Effect of cryopreservation on human granulosa cell viability and responsiveness to gonadotropin

  • Céline Bouillon
  • Fabrice Guérif
  • Philippe Monget
  • Marie-Christine Maurel
  • Elodie KaraEmail author
Regular Article


In human, the use of freshly recovered granulosa cells for experiments remains difficult. Because of the single use of human cells, the experiments cannot be repeated, and no additional conditions can be tested afterwards with the cells of the same patient. Therefore, granulosa cell cryopreservation could be a good alternative to keep part of these cells for later controls or experiments. The aim of this study is to compare the responsiveness to FSH of fresh and frozen-thawed human primary granulosa-lutein cells (hGLC) and determine if cryopreserved granulosa cells can be used in place of fresh cells. Two cryopreservation methods were also compared: a conventional versus a simplified freezing method. This experimental study was undertaken at Igyxos S.A., Nouzilly, France. Seventy women undergoing oocyte retrieval at the IVF Unit from Bretonneau University Hospital (Tours, France) were recruited in 2016. Fresh and frozen-thawed hGLC were cultured for 7 days and then stimulated by r-FSH for 48 h. To assess r-FSH efficacy and potency, extracellular cAMP accumulated in the supernatant for each stimulation point was measured. We demonstrated that hGLC remain responsive to FSH stimulation after freezing-thawing and 7 days of pre-culture. They are able to secrete cAMP with a similar EC50 value as fresh hGLC, but FSH efficacy is lowered. As our study did not show any significant difference between the two freezing methods concerning the sensitivity of hGLC to FSH, hGLC could be cryopreserved with the simplified freezing method without taking up too much time for IVF laboratories.


Human granulosa cells Cryopreservation cAMP FSH IVF 



We warmly thank the technicians of the IVF Unit (Bretonneau University Hospital, Tours, France) for putting follicular fluids aside for our experiments. The technical assistance of Anaïs Benon is gratefully acknowledged.

Funding statement

This work was funded by the “Agence de la Biomédecine.”

Compliance with ethical standards

Conflict of interest

Marie-Christine Maurel and Elodie Kara were employed by Igyxos S.A. Igyxos aims to develop innovative medical technologies in the field of fertility, but declares no conflict of interest regarding the technique presented herein because it has been developed for research purposes only. It is not, and will never be, part of a technology developed by the company. The academic authors, Céline Bouillon, Philippe Monget, and Fabrice Guérif, declare no competing interests.


  1. Agrawal R, Jacobs H, Payne N, Conway G (2002) Concentration of vascular endothelial growth factor released by cultured human luteinized granulosa cells is higher in women with polycystic ovaries than in women with normal ovaries. Fertil Steril 78:1164–1169. CrossRefPubMedGoogle Scholar
  2. Benkhalifa M, Demirol A, Sari T et al (2012) Autologous embryo–cumulus cells co-culture and blastocyst transfer in repeated implantation failures: a collaborative prospective randomized study. Zygote 20:173–180. CrossRefPubMedGoogle Scholar
  3. Broussard JR, Thibodeaux JK, Myers MW et al (1994) Frozen-thawed cumulus-granulosa cells support bovine embryo development during coculture. Fertil Steril 62:176–180CrossRefGoogle Scholar
  4. Casarini L, Lispi M, Longobardi S et al (2012) LH and hCG action on the same receptor results in quantitatively and qualitatively different intracellular signalling. PLoS One 7. CrossRefGoogle Scholar
  5. Casarini L, Moriondo V, Marino M et al (2014) FSHR polymorphism p.N680S mediates different responses to FSH in vitro. Mol Cell Endocrinol 393:83–91. CrossRefPubMedGoogle Scholar
  6. Casarini L, Riccetti L, De Pascali F et al (2016) Follicle-stimulating hormone potentiates the steroidogenic activity of chorionic gonadotropin and the anti-apoptotic activity of luteinizing hormone in human granulosa-lutein cells in vitro. Mol Cell Endocrinol 422:103–114. CrossRefPubMedGoogle Scholar
  7. Casillas F, Ducolomb Y, Lemus AE et al (2015) Porcine embryo production following in vitro fertilization and intracytoplasmic sperm injection from vitrified immature oocytes matured with a granulosa cell co-culture system. Cryobiology 71:299–305. CrossRefPubMedGoogle Scholar
  8. Chabrolle C, Tosca L, Ramé C et al (2009) Adiponectin increases insulin-like growth factor I-induced progesterone and estradiol secretion in human granulosa cells. Fertil Steril 92:1988–1996. CrossRefPubMedGoogle Scholar
  9. Deng W, Yu XL, Pang YZ, Zan LS (2008) Effects of granulosa cells treatments and follicular fluid on cleavage rate and blastocyst rate of bovine oocyte after in vitro fertilization and culture. Fen Zi Xi Bao Sheng Wu Xue Bao 41:393–402PubMedGoogle Scholar
  10. Dirnfeld M, Goldman S, Gonen Y et al (1997) A simplified coculture system with luteinized granulosa cells improves embryo quality and implantation rates: a controlled study. Fertil Steril 67:120–122CrossRefGoogle Scholar
  11. Fabbri R, Porcu E, Marsella T et al (2000) Human embryo development and pregnancies in an homologous granulosa cell coculture system. J Assist Reprod Genet 17:1–12. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Goovaerts IGF, Leroy JLMR, Rizos D et al (2011) Single in vitro bovine embryo production: Coculture with autologous cumulus cells, developmental competence, embryo quality and gene expression profiles. Theriogenology 76:1293–1303. CrossRefPubMedGoogle Scholar
  13. Guerif F, Bidault R, Gasnier O et al (2004) Efficacy of blastocyst transfer after implantation failure. Reprod BioMed Online 9:630–636CrossRefGoogle Scholar
  14. Lin T, Oqani RK, Lee JE et al (2016) Coculture with good-quality COCs enhances the maturation and development rates of poor-quality COCs. Theriogenology 85:396–407. CrossRefPubMedGoogle Scholar
  15. May JV, Gilliam FR, Rein MS et al (1982) Monolayer growth and differentiated function of porcine and rat granulosa cells following cryopreservation. Biol Reprod 27:641–651. CrossRefPubMedGoogle Scholar
  16. Nordhoff V, Sonntag B, von Tils D et al (2011) Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells. Reprod BioMed Online 23:196–203. CrossRefPubMedGoogle Scholar
  17. Plachot M, Antoine JM, Alvarez S et al (1993) Granulosa cells improve human embryo development in vitro. Hum Reprod Oxf Engl 8:2133–2140CrossRefGoogle Scholar
  18. Quinn MCJ, McGregor SB, Stanton JL et al (2006) Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates. Reprod Fertil Dev 18:501–508CrossRefGoogle Scholar
  19. Saint-Dizier M, Chastant-Maillard S (2014) La reproduction animale et humaine. Editions QuaeGoogle Scholar
  20. Scaramuzzi RJ, Baird DT, Campbell BK et al (2011) Regulation of folliculogenesis and the determination of ovulation rate in ruminants. Reprod Fertil Dev 23:444–467. CrossRefPubMedGoogle Scholar
  21. Shin S-Y, Lee J-Y, Lee E et al (2006) Protective effect of vascular endothelial growth factor (VEGF) in frozen-thawed granulosa cells is mediated by inhibition of apoptosis. Eur J Obstet Gynecol Reprod Biol 125:233–238. CrossRefPubMedGoogle Scholar
  22. Sluss PM, Lee K, Mattox JH et al (1994) Estradiol and progesterone production by cultured granulosa cells cryopreserved from in vitro fertilization patients. Eur J Endocrinol 130:259–264CrossRefGoogle Scholar
  23. Tirelli M, Basini G, Grasselli F et al (2005) Cryopreservation of pig granulosa cells: effect of FSH addition to freezing medium. Domest Anim Endocrinol 28:17–33. CrossRefPubMedGoogle Scholar
  24. Zeyneloglu HB, Kahraman S (2009) The use of coculture in assisted reproductive technology: does it have any impact? Curr Opin Obstet Gynecol 21:253–259. CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Service de médecine et Biologie de la Reproduction, CHRU de ToursToursFrance
  2. 2.PRC, INRA, CNRS, IFCE, Université de ToursNouzillyFrance
  3. 3.Igyxos S.A.NouzillyFrance

Personalised recommendations