Granulosa Cell Conditioned Medium Enhances The Rate of Mouse Oocyte In Vitro Maturation and Embryo Formation

Document Type : Original Article


1 Department Of Animal Sciences , Faculty Of life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran

2 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

3 School of pharmacy and Bioengineering , Keele University, ST58G, UK


Objective: In vitro maturation (IVM) and cryopreservation of oocytes are two important parts of assisted reproductive technology (ART), but their efficacy is low. This study aimed to improve the quality of in vitro vitrified-warmed maturated oocytes using granulosa cell conditioned medium (GCCM).
Materials and Methods: In the experimental study, fresh/non-vitrified and vitrified-warmed mouse germinal vesicle (GV)
oocytes (as F and V) were in vitro maturated using basal medium (BM) and also BM supplemented with 50% GCCM as treated groups (GM), and categorized as FBM, FGM, VBM and VGM groups, respectively. The rate of successful IVM (MII oocyte formation), mitochondrial membrane potential and the viability of MII oocytes were determined using inverted microscopy, JC-1 and trypan blue staining. Then, the rate of in vitro fertilization (IVF) and subsequent two-cell embryo formation was calculated. Finally, the expression levels of Oct4, Sox2, Cdk-2, Gdf9, Integrin beta1 and Igf2 were analyzed using real-time polymerase chain reaction (PCR) in MII oocytes and two-cell embryos.
Results: These analyses showed that GCCM significantly increased the IVM rate, oocyte meiotic resumption and mitochondrial membrane potential (P<0.05). In addition, the rate of IVF and two-cell embryo formation was significantly
higher in FGM and VGM compared to FBM and VBM (P<0.05). Interestingly, GCCM significantly affected the expression
of the studied genes.
Conclusion: Our findings suggest that GCCM might be useful for improving the efficiency of IVM and the subsequent
IVF outcomes.


  1. Talaulikar VS, Conway GS, Pimblett A, Davies MC. Outcome of ovarian stimulation for oocyte cryopreservation in women with Turner syndrome. Fertil Steril. 2019; 111(3): 505-509.
  2. Iussig B, Maggiulli R, Fabozzi G, Bertelle S, Vaiarelli A, Cimadomo D, et al. A brief history of oocyte cryopreservation: arguments and facts. Acta Obstet Gynecol Scand. 2019; 98(5): 550-558.
  3. Khalili MA, Nottola SA, Shahedi A, Macchiarelli G. Contribution of human oocyte architecture to success of in vitro maturation technology. Iran J Reprod Med. 2013; 11(1): 1-10.
  4. Delvigne A, Rozenberg S. Epidemiology and prevention of ovarian hyperstimulation syndrome (OHSS): a review. Hum Reprod Update. 2002; 8(6): 559-577.
  5. Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence. Reproduction. 2001; 121(1): 51-75.
  6. Dekel N. Molecular control of meiosis. Trends Endocrinol Metab. 1995; 6(5): 165-169.
  7. Liu W, Xin Q, Wang X, Wang S, Wang H, Zhang W, et al. Estrogen receptors in granulosa cells govern meiotic resumption of preovulatory oocytes in mammals. Cell Death Dis. 2017; 8(3): e2662- e2662.
  8. Jahromi BN, Mosallanezhad Z, Matloob N, Davari M, Ghobadifar MA. The potential role of granulosa cells in the maturation rate of immature human oocytes and embryo development: a co-culture study. Clin Exp Reprod Med. 2015; 42(3): 111-117.
  9. Wigglesworth K, Lee KB, O’Brien MJ, Peng J, Matzuk MM, Eppig JJ. Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes. Proc Natl Acad Sci USA. 2013; 110(39): E3723-E3729.
  10. Webb RJ, Marshall F, Swann K, Carroll J. Follicle-stimulating hormone induces a gap junction-dependent dynamic change in [cAMP] and protein kinase a in mammalian oocytes. Dev Biol. 2002; 246(2): 441-454.
  11. 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): 120-122.
  12. Malekshah AK, Moghaddam AE, Daraka SM. Comparison of conditioned medium and direct co-culture of human granulosa cells on mouse embryo development. Indian J Exp Biol. 2006; 44(3): 189-192.
  13. Zand E, Fathi R, Nasrabadi MH, Atrabi MJ, Spears N, Akbarinejad V. Maturational gene upregulation and mitochondrial activity enhancement in mouse in vitro matured oocytes and using granulosa cell conditioned medium. Zygote. 2018; 26(5): 366-371.
  14. Abdel-Ghani MA, Abe Y, Asano T, Hamano S, Suzuki H. Effect ofbovine cumulus–oocyte complexes-conditioned medium on in vitro maturation of canine oocytes. Reprod Med Biol. 2011; 10(1): 43-49.
  15. Zhong H, Sun Q, Chen P, Xiong F, Li G, Wan C, et al. Detection of IL-6, IL-10, and TNF-α level in human single-blastocyst conditioned medium using ultrasensitive Single Molecule Array platform and its relationship with embryo quality and implantation: a pilot study. J Assist Reprod Genet. 2020; 37(7): 1695-1702.
  16. Akbari H, Eftekhar Vaghefi SH, Shahedi A, Habibzadeh V, Mirshekari TR, Ganjizadegan A, et al. Mesenchymal stem cell-conditioned medium modulates apoptotic and stress-related gene expression, ameliorates maturation and allows for the development of immature human oocytes after artificial activation. Genes. 2017; 8(12): 371.
  17. Succu S, Bebbere D, Bogliolo L, Ariu F, Fois S, Leoni GG, et al. Vitrification of in vitro matured ovine oocytes affects in vitro pre-implantation development and mRNA abundance. Mol Reprod Dev. 2008; 75(3): 538-546.
  18. Massip A. Cryopreservation of bovine oocytes: current status and recent developments. Reprod Nutr Dev. 2003; 43(4): 325-330.
  19. Lei T, Guo N, Tan MH, Li YF. Effect of mouse oocyte vitrification on mitochondrial membrane potential and distribution. J Huazhong Univ Sci Technol Med Sci. 2014; 34(1): 99-102.
  20. Garzo VG, Dorrington JH. Aromatase activity in human granulosa cells during follicular development and the modulation by folliclestimulating hormone and insulin. Am J Obstet Gynecol. 1984; 148(5): 657-662.
  21. Nishi Y, Yanase T, Mu Y, Oba K, Ichino I, Saito M, et al. Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor. Endocrinology. 2001; 142(1): 437-445.
  22. De Leo V, Musacchio MC, Cappelli V, Massaro MG, Morgante G, Petraglia F. Genetic, hormonal and metabolic aspects of PCOS: an update. Reprod Biol Endocrinol. 2016; 14(1): 38.
  23. Azari M, Kafi M, Ebrahimi B, Fatehi R, Jamalzadeh M. Oocyte maturation, embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification. Vet Res Commun. 2017; 41(1): 49-56.
  24. Niu HR, Zi XD, Xiao X, Xiong XR, Zhong JC, Li J, et al. Cloning of cDNAs for H1F0, TOP1, CLTA and CDK1 and the effects of cryopreservation on the expression of their mRNA transcripts in yak (Bos grunniens) oocytes. Cryobiology. 2014; 69(1): 55-60.
  25. Monte AP, Santos JM, Menezes VG, Gouveia BB, Lins TL, Barberino RS, et al. Growth differentiation factor-9 improves development, mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles. Reprod Domest Anim. 2019; 54(9): 1169-1176.
  26. Kim SS, Olsen R, Kim DD, Albertini DF. The impact of vitrification on immature oocyte cell cycle and cytoskeletal integrity in a rat model. J Assist Reprod Genet. 2014; 31(6): 739-747.
  27. Ebrahimi B, Valojerdi MR, Eftekhari-Yazdi P, Baharvand H. In vitro maturation, apoptotic gene expression and incidence of numerical chromosomal abnormalities following cryotop vitrification of sheep cumulus-oocyte complexes. J Assist Reprod Genet. 2010; 27(5): 239-246.
  28. Succu S, Leoni GG, Berlinguer F, Madeddu M, Bebbere D, Mossa F, et al. Effect of vitrification solutions and cooling upon in vitro matured prepubertal ovine oocytes. Theriogenology. 2007; 68(1): 107-114.
  29. Talreja D, Gupta C, Pai H, Palshetkar N. Oocyte vitrification: a comparative analysis between fresh and cryopreserved oocytes in an oocyte donation program. Fertil Reprod. 2020; 2(01): 9-13.
  30. Elnahas A, Alcolak E, Marar EA, Elnahas T, Elnahas K, Palapelas V, et al. Vitrification of human oocytes and different development stages of embryos: an overview. Middle East Fertil Soc J. 2010; 15(1): 2-9.
  31. Rodriguez-Wallberg KA, Waterstone M, Anastácio A. Ice age: cryopreservation in assisted reproduction - an update. Reprod Biol. 2019; 19(2): 119-126.
  32. Miraki S, Mokarizadeh A, Banafshi O, Assadollahi V, Abdi M, Roshani D, et al. Embryonic stem cell conditioned medium supports in vitro maturation of mouse oocytes. Avicenna J Med Biotechnol. 2017; 9(3): 114-119.
  33. Uhde K, van Tol HT, Stout TA, Roelen BA. Metabolomic profiles of bovine cumulus cells and cumulus-oocyte-complex-conditioned medium during maturation in vitro. Sci Rep. 2018; 8(1): 1-14.
  34. Maeda J, Kotsuji F, Negami A, Kamitani N, Tominaga T. In vitro development of bovine embryos in conditioned media from bovine granulosa cells and vero cells cultured in exogenous protein- and amino acid-free chemically defined human tubal fluid medium. Biol Reprod. 1996; 54(4): 930-936.
  35. Wu G, Schöler HR. Role of Oct4 in the early embryo development. Cell Regen. 2014; 3(1): 7.
  36. Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 2003; 17(1): 126-140.
  37. Stephens LE, Sutherland AE, Klimanskaya IV, Andrieux A, Meneses J, Pedersen RA, et al. Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethality. Genes Dev. 1995; 9(15): 1883-1895.
  38. Toori MA, Mosavi E, Nikseresht M, Barmak MJ, Mahmoudi R. Influence of insulin-like growth factor-i on maturation and fertilization rate of immature oocyte and embryo development in NMRI mouse with TCM199 and α-MEM medium. JCDR. 2014; 8(12): AC05.
  39. Kelley RL, Gardner DK. Addition of interleukin-6 to mouse embryo culture increases blastocyst cell number and influences the inner cell mass to trophectoderm ratio. Clin Exp Reprod Med. 2017; 44(3): 119-125.
  40. Kirkegaard K, Yan Y, Sørensen BS, Hardarson T, Hanson C, Ingerslev HJ, et al. Comprehensive analysis of soluble RNAs in human embryo culture media and blastocoel fluid. J Assist Reprod Genet. 2020; 37(9): 2199-2209.