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Does the number of embryos loaded on a single cryo-carrier affect post-vitrification survival rate?

  • Adva Aizer (a1), Meirav Noach-Hirsh (a1), Olga Dratviman-Storobinsky (a1), Jigal Haas (a1) and Raoul Orvieto (a1) (a2)...



We aimed to assess whether the survival rate of embryos is influenced by the number of embryos/oocytes loaded on a single cryo-carrier during vitrification.


This was a retrospective study that included 974 patients who underwent thawing of 1896 embryo-warming cycles between September 2016 and January 2020. A distinct analysis was made for cleavage stage embryos (2–10-cell stage) and blastocysts. For vitrification, embryos were placed in a Cryotop™ open device using a SAGE vitrification kit following the manufacturer’s instructions. Warming was carried using a SAGE warming vitrification kit according the manufacturer’s instructions.


Total post-vitrification survival rates of embryos at the cleavage stage or blastocyst stage was 94.8%. At the cleavage stage, cryo-preserving three embryos per single cryo-carrier gave the highest full intact embryo survival rate (91.5%) compared with one or two embryo(s) per single cryo-carrier (85.7%, P < 0.0002 and 87.3%, P < 0.004). Conversely, post warmed full intact blastocyst survival rate for two blastocysts was significantly lower compared with one blastocyst (76.7% vs. 87.9%, P < 0.0193) per single cryo-carrier.


Post-thawing survival rate following vitrification is affected by the number of embryos per single cryo-carrier undergoing the vitrification equilibration phase, with the optimum number of three cleaved embryos or one blastocyst per single cryo-carrier. Further studies are required to determine the optimum number of cleaved embryos or blastocysts that should be loaded onto a single cryo-carrier vitrification device.


Corresponding author

Author for correspondence: Adva Aizer. Infertility and IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer, Tel Aviv University, Tel Aviv, Israel. E-mail:


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Alpha Scientists In Reproductive Medicine (2012). The Alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reprod Biomed Online 25, 146–67.
Arav, A (2014). Cryopreservation of oocytes and embryos. Theriogenology 81, 96102.
Donnay, I, Van Langendonckt, A, Auquier, P, Grisart, B, Vansteenbrugge, A, Massip, A and Dessy, F (1997). Effects of co-culture and embryo number on the in vitro development of bovine embryos. Theriogenology 47, 1549–61.
Ebner, T, Shebl, O, Moser, M, Mayer, RB, Arzt, W and Tews, G (2010). Group culture of human zygotes is superior to individual culture in terms of blastulation, implantation and life birth. Reprod Biomed Online 21, 762–8.
ESHRE Special Interest Group of Embryology and Alpha Scientists in Reproductive Medicine (2017). The Vienna consensus: report of an expert meeting on the development of ART laboratory performance indicators. Reprod Biomed Online 35, 494510.
Fuller, BJ (2004). Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo Lett 25, 375–88.
Gardner, DK, Lane, M, Stevens, J, Schlenker, T and Schoolcraft, WB (2000). Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 73, 1155–8.
Hendriks, WK, Roelen, BA, Colenbrander, B and Stout, TAE (2015). Cellular damage suffered by equine embryos after exposure to cryoprotectants or cryopreservation by slow freezing or vitrification. Equine Vet J 47, 701–7.
Kleinhans, FW (1998). Membrane permeability modeling: Kedem-Katchalsky vs a two-parameter formalism. Cryobiology 37, 271–89.
Kuwayama, M, Vajta, G, Kato, O and Leibo, SP (2005). Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 11, 300–8.
Lane, M, Bavister, BD, Lyons, EA and Forest, KT (1999). Containerless vitrification of mammalian oocytes and embryos. Nat Biotechnol 17, 1234–6.
Lehner, A, Kaszas, Z, Murber, A, Rigo, J Jr, Urbancsek, J and Fancsovits, P (2017). Embryo density may affect embryo quality during in vitro culture in a microwell group culture dish. Arch Gynecol Obstet 296, 345–53.
Lin, TK, Su, JT, Lee, FK, Lin, Y-R and Lo, H-C (2010). Cryotop vitrification as compared with conventional slow freezing for human embryos at the cleavage stage: survival and outcomes. Taiwan J Obstet Gynecol 49, 272–8.
O’Doherty, EM, Wade, MG, Hill, JL and Boland, MP (1997). Effects of culturing bovine oocytes either singly or in groups on development to blastocysts. Theriogenology 48, 161–9.
Reed, ML (2012). Culture systems: embryo density. In Smith, GD, Swain, JE and Pool, TB (eds) Embryo Culture. Humana Press, New York City, pp 273312.
Rienzi, L, Gracia, C, Maggiulli, R, LaBarbera, A, Kaser, DJ, Ubaldi, FM, Vanderpoel, S and Racowsky, C (2016). Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Hum Reprod Update 23, 139–55.
Saragusty, J and Arav, A (2011). Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction 141, 119.
Trounson, A and Mohr, L (1983). Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305(5936), 707–9.
Vajta, G and Kuwayama, M (2006). Improving cryopreservation systems. Theriogenology 65, 236–44.
Zacà, C and Borini, A (2017). Chapter 8 Human oocytes slow-rate freezing: methodology. Methods Mol Biol 1568, 105–17.
Zeilmaker, GH, Alberda, AT, van Gent, I, Rijkmans, CM and Drogendijk, AC (1984). Two pregnancies following transfer of intact frozen–thawed embryos. Fertil Steril 42, 293–6.


Does the number of embryos loaded on a single cryo-carrier affect post-vitrification survival rate?

  • Adva Aizer (a1), Meirav Noach-Hirsh (a1), Olga Dratviman-Storobinsky (a1), Jigal Haas (a1) and Raoul Orvieto (a1) (a2)...


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