Journal of Assisted Reproduction and Genetics

, Volume 33, Issue 4, pp 473–480 | Cite as

Effect of cryopreservation on the properties of human endometrial stromal cells used in embryo co-culture systems

  • Ivan BochevEmail author
  • Kalina Belemezova
  • Atanas Shterev
  • Stanimir Kyurkchiev
Embryo Biology



Along with comparative investigation of the decidualization potential and IL-6 secretion by fresh and frozen ESCs, we also aimed to evaluate the effectiveness of co-culture systems based on fresh or frozen ESCs in terms of clinical pregnancy rates.


Outcome analysis of a total of 215 IVF cycles with co-culture with fresh or frozen ESCs was performed. Endometrial tissue was obtained from 17 healthy donors. Concentrations of secreted prolactin, IGFBP-1, and IL-6 in conditioned media from cultured fresh and frozen ESCs (decidualized or not) were measured using ELISA or ECLIA.


Embryo co-culture with frozen ESCs resulted in a much lower pregnancy rate compared to the alternative system using fresh ESCs. Furthermore, cultivated frozen ESCs showed considerably decreased release of prolactin, IGFBP-1, and IL-6 compared to fresh ESCs, indicating that cryopreservation negatively affects their decidualization potential and cytokine production.


Altogether, this data illustrates the need for optimization and improvement of the existing autologous endometrial co-culture systems.


Embryo co-culture Endometrial stromal cells Cryopreservation Decidualization Cytokine production Pregnancy rate 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Menezo YJ, Servy E, Veiga A, Hazout A, Elder K. Culture systems: embryo co-culture. Methods Mol Biol. 2012;912:231–47.PubMedGoogle Scholar
  2. 2.
    Menezo YJ, Sakkas D, Janny L. Co-culture of the early human embryo: factors affecting human blastocyst formation in vitro. Microsc Res Tech. 1995;32(1):50–6.CrossRefPubMedGoogle Scholar
  3. 3.
    Spandorfer SD, Navarro J, Levy D, Black AR, Liu HC, Veeck L, et al. Autologous endometrial coculture in patients with in vitro-fertilization (IVF) failure: correlations of outcome with leukemia inhibiting factor (LIF) production. Am J Reprod Immunol. 2001;46(6):375–80.CrossRefPubMedGoogle Scholar
  4. 4.
    Spandorfer SD, Barmat LI, Liu HC, Mele C, Veeck L, Rosenwaks Z. Granulocyte macrophage-colony stimulating factor production by autologous endometrial co-culture is associated with outcome for in vitro fertilization patients with a history of multiple implantation failures. Am J Reprod Immunol. 1998;40(5):377–81.CrossRefPubMedGoogle Scholar
  5. 5.
    Spandorfer SD, Neuer A, Liu HC, Bivis L, Clarke R, Veeck L, et al. Interleukin-1 levels in the supernatant of conditioned media of embryos grown in autologous endometrial coculture: correlation with outcome after in vitro fertilization. Am J Reprod Immunol. 2000;43(1):6–11.CrossRefPubMedGoogle Scholar
  6. 6.
    Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update. 1995;1(2):91–148.CrossRefPubMedGoogle Scholar
  7. 7.
    Feng HL, Wen XH, Presser SC. Effect of different co-culture systems in early human embryo development. Hum Reprod. 1996;11(7):1525–8.CrossRefPubMedGoogle Scholar
  8. 8.
    Dirnfeld M, Goldman S, Gonen Y, Koifman M, Calderon I, Abramovici H. A simplified coculture system with luteinized granulosa cells improves embryo quality and implantation rates: a controlled study. Fertil Steril. 1997;67(1):120–2.CrossRefPubMedGoogle Scholar
  9. 9.
    Bongso A, Soon-Chye N, Sathananthan H, Lian NP, Rauff M, Ratnam S. Improved quality of human embryos when co-cultured with human ampullary cells. Hum Reprod. 1989;4(6):706–13.PubMedGoogle Scholar
  10. 10.
    Wiemer KE, Cohen J, Wiker SR, Malter HE, Wright G, Godke RA. Coculture of human zygotes on fetal bovine uterine fibroblasts: embryonic morphology and implantation. Fertil Steril. 1989;52(3):503–8.PubMedGoogle Scholar
  11. 11.
    Mercader A, Garcia-Velasco JA, Escudero E, Remohi J, Pellicer A, Simon C. Clinical experience and perinatal outcome of blastocyst transfer after coculture of human embryos with human endometrial epithelial cells: a 5-year follow-up study. Fertil Steril. 2003;80(5):1162–8.CrossRefPubMedGoogle Scholar
  12. 12.
    Barmat LI, Liu HC, Spandorfer SD, Xu K, Veeck L, Damario MA, et al. Human preembryo development on autologous endometrial coculture versus conventional medium. Fertil Steril. 1998;70(6):1109–13.CrossRefPubMedGoogle Scholar
  13. 13.
    Xu J, Cheung TM, Chan ST, Ho PC, Yeung WS. Human oviductal cells reduce the incidence of apoptosis in cocultured mouse embryos. Fertil Steril. 2000;74(6):1215–9.CrossRefPubMedGoogle Scholar
  14. 14.
    Eyheremendy V, Raffo FG, Papayannis M, Barnes J, Granados C, Blaquier J. Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure. Fertil Steril. 2010;93(3):769–73.CrossRefPubMedGoogle Scholar
  15. 15.
    Menezo YJ, Guerin JF, Czyba JC. Improvement of human early embryo development in vitro by coculture on monolayers of Vero cells. Biol Reprod. 1990;42(2):301–6.CrossRefPubMedGoogle Scholar
  16. 16.
    Menezo Y, Hazout A, Dumont M, Herbaut N, Nicollet B. Coculture of embryos on Vero cells and transfer of blastocysts in humans. Hum Reprod. 1992;7 Suppl 1:101–6.CrossRefPubMedGoogle Scholar
  17. 17.
    Schillaci R, Ciriminna R, Cefalu E. Vero cell effect on in-vitro human blastocyst development: preliminary results. Hum Reprod. 1994;9(6):1131–5.PubMedGoogle Scholar
  18. 18.
    Veiga A, Torello MJ, Menezo Y, Busquets A, Sarrias O, Coroleu B, et al. Use of co-culture of human embryos on Vero cells to improve clinical implantation rate. Hum Reprod. 1999;14 Suppl 2:112–20.CrossRefPubMedGoogle Scholar
  19. 19.
    Saito H, Hirayama T, Koike K, Saito T, Nohara M, Hiroi M. Cumulus mass maintains embryo quality. Fertil Steril. 1994;62(3):555–8.PubMedGoogle Scholar
  20. 20.
    Quinn P, Margalit R. Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos. J Assist Reprod Genet. 1996;13(1):9–14.CrossRefPubMedGoogle Scholar
  21. 21.
    Quinn P. Use of coculture with cumulus cells in insemination medium in human in vitro fertilization (IVF). J Assist Reprod Genet. 1994;11(5):270–7.CrossRefPubMedGoogle Scholar
  22. 22.
    Freeman MR, Whitworth CM, Hill GA. Granulosa cell co-culture enhances human embryo development and pregnancy rate following in-vitro fertilization. Hum Reprod. 1995;10(2):408–14.PubMedGoogle Scholar
  23. 23.
    Plachot M, Antoine JM, Alvarez S, Firmin C, Pfister A, Mandelbaum J, et al. Granulosa cells improve human embryo development in vitro. Hum Reprod. 1993;8(12):2133–40.PubMedGoogle Scholar
  24. 24.
    Jayot S, Parneix I, Verdaguer S, Discamps G, Audebert A, Emperaire JC. Coculture of embryos on homologous endometrial cells in patients with repeated failures of implantation. Fertil Steril. 1995;63(1):109–14.PubMedGoogle Scholar
  25. 25.
    Dominguez F, Gadea B, Mercader A, Esteban FJ, Pellicer A, Simon C. Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system. Fertil Steril. 2010;93(3):774–82. e1.CrossRefPubMedGoogle Scholar
  26. 26.
    Liu HC, He ZY, Mele CA, Veeck LL, Davis O, Rosenwaks Z. Human endometrial stromal cells improve embryo quality by enhancing the expression of insulin-like growth factors and their receptors in cocultured human preimplantation embryos. Fertil Steril. 1999;71(2):361–7.CrossRefPubMedGoogle Scholar
  27. 27.
    Nieto FS, Watkins WB, Lopata A, Baker HW, Edgar DH. The effects of coculture with autologous cryopreserved endometrial cells on human in vitro fertilization and early embryo morphology: a randomized study. J Assist Reprod Genet. 1996;13(5):386–9.CrossRefPubMedGoogle Scholar
  28. 28.
    Gellersen B, Brosens IA, Brosens JJ. Decidualization of the human endometrium: mechanisms, functions, and clinical perspectives. Semin Reprod Med. 2007;25(6):445–53.CrossRefPubMedGoogle Scholar
  29. 29.
    Lessey BA. Endometrial receptivity and the window of implantation. Baillieres Best Pract Res Clin Obstet Gynaecol. 2000;14(5):775–88.CrossRefPubMedGoogle Scholar
  30. 30.
    Dunn CL, Kelly RW, Critchley HO. Decidualization of the human endometrial stromal cell: an enigmatic transformation. Reprod Biomed Online. 2003;7(2):151–61.CrossRefPubMedGoogle Scholar
  31. 31.
    Maruyama T, Yoshimura Y. Molecular and cellular mechanisms for differentiation and regeneration of the uterine endometrium. Endocr J. 2008;55(5):795–810.CrossRefPubMedGoogle Scholar
  32. 32.
    Chen DB, Hilsenrath R, Yang ZM, Le SP, Kim SR, Chuong CJ, et al. Leukaemia inhibitory factor in human endometrium during the menstrual cycle: cellular origin and action on production of glandular epithelial cell prostaglandin in vitro. Hum Reprod. 1995;10(4):911–8.PubMedGoogle Scholar
  33. 33.
    Chegini N, Tang XM, Dou Q. The expression, activity and regulation of granulocyte macrophage-colony stimulating factor in human endometrial epithelial and stromal cells. Mol Hum Reprod. 1999;5(5):459–66.CrossRefPubMedGoogle Scholar
  34. 34.
    Rutanen EM. Insulin-like growth factors in endometrial function. Gynecol Endocrinol. 1998;12(6):399–406.CrossRefPubMedGoogle Scholar
  35. 35.
    Chegini N, Rossi MJ, Masterson BJ. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and EGF and PDGF beta-receptors in human endometrial tissue: localization and in vitro action. Endocrinology. 1992;130(4):2373–85.PubMedGoogle Scholar
  36. 36.
    Tabibzadeh SS, Santhanam U, Sehgal PB, May LT. Cytokine-induced production of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells. Modulation by estradiol-17 beta. J Immunol. 1989;142(9):3134–9.PubMedGoogle Scholar
  37. 37.
    Laird SM, Li TC, Bolton AE. The production of placental protein 14 and interleukin 6 by human endometrial cells in culture. Hum Reprod. 1993;8(6):793–8.PubMedGoogle Scholar
  38. 38.
    Lonergan P, Carolan C, Van Langendonckt A, Donnay I, Khatir H, Mermillod P. Role of epidermal growth factor in bovine oocyte maturation and preimplantation embryo development in vitro. Biol Reprod. 1996;54(6):1420–9.CrossRefPubMedGoogle Scholar
  39. 39.
    Desai N, Scarrow M, Lawson J, Kinzer D, Goldfarb J. Evaluation of the effect of interleukin-6 and human extracellullar matrix on embryonic development. Hum Reprod. 1999;14(6):1588–92.CrossRefPubMedGoogle Scholar
  40. 40.
    Naugler WE, Karin M. The wolf in sheep’s clothing: the role of interleukin-6 in immunity, inflammation and cancer. Trends Mol Med. 2008;14(3):109–19.CrossRefPubMedGoogle Scholar
  41. 41.
    Kishimoto T. IL-6: from its discovery to clinical applications. Int Immunol. 2010;22(5):347–52.CrossRefPubMedGoogle Scholar
  42. 42.
    Tovey MG, Content J, Gresser I, Gugenheim J, Blanchard B, Guymarho J, et al. Genes for IFN-beta-2 (IL-6), tumor necrosis factor, and IL-1 are expressed at high levels in the organs of normal individuals. J Immunol. 1988;141(9):3106–10.PubMedGoogle Scholar
  43. 43.
    Calabrese LH, Rose-John S. IL-6 biology: implications for clinical targeting in rheumatic disease. Nat Rev Rheumatol. 2014;10(12):720–7.CrossRefPubMedGoogle Scholar
  44. 44.
    Tabibzadeh S, Kong QF, Babaknia A, May LT. Progressive rise in the expression of interleukin-6 in human endometrium during menstrual cycle is initiated during the implantation window. Hum Reprod. 1995;10(10):2793–9.PubMedGoogle Scholar
  45. 45.
    von Wolff M, Thaler CJ, Zepf C, Becker V, Beier HM, Strowitzki T. Endometrial expression and secretion of interleukin-6 throughout the menstrual cycle. Gynecol Endocrinol. 2002;16(2):121–9.CrossRefGoogle Scholar
  46. 46.
    Sharkey AM, Dellow K, Blayney M, Macnamee M, Charnock-Jones S, Smith SK. Stage-specific expression of cytokine and receptor messenger ribonucleic acids in human preimplantation embryos. Biol Reprod. 1995;53(4):974–81.CrossRefPubMedGoogle Scholar
  47. 47.
    Desai NN, Goldfarb JM. Growth factor/cytokine secretion by a permanent human endometrial cell line with embryotrophic properties. J Assist Reprod Genet. 1996;13(7):546–50.CrossRefPubMedGoogle Scholar
  48. 48.
    Desai N, Goldfarb J. Co-cultured human embryos may be subjected to widely different microenvironments: pattern of growth factor/cytokine release by Vero cells during the co-culture interval. Hum Reprod. 1998;13(6):1600–5.CrossRefPubMedGoogle Scholar
  49. 49.
    Dariolli R, Bassaneze V, Nakamuta JS, Omae SV, Campos LC, Krieger JE. Porcine adipose tissue-derived mesenchymal stem cells retain their proliferative characteristics, senescence, karyotype and plasticity after long-term cryopreservation. PLoS One. 2013;8(7), e67939.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Bambang LS, Mazzucotelli JP, Moczar M, Beaujean F, Loisance D. Effects of cryopreservation on the proliferation and anticoagulant activity of human saphenous vein endothelial cells. J Thorac Cardiovasc Surg. 1995;110(4 Pt 1):998–1004.CrossRefPubMedGoogle Scholar
  51. 51.
    Hengstler JG, Utesch D, Steinberg P, Platt KL, Diener B, Ringel M, et al. Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction. Drug Metab Rev. 2000;32(1):81–118.CrossRefPubMedGoogle Scholar
  52. 52.
    Grondin M, Hamel F, Sarhan F, Averill-Bates DA. Metabolic activity of cytochrome p450 isoforms in hepatocytes cryopreserved with wheat protein extract. Drug Metab Dispos. 2008;36(10):2121–9.CrossRefPubMedGoogle Scholar
  53. 53.
    Bujia J, Kremer D, Sudhoff H, Viviente E, Sprekelsen C, Wilmes E. Determination of viability of cryopreserved cartilage grafts. Eur Arch Otorhinolaryngol. 1995;252(1):30–4.CrossRefPubMedGoogle Scholar
  54. 54.
    Boonlayangoor P, Telischi M, Boonlayangoor S, Sinclair TF, Millhouse EW. Cryopreservation of human granulocytes: study of granulocyte function and ultrastructure. Blood. 1980;56(2):237–45.PubMedGoogle Scholar
  55. 55.
    Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26(6):1270–83.Google Scholar
  56. 56.
    Tseng L, Gao JG, Chen R, Zhu HH, Mazella J, Powell DR. Effect of progestin, antiprogestin, and relaxin on the accumulation of prolactin and insulin-like growth factor-binding protein-1 messenger ribonucleic acid in human endometrial stromal cells. Biol Reprod. 1992;47(3):441–50.CrossRefPubMedGoogle Scholar
  57. 57.
    Reinach B, de Sousa G, Dostert P, Ings R, Gugenheim J, Rahmani R. Comparative effects of rifabutin and rifampicin on cytochromes P450 and UDP-glucuronosyl-transferases expression in fresh and cryopreserved human hepatocytes. Chem Biol Interact. 1999;121(1):37–48.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ivan Bochev
    • 1
    Email author
  • Kalina Belemezova
    • 2
  • Atanas Shterev
    • 1
  • Stanimir Kyurkchiev
    • 2
    • 3
  1. 1.IVF DepartmentOb/Gyn Hospital Dr. ShterevSofiaBulgaria
  2. 2.Tissue bank BULGENSofiaBulgaria
  3. 3.Institute of Reproductive HealthSofiaBulgaria

Personalised recommendations