Original Articles

By Ms. Trina Shah , Mr. Hamish Barblett , Dr. Phillip Matson
Corresponding Author Ms. Trina Shah
Hollywood Fertility Centre, Hollywood Private Hospital, Monash Avenue, Nedlands - Australia WA 6009
Submitting Author Dr. Phillip L Matson
Other Authors Mr. Hamish Barblett
Hollywood Fertility Centre, Hollywood Private Hospital, Monash Avenue, Nedlands - Australia WA 6009

Dr. Phillip Matson
Hollywood Fertility Centre, Monash Avenue - Australia WA 6009


Zygotes, Refrozen blastocysts, Human IVF

Shah T, Barblett H, Matson P. Human Blastocysts Developed From Thawed Zygotes: Refreezing Does Not Affect Their Survival Or Implantation. WebmedCentral REPRODUCTION 2010;1(11):WMC001063
doi: 10.9754/journal.wmc.2010.001063
Submitted on: 02 Nov 2010 06:10:46 AM GMT
Published on: 02 Nov 2010 09:23:24 PM GMT


The thawing of frozen human zygotes and culture to the blastocyst stage often generates supernumerary blastocysts which can be refrozen. A total of 9 recipients of donated oocytes and 35 women originally at risk of ovarian hyperstimulation syndrome had such blastocysts thawed. There were respective survival rates of 72.0% and 66.7%, and on-going pregnancy rates of 23.5% and 18.8% after transfer, which did not differ from the general IVF population of women receiving thawed blastocysts generated from fresh zygotes. The refreezing of blastocysts from thawed zygotes can therefore be undertaken with confidence.


Human embryos within in vitro fertilisation (IVF) programmes have often been frozen in the past as zygotes (ie pronuclear oocytes) because of their apparent superior survival rates [4, 9]. This stage is particularly suited to the “freeze-all” strategy in which all embryos are frozen and the transfer is postponed, as in cases at risk of ovarian hyperstimulation syndrome (OHSS) [3] or where fertilised donated oocytes are to be quarantined [8]. However, the relatively recent availability of commercially prepared sequential media and the extended culture of embryos to the blastocyst stage has resulted in increased pregnancy rates per embryo transfer [10], and the improved selection of high quality embryos to maintain an effective single embryo transfer policy to reduce the risks of multiple pregnancy [7]. An increase in the proportion of laboratories undertaking blastocyst culture has occurred over the last 5 years [21].
Laboratories that have frozen zygotes in storage but now use extended culture are presented with a dilemma at thawing: how many zygotes should be thawed and cultured on? Unfortunately, less than half of the embryos will become good quality blastocysts [14, 20] and some patients may even have none form for transfer [19]. It would therefore seem prudent to thaw a sufficient number of zygotes to ensure a reasonable chance of getting at least one blastocyst for transfer, although there is then risk of having too many blastocysts formed if a maximum of only one or two are transferred [15]. In such cases, the refreezing of supernumerary blastocysts would seem a viable option as indicated by an isolated case report [5]. The present report describes our clinical experience of thawing frozen blastocysts derived from thawed zygotes for women that were initially at risk of OHSS or recipients of donated oocytes.


Women treated between 2000 and 2007 were included if their frozen/thawed embryos were cultured to the blastocyst stage and supernumerary blastocysts refrozen. These were either:
a) Fertility patients who had all embryos frozen because the serum estradiol was >20,000 pmol/l or >20 oocytes were collected, putting them at risk of developing OHS.
b) Oocyte recipients whose donated oocytes had been fertilised, cryopreserved and stored for at least six months before use due to quarantine restrictions.
All embryos were stored with standard operating procedures and according to West Australian law. Embryos were initially frozen and thawed as zygotes using a previously published slow rate freezing protocol with sucrose and propoanediol as the main cryoprotectants [11]. Up to 7 zygotes were thawed at any one time. Following culture for a further 4-5 days and the transfer of a maximum of two embryos, supernumerary blastocysts were frozen using glycerol as the principle cryoprotectant [2]. In subsequent cycles, blastocysts were thawed on the day of transfer and embryo transfers were performed under ultrasound-guidance when the patient had a half-full bladder. Serum hCG was measured 12 days after the embryo transfer, with a positive pregnancy test result being recorded if the hCG concentration >25iu/l. The pregnancies were monitored weekly by serum hCG, estradiol and progesterone, and an ultrasound performed at 7 weeks to confirm the presence of a fetal heart.
Proportions were compared with the χ2 test, and Yates’ correction for continuity was applied if >20% of expected frequencies were


The fate of the refrozen supernumerary blastocysts upon thawing is shown in Illustration 1. Comparison of the performance of refrozen blastocyst from donated oocyte recipients and patients originally at risk of OHS showed that there was a similar survival rate of blastocysts (χ2=0.3, p=0.62) and incidence of pregnancy after transfer (χ2=0.0, p=0.94). There were also no significant differences in survival and pregnancy rates when compared to patients with once frozen blastocysts.


Many IVF laboratories will have cryopreserved zygotes in storage, largely from cases at risk of developing OHSS or from recipients of donated oocytes. Furthermore, there are some countries whereby the local legislation prevents the cryopreservation of embryos and so freezing of zygotes is undertaken prior to syngamy, eg Germany [12] and Italy [13]. The technology for cryopreservation of zygotes is fairly robust and effective, but the difficulty really comes at thawing when deciding the number to thaw for any given woman. It is important for the embryos and the uterine environment to be synchronised and so more embryos cannot be thawed in that cycle should the thawed zygotes fail to develop to the blastocyst stage. One solution is to thaw several zygotes, culture, choose the best for transfer, and then refreeze the remainder. Such an approach would seem feasible based on an earlier case report in which supernumerary blastocysts following the thaw and culture of cryopreserved zygotes can be refrozen [5].
The present study has confirmed that freezing zygotes with propanediol as the principle cryoprotectant, followed by refreezing the blastocysts using glycerol, gives good results in a cohort of women attending the Hollywood Fertility Centre. The refreezing strategy should also be suitable for other combinations of developmental stages and cryoprotectants as has been suggested by a number of isolated case reports, although they should be confirmed by larger studies. These case reports include thawed zygotes refrozen [1], early cleavage embryos thawed and refrozen at blastocyst [6], thawed blastocysts refrozen [16], thawed zygotes refrozen at the morula or blastocyst stage [22], or thawed early cleavage embryos refrozen as blastocysts [18]. The increased efficiency of vitrification of blastocysts [17] will make this strategy more attractive.
In conclusion, the freezing of zygotes using propanediol and then the refreezing of blastocysts with glycerol can be undertaken with confidence.


The efforts of all staff at the Hollywood Fertility Centre over the years is gratefully acknowledged.


1. Baker A, Check J, Lurie D, Hourani C, Hoover L. Pregnancy achieved with pronuclear-stage embryos that were cryopreserved and thawed twice: a case report. J Assist Reprod Genet 1996; 13: 713-715.
2. Cohen J, Simons R, Edwards R, Fehilly C, Fishel S. Pregnancies following storage of expanding human blastocysts. J In Vitro Fert Embryo Transf 1985; 2: 59-64.
3. D'Angelo A, Amso NN. Embryo freezing for preventing ovarian hyperstimulation syndrome. Cochrane Database of Systematic Reviews 2007; Issue 3. Art. No.:CD002806.
4. Demoulin A, Jouan C, Gerday C, Dubois M. Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages. Hum Reprod 1991; 6: 799-804.
5. Estes S, Laky D, Hoover L, Smith S, Schinfeld J, Somkuti S. Successful pregnancy resulting from cryopreserved pronuclear and cleaved embryos thawed and cultured to blastocysts, refrozen and transferred. A case report. J Reprod Med 2003; 48: 46-48.
6. Farhat M, Zentner B-S, Lossos F, Bdolah Y, Holtzer H, Hurwitz A. Successful pregnancy following replacement of embryos previously refrozen at blastocyst stage. Hum Reprod 2001; 16: 337-339.
7. Gardner D, Lane M, Stevens J, Schlenker T, Schoolcraft W. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000; 73: 1155-1158.
8. Hamer F, Horne G, Pease E, Matson P, Lieberman B. The quarantine of fertilized donated oocytes. Hum Reprod 1995; 10: 1194-1196.
9. Horne G, Critchlow J, Newman M, Edozien L, Matson P, Lieberman B. A prospective evaluation of cryopreservation strategies in a two-embryo transfer programme. Hum Reprod 1997; 12: 542-547.
10. Langley M, Marek D, Gardner D, Doody K, Doody K. Extended embryo culture in human assisted reproduction treatments. Hum Reprod 2001; 16: 902-908.
11. Lassalle B, Testart J, Renard J. Human embryo features that influence the success of cryopreservation with the use of 1,2-propanediol. Fertil Steril 1985; 44: 645-651.
12. Robertson J. Reproductive technology in Germany and the United States: an essay in comparative law and bioethics. Columbia Journal of Transnational Law 2004; 43: 189-227.
13. Robertson J. Protecting embryos and burdening women: assisted reproduction in Italy. Hum Reprod 2004; 19: 1693-1696.
14. Schoolcraft W, Gardner D, Lane M, Schlenker T, Hamilton F, Meldrum D. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril 1999; 72: 604-609.
15. Shapiro B, Daneshmand S, Garner F, Aguirre M, Hudson C, Thomas S. High ongoing pregnancy rates after deferred transfer through bipronuclear oocyte cryopreservation and post-thaw extended culture. Fertil Steril 2009; 92: 1594-1599.
16. Smith L, Roots E, Dorsett M. Live birth of a normal healthy baby after a frozen embryo transfer with blastocysts that were frozen and thawed twice. Fertil Steril 2005; 83: 198-200.
17. Stehlik E, Stehlik J, Katayama K, Kuwayama M, Jambor V, Brohammer R, Kato O. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reproductive Biomedicine Online 2005; 11: 53-57.
18. Takahashi T, Araki Y. Successfully healthy baby delivery from human refrozen blastocyst embryos by vitrification. Journal of Mammalian Ova Research 2004; 21: 162-165.
19. Thomas M, AESparks, Ryan G, Van Voorhis J. Clinical predictors of human blastocyst formation and pregnancy after extended embryo culture and transfer. Fertil Steril 2009; in press.
20. Van Landuyt L, De Vos A, Joris H, Verheyen G, Devroey P, Van Steirteghem A. Blastocyst formation in in vitro fertilization versus intracytoplasmic sperm injection cycles: Influence of the fertilization procedure. Fertil Steril 2005; 83: 1397-1403.
21. Wang Y, Chambers G, Sullivan E. Assisted reproduction technology in Australia and New Zealand 2008. Assisted reproduction technology series no. 14. Cat. no. PER 49. Canberra: AIHW. 2010.
22. Yokota Y, Yokota H, Yokota M, Sato S, Araki Y. Birth of healthy twins from in vitro development of human refrozen embryos. Fertil Steril 2001; 76: 1063-1065.

Source(s) of Funding

Work funded by the Hollywood Fertility Centre.

Competing Interests



This article has been downloaded from WebmedCentral. With our unique author driven post publication peer review, contents posted on this web portal do not undergo any prepublication peer or editorial review. It is completely the responsibility of the authors to ensure not only scientific and ethical standards of the manuscript but also its grammatical accuracy. Authors must ensure that they obtain all the necessary permissions before submitting any information that requires obtaining a consent or approval from a third party. Authors should also ensure not to submit any information which they do not have the copyright of or of which they have transferred the copyrights to a third party.
Contents on WebmedCentral are purely for biomedical researchers and scientists. They are not meant to cater to the needs of an individual patient. The web portal or any content(s) therein is neither designed to support, nor replace, the relationship that exists between a patient/site visitor and his/her physician. Your use of the WebmedCentral site and its contents is entirely at your own risk. We do not take any responsibility for any harm that you may suffer or inflict on a third person by following the contents of this website.

0 comments posted so far

Please use this functionality to flag objectionable, inappropriate, inaccurate, and offensive content to WebmedCentral Team and the authors.


Author Comments
0 comments posted so far


What is article Popularity?

Article popularity is calculated by considering the scores: age of the article
Popularity = (P - 1) / (T + 2)^1.5
P : points is the sum of individual scores, which includes article Views, Downloads, Reviews, Comments and their weightage

Scores   Weightage
Views Points X 1
Download Points X 2
Comment Points X 5
Review Points X 10
Points= sum(Views Points + Download Points + Comment Points + Review Points)
T : time since submission in hours.
P is subtracted by 1 to negate submitter's vote.
Age factor is (time since submission in hours plus two) to the power of 1.5.factor.

How Article Quality Works?

For each article Authors/Readers, Reviewers and WMC Editors can review/rate the articles. These ratings are used to determine Feedback Scores.

In most cases, article receive ratings in the range of 0 to 10. We calculate average of all the ratings and consider it as article quality.

Quality=Average(Authors/Readers Ratings + Reviewers Ratings + WMC Editor Ratings)