Impact of prolonged oocyte incubation time before vitrification on oocyte survival, embryo formation, and embryo quality in mice

  • Azade Karami
  • Mitra BakhtiariEmail author
  • Mehri Azadbakht
  • Rostam Ghorbani
  • Mozafar Khazaei
  • Mansour Rezaei


Oocyte incubation time before freezing is one of the factors affecting oocyte vitrification. In the assisted reproductive technology (ART) clinics, it is sometimes decided to perform oocyte vitrification after a long period of incubation time due to various conditions, such as inability to collect semen samples, unsuccessful urological interventions (PESA, TESE, etc.), or unexpected conditions. A time factor of up to 6 h has been studied in the available reports. Therefore, this study was designed to evaluate oocyte incubation time before freezing at 0, 6, 12, 18, and 24 h after retrieval. Metaphase II (MII) oocytes were obtained from NMRI female mice after being randomly divided into the five groups of 0, 6, 12, 18, and 24 h of freezing via hormonal stimulation following retrieval and entered into the vitrification-warming process. The thawed oocytes were evaluated according to the survival criteria and then inseminated with the sperms of male mice for in vitro fertilization. The next day, the embryo formation rate and embryo quality were assessed. Our results demonstrated that even after 24 h of incubation, the survival rate of oocytes was 51.35% with the embryo formation rate of 73.21%. However, the survival and embryo formation rates significantly decreased within 12, 18, and 24 h after retrieval compared to the groups vitrified at 0 h. The embryo quality was significantly reduced by vitrification at 0 to 24 h after retrieval. According to our data, although a prolonged incubation time before freezing reduced the survival rate, there was still a chance for oocytes to stay alive with acceptable embryo formation and quality rates after vitrification warming of oocytes.


Oocyte vitrification Prolonged incubation time Survival Embryo formation Embryo quality 



This study was supported by Kermanshah University of Medical Sciences School of Medicine. We should warmly appreciate the valuable contribution of those responsible in the Histology-Embryology Laboratory of the Department of Biology, Faculty of Sciences, Razi University, Kermanshah. The animals were taken care of according to the institutional guidelines of Kermanshah University of Medical Sciences and the National Institutes of Health (NIH) guidelines, including a 12/12 light/dark cycle, a temperature of 25°C, and free access to food and water.

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Chen S, Lien Y, Chao K, Ho H, Yang Y, Lee T (2003) Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing—a review article. Mol Cell Endocrinol 202:101–107CrossRefPubMedGoogle Scholar
  2. Dozortsev D, Nagy P, Abdelmassih S, Oliveira F, Brasil A, Abdelmassih V, Diamond M, Abdelmassih R (2004) The optimal time for intracytoplasmic sperm injection in the human is from 37 to 41 hours after administration of human chorionic gonadotropin. Fertil Steril 82:1492–1496CrossRefPubMedGoogle Scholar
  3. Ducibella T, Fissore R (2008) The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development. Dev Biol 315:257–279CrossRefPubMedPubMedCentralGoogle Scholar
  4. Fujino Y, Ozaki K, Yamamasu S, Ito F, Matsuoka I, Hayashi E, Nakamura H, Ogita S, Sato E, Inoue M (1996) Ovary and ovulation: DNA fragmentation of oocytes in aged mice. Hum Reprod 11:1480–1483CrossRefPubMedGoogle Scholar
  5. Fukuda A, Roudebush WE, Thatcher SS (1992) Influences of in vitro oocyte aging on microfertilization in the mouse with reference to zona hardening. J Assist Reprod Genet 9:378–383CrossRefPubMedGoogle Scholar
  6. Gook DA, Edgar DH (2007) Human oocyte cryopreservation. Hum Reprod Update 13:591–605CrossRefPubMedGoogle Scholar
  7. Gook DA, Osborn SM, Bourne H, Johnston W (1994) Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Hum Reprod 9:684–691CrossRefPubMedGoogle Scholar
  8. Gook DA, Osborn SM, Johnston W (1995) Fertilization and early embryology: parthenogenetic activation of human oocytes following cryopreservation using 1, 2-propanediol. Hum Reprod 10:654–658CrossRefPubMedGoogle Scholar
  9. Gordo AC, Rodrigues P, Kurokawa M, Jellerette T, Exley GE, Warner C, Fissore R (2002) Intracellular calcium oscillations signal apoptosis rather than activation in in vitro aged mouse eggs. Biol Reprod 66:1828–1837CrossRefPubMedGoogle Scholar
  10. Grynberg M, Hesters L, Thubert T, Izard V, Fanchin R, Frydman R, Frydman N (2012) Oocyte cryopreservation following failed testicular sperm extraction: a French case report with implications for the management of non-obstructive azoospermia. Reprod BioMed Online 24:611–613CrossRefPubMedGoogle Scholar
  11. Hunter J, Bernard A, Fuller B, Mcgrath J, Shaw R (1992) Measurements of the membrane water permeability (Lp) and its temperature dependence (activation energy) in human fresh and failed-to-fertilize oocytes and mouse oocyte. Cryobiology 29:240–249CrossRefPubMedGoogle Scholar
  12. Hutt KJ, Albertini DF (2007) An oocentric view of folliculogenesis and embryogenesis. Reprod BioMed Online 14:758–764CrossRefPubMedGoogle Scholar
  13. Igarashi H, Takahashi E, Hiroi M (1997) Aging-related changes in calcium oscillations in fertilized mouse oocytes. Mol Reprod Dev 48:383–390CrossRefPubMedGoogle Scholar
  14. Jain JK, Paulson RJ (2006) Oocyte cryopreservation. Fertil Steril 86:1037–1046CrossRefPubMedGoogle Scholar
  15. Jung YJ, Cheon YP (2014) Improvement of the vitrification method suppressing the disturbance of meiotic spindle and chromosome systems in mature oocytes. Dev & Reprod 18:117–125CrossRefGoogle Scholar
  16. Khalili MA, Maione M, Palmerini MG, Bianchi S, Macchiarelli G, Nottola SA (2012) Ultrastructure of human mature oocytes after vitrification. Eur J Histochem 56:236–242CrossRefGoogle Scholar
  17. Koutlaki N, Schoepper B, Maroulis G, Diedrich K, Al Hasani S (2006) Human oocyte cryopreservation: past, present and future. Reprod BioMed Online 13:427–436CrossRefPubMedGoogle Scholar
  18. Lee HJ, Elmoazzen H, Wright D, Biggers J, Rueda BR, Heo YS, Toner M, Toth TL (2010) Ultra-rapid vitrification of mouse oocytes in low cryoprotectant concentrations. Reprod BioMed Online 20:201–208CrossRefPubMedGoogle Scholar
  19. Li XH, Chen SU, Zhang X, Tang M, Kui YR, Wu X, Wang S, Guo YL (2005) Cryopreserved oocytes of infertile couples undergoing assisted reproductive technology could be an important source of oocyte donation: a clinical report of successful pregnancies. Hum Reprod 20:3390–3394CrossRefPubMedGoogle Scholar
  20. Liow SL, Foong LC, Chen NQ, Yip WY, Khaw CL, Kumar J, Vajta G, Ng SC (2009) Live birth from vitrified–warmed human oocytes fertilized with frozen–thawed testicular spermatozoa. Reprod BioMed Online 19:198–201CrossRefPubMedGoogle Scholar
  21. Monzo C, Haouzi D, Roman K, Assou S, Dechaud H, Hamamah S (2012) Slow freezing and vitrification differentially modify the gene expression profile of human metaphase II oocytes. Hum Reprod 27:2160–2168CrossRefPubMedGoogle Scholar
  22. Nakagata N, Takeo T, Fukumoto K, Kondo T, Haruguchi Y, Takeshita Y, Nakamuta Y, Matsunaga H, Tsuchiyama S, Ishizuka Y (2013) Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization. Cryobiology 67:188–192CrossRefPubMedGoogle Scholar
  23. Parmegiani L, Cognigni G, Bernardi S, Ciampaglia W, Infante F, Pocognoli P, De Fatis CT, Troilo E, Filicori M (2008) Freezing within 2 h from oocyte retrieval increases the efficiency of human oocyte cryopreservation when using a slow freezing/rapid thawing protocol with high sucrose concentration. Hum Reprod 23:1771–1777CrossRefPubMedGoogle Scholar
  24. Porcu E, Venturoli S, Damiano G, Ciotti P, Notarangelo L, Paradisi R, Moscarini M, Ambrosini G (2008) Healthy twins delivered after oocyte cryopreservation and bilateral ovariectomy for ovarian cancer. Reprod BioMed Online 17:265–267CrossRefPubMedGoogle Scholar
  25. Song W, Xin Z, Jin H, Peng Z, Chen X, Shi S, Dai S, Sun Y (2013) Effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection in mouse mature oocytes. Cell Biol In 37:561–567Google Scholar
  26. Takahashi T, Igarashi H, Kawagoe J, Amita M, Hara S, Kurachi H (2009) Poor embryo development in mouse oocytes aged in vitro is associated with impaired calcium homeostasis. Biol Reprod 80:493–502CrossRefPubMedGoogle Scholar
  27. Tao J, Tamis R, Fink K, Williams B, Nelson White T, Craig R (2002) The neglected morula/compact stage embryo transfer. Hum Reprod 17:1513–1518CrossRefPubMedGoogle Scholar
  28. Tarín JJ, Pérez Albalá S, Cano A (2001) Cellular and morphological traits of oocytes retrieved from aging mice after exogenous ovarian stimulation. Biol Reprod 65:141–150CrossRefPubMedGoogle Scholar
  29. Tatone C, Carbone MC, Gallo R, Delle Monache S, Di Cola M, Alesse E, Amicarelli F (2006) Age-associated changes in mouse oocytes during postovulatory in vitro culture: possible role for meiotic kinases and survival factor BCL2. Biol Reprod 74:395–402CrossRefPubMedGoogle Scholar
  30. Tatone C, Di Emidio G, Barbaro R, Vento M, Ciriminna R, Artini PG (2011) Effects of reproductive aging and postovulatory aging on the maintenance of biological competence after oocyte vitrification: insights from the mouse model. Theriogenology 76:864–873CrossRefPubMedGoogle Scholar
  31. Yanagida K, Yazawa H, Katayose H, Suzuki K, Hoshi K, Sato A (1998) Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection. Hum Reprod 13:2223–2226CrossRefPubMedGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2017

Authors and Affiliations

  • Azade Karami
    • 1
  • Mitra Bakhtiari
    • 2
    Email author
  • Mehri Azadbakht
    • 3
  • Rostam Ghorbani
    • 2
  • Mozafar Khazaei
    • 2
  • Mansour Rezaei
    • 4
  1. 1.Students Research CommitteeKermanshah University of Medical SciencesKermanshahIran
  2. 2.Fertility & Infertility Research CenterKermanshah University of Medical SciencesKermanshahIran
  3. 3.Department of Biology, Faculty of SciencesRazi UniversityKermanshahIran
  4. 4.Department of Biostatistics & EpidemiologyKermanshah University of Medical SciencesKermanshahIran

Personalised recommendations