed  Google Scholar 

  15. Bungum M, Humaidan P, Bungum L. Recombinant human albumin as protein source in culture media used for IVF: a prospective randomized study. Reprod Biomed Online. 2002;4:233–6.

    Article  CAS  PubMed  Google Scholar 

  16. Otsuki J, Nagai Y, Matsuyama Y, Terada T, Era S. The redox state of recombinant human serum albumin and its optimal concentration for mouse embryo culture. Syst Biol Reprod Med. 2013;59:48–52.

    Article  CAS  PubMed  Google Scholar 

  17. Paczkowski M, Yuan Y, Fleming-Waddell J, Bidwell CA, Spurlock D, Krisher RL. Alterations in the transcriptome of porcine oocytes derived from prepubertal and cyclic females is associated with developmental potential. J Anim Sci. 2011;89:3561–71.

    Article  CAS  PubMed  Google Scholar 

  18. Paczkowski M, Silva E, Schoolcraft WB, Krisher RL. Comparative importance of fatty acid beta-oxidation to nuclear maturation, gene expression, and glucose metabolism in mouse, bovine, and porcine cumulus oocyte complexes. Biol Reprod. 2013;88:111.

    Article  PubMed  Google Scholar 

  19. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365–86.

    CAS  PubMed  Google Scholar 

  20. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002;30:e36.

    Article  PubMed Central  PubMed  Google Scholar 

  21. Yuan Y, Ida JM, Paczkowski M, Krisher RL. Identification of developmental competence-related genes in mature porcine oocytes. Mol Reprod Dev. 2011;78:565–75.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Silva E, Paczkowski M, Krisher RL. The effect of leptin on maturing porcine oocytes is dependent on glucose concentration. Mol Reprod Dev. 2012;79:296–307.

    Article  CAS  PubMed  Google Scholar 

  23. Pool TB, Martin JE. High continuing pregnancy rates after in vitro fertilization-embryo transfer using medium supplemented with a plasma protein fraction containing alpha- and beta-globulins. Fertil Steril. 1994;61:714–9.

    CAS  PubMed  Google Scholar 

  24. Kaspar H, Dettmer K, Gronwald W, Oefner PJ. Automated GC-MS analysis of free amino acids in biological fluids. J Chromatogr B Analyt Technol Biomed Life Sci. 2008;870:222–32.

    Article  CAS  PubMed  Google Scholar 

  25. Morbeck DE, Krisher RL, Herrick JR, Baumann NA, Matern D, Moyer T. Composition of commercial media used for human embryo culture. Fertil Steril. 2014;102:759–66 e9.

    Article  CAS  PubMed  Google Scholar 

  26. Wolff HS, Fredrickson JR, Walker DL, Morbeck DE. Advances in quality control: mouse embryo morphokinetics are sensitive markers of in vitro stress. Hum Reprod. 2013;28:1776–82.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Meuter A, Rogmann LM, Winterhoff BJ, Tchkonia T, Kirkland JL, Morbeck DE. Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen. J Assist Reprod Genet. 2014 epub.

  28. Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigo R, et al. Characterization of mammalian selenoproteomes. Science. 2003;300:1439–43.

    Article  CAS  PubMed  Google Scholar 

  29. Halliwell B. The wanderings of a free radical. Free Radic Biol Med. 2009;46:531–42.

    Article  CAS  PubMed  Google Scholar 

  30. Halliwell B. Cell culture, oxidative stress, and antioxidants: avoiding pitfalls. Biomed J. 2014;37:99–105.

    PubMed  Google Scholar 

  31. Fraga CG. Relevance, essentiality and toxicity of trace elements in human health. Mol Aspects Med. 2005;26:235–44.

    Article  CAS  PubMed  Google Scholar 

  32. Bal W, Kasprzak KS. Induction of oxidative DNA damage by carcinogenic metals. Toxicol Lett. 2002;127:55–62.

    Article  CAS  PubMed  Google Scholar 

  33. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem. 2005;12:1161–208.

    Article  CAS  PubMed  Google Scholar 

  34. Quinlan GJ, Coudray C, Hubbard A, Gutteridge JM. Vanadium and copper in clinical solutions of albumin and their potential to damage protein structure. J Pharm Sci. 1992;81:611–4.

    Article  CAS  PubMed  Google Scholar 

  35. Balla J, Vercellotti GM, Jeney V, Yachie A, Varga Z, Jacob HS, et al. Heme, heme oxygenase, and ferritin: how the vascular endothelium survives (and dies) in an iron-rich environment. Antioxid Redox Signal. 2007;9:2119–37.

    Article  CAS  PubMed  Google Scholar 

  36. Breuer W, Shvartsman M, Cabantchik ZI. Intracellular labile iron. Int J Biochem Cell Biol. 2008;40:350–4.

    Article  CAS  PubMed  Google Scholar 

  37. Halliwell B, Whiteman M. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol. 2004;142:231–55.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Halliwell B. Oxidative stress in cell culture: an under-appreciated problem? FEBS Lett. 2003;540:3–6.

    Article  CAS  PubMed  Google Scholar 

  39. Combelles CM, Hennet ML. Media composition: antioxidants/chelators and cellular function. Methods Mol Biol. 2012;912:129–59.

    CAS  PubMed  Google Scholar 

  40. Roche M, Rondeau P, Singh NR, Tarnus E, Bourdon E. The antioxidant properties of serum albumin. FEBS Lett. 2008;582:1783–7.

    Article  CAS  PubMed  Google Scholar 

  41. Rosenbluth EM, Shelton DN, Wells LM, Sparks AE, Van Voorhis BJ. Human embryos secrete microRNAs into culture media-a potential biomarker for implantation. Fertil Steril. 2014;101:1493–500.

    Article  CAS  PubMed  Google Scholar 

  42. Menezo Y, Khatchadourian C. Peptides bound to albumin. Life Sci. 1986;39:1751–3.

    Article  CAS  PubMed  Google Scholar 

  43. Akopian V, Andrews PW, Beil S, Benvenisty N, Brehm J, Christie M, et al. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells. In Vitro Cell Dev Biol Anim. 2010;46:247–58.

    Article  PubMed Central  PubMed  Google Scholar 

  44. Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8:424–9.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Garcia-Gonzalo FR, Izpisua Belmonte JC. Albumin-associated lipids regulate human embryonic stem cell self-renewal. PLoS One. 2008;3:e1384.

    Article  PubMed Central  PubMed  Google Scholar 

  46. Skottman H, Hovatta O. Culture conditions for human embryonic stem cells. Reproduction. 2006;132:691–8.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Mayo Clinic, Charlton 3A, Rochester, MN, 55905, USA

    Dean E. Morbeck & Heather S. Hoff

  2. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA

    Dean E. Morbeck, Jolene R. Fredrickson, Nikola A. Baumann, Thomas Moyer & Dietrich Matern

  3. National Foundation for Fertility Research, Lone Tree, CO, 80124, USA

    Melissa Paczkowski & Rebecca L. Krisher

Authors
  1. Dean E. Morbeck
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Melissa Paczkowski
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Jolene R. Fredrickson
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Rebecca L. Krisher
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Heather S. Hoff
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Nikola A. Baumann
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Thomas Moyer
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Dietrich Matern
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence to Dean E. Morbeck.

Additional information

Capsule

Protein supplements for IVF and embryo culture are highly heterogeneous and poorly defined. Some components of supplements adversely affect mouse embryo development and these effects are dependent on oxygen concentration used for culture.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 38 kb)

ESM 2

(DOC 39 kb)

ESM 3

(DOC 34 kb)

ESM 4

(DOC 34 kb)

ESM 5

(DOC 33 kb)

ESM 6

(DOC 35 kb)

ESM 7

(DOC 35 kb)

Supplemental Figure 1

Specific cell division timings (hours; mean±se) from the 2-cell stage to 3-, 4-, 5- and 8 cells for embryos cultured with different protein supplements. Time to the 3-cell stage relative to AlbIX was longer for SPS (p<0.01) and Buminate and SSS (p<0.001). Time to 8-cell was delayed relative to AlbIX for Buminate, SSS, SPS and LGPS (p<0.01). (PPT 201 kb)

Supplemental Figure 2

Gene expression analyses, relative to 18s rRNA, for hatching mouse blastocysts cultured in 5 mg/mL AlbIX,10% Buminate, 5 mg/mL HSA, 10% LGPS, 10% SPS, or 10% SSS at 5% O2 compared to 20% O2. Positive fold changes indicate an increase in transcript abundance in embryos cultured at 20% O2 as compared to 5% O2, whereas a negative fold change indicates a decrease in transcript abundance in embryos cultured at 20% O2 as compared to 5% O2. *, P < 0.05; ‡, P < 0.1. (DOC 190 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Morbeck, D.E., Paczkowski, M., Fredrickson, J.R. et al. Composition of protein supplements used for human embryo culture. J Assist Reprod Genet 31, 1703–1711 (2014). https://doi.org/10.1007/s10815-014-0349-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-014-0349-2

Keywords