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Hum. Reprod. Advance Access originally published online on March 3, 2005
Human Reproduction 2005 20(6):1615-1618; doi:10.1093/humrep/deh808
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© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions{at}oupjournals.org

Comparison of the survival of human biopsied embryos after cryopreservation with four different methods using non-transferable embryos

W.T. Zheng1,2, G.L. Zhuang1, C.Q. Zhou1, C. Fang1, J.P. Ou1, T. Li1, M.F. Zhang1 and X.Y. Liang1

1 Center for Reproductive Medicine, Department of Obstetrics and Gynecology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China

2 To whom correspondence should be addressed. Email: sunnnyjenny{at}yahoo.com.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The standard embryo cryopreservation method is still less than optimal for biopsied embryos. The aim of this study was to compare the survival of biopsied embryos cryopreserved with four different methods using non-transferable embryos. METHODS: Abnormal embryos from one or three pronuclei and spare embryos of grade 3 and 4 were used for this study. Non-biopsied embryos were cryopreserved using the standard method as control. Biopsied embryos were cryopreserved using four methods as follows: standard method, modified freezing method, modified thawing method and vitrification. Blastomere survival and blastulation of frozen–thawed embryos were compared between the different methods. RESULTS: The proportion of embryos with ≥50% blastomere survival and total blastomere survival rate of biopsied embryos were significantly higher with vitrification than the other three methods. Both the modified freezing and modified thawing methods had significantly higher embryo survival and total blastomere survival rates than standard methods. However, there was no significant difference in blastulation of surviving embryos in all the five groups. CONCLUSIONS: Non-transferable embryos derived from clinical IVF/ICSI are useful for evaluation of the optimal freezing procedures for biopsied embryos. Vitrification increases the survival rate of human biopsied embryos above standard and modified cryopreservation methods.

Key words: biopsied embryo/cryopreservation/vitrification


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Preimplantation genetic diagnosis (PGD) has now been widely used for screening abnormal embryos in couples with a high risk of genetic disease especially for single gene disorders (Verlinsky et al., 2004Go). In some clinics, routine PGD has been used to screen all embryos to be able to transfer only chromosomally normal embryos to improve implantation and pregnancy rates in couples with previous persistent failure of implantation (Geraedts et al., 2000Go). Thus cryopreservation of biopsied embryos will be important for routine PGD since many couples will have some excessive biopsied embryos to be stored frozen for subsequent transfers. However, the survival rate of biopsied human embryos is significantly lower than non-biopsied embryos when conventional cryopreservation methods are used (Joris et al., 1999Go; Magli, 1999Go; Ciotti et al., 2000Go). In a recent report by Jericho et al. (2003)Go, it was shown that a modified cryopreservation method with increased sucrose concentration up to 0.2 mol/l could significantly improve the survival rate of biopsied embryos compared with standard freezing methods. Another alternative method is vitrification which has been reported to be a simple, low cost method which is efficient for cryopreservation of mammalian and human oocytes, embryos and blastocysts (Kasai et al., 1996Go; Kasai and Mukaida, 2004Go; Liebermann, 2004Go). However, in the current literature, it is still unclear which cryopreservation method is optimal for biopsied human embryos. Therefore, we performed this study to compare survival of human biopsied embryos after cryopreservation with four different methods including vitrification using non-transferable embryos obtained from clinical IVF/ICSI.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Human embryos
On the day of embryo transfer (day 3), the embryos were scored according to our grading system. This system consists of four main embryo morphological grades, where a developmental rate of >6 blastomeres (cells) on day 3 is considered normal. Grade I is considered the ‘optimal’ embryo, with no fragments, even sized blastomeres and light, homogeneous cytoplasm. Grade II is divided into three categories: <20% fragments, uneven sized blastomeres and/or non-homogeneous cytoplasm. The remaining embryos fall into the third and fourth grade groups. These poor embryos are only rarely used for transfer and never frozen, and will hereafter be discarded. Also IVF occasionally produces abnormal embryos from zygotes with one or three pronuclei (PN) which routinely are also discarded. With the consent of patients, 256 discarded embryos were included in this study (46 from 3 PN, four from 1 PN and 206 of poor quality, with ≥4 cells, ≤12 even sized cells and ≤30% fragmentation) The embryos were randomly allocated to five groups: standard programmed slow cryopreservation without biopsy (group I), standard programmed slow cryopreservation after biopsy (group II), a modified cryopreservation method (Jericho et al., 2003Go) after biopsy (group III), programmed slow cryopreservation with modified thawing steps after biopsy (group IV), and vitrification after biopsy (group V).

Embryo biopsy
Partial zona dissection (PZD) was carried out by creating an opening in the zona pellucida mechanically. Compacted embryos were incubated for 5 min in Ca2 + - and Mg2 + -free medium (SAGE, BioPharma, USA) before PZD. One blastomere was removed from the opening of each embryo, and the fragments were also removed as much as possible. After biopsy, the embryos were incubated for 6–8 h before freezing with the four methods described below. Frozen embryos were stored in liquid nitrogen for 1–7 days before thawing. All the freezing and thawing fluids were based on phosphate-buffered saline (PBS; Sigma St Louis, MO) supplemented with 20% (v/v) inactivated fetal bovine serum (FBS, Si Ji Qing Company, China).

Embryo cryopreservation
Standard freezing method. Embryos were equilibrated in 1.5 mol/l PROH (1, 2-propanediol; Sigma) for 10 min at room temperature. Then they were transferred to 1.5 mol/l PROH with 0.1 mol/l sucrose (Sigma) and loaded into plastic straws. Cooling was conducted in a programmable freezer (Kryo 10 Series, Planar Products, Sunbury Thames, UK) at a rate of –2 °C/min from room temperature to –7 °C, at which point seeding was carried out manually. Cooling was then continued at rates of –0.3 °C/min to –30 °C and –50 °C/min to –150 °C before plunging and storing in liquid nitrogen. For the thawing process, the straw was removed from storage, held in the air for 30 s and immersed in a water bath at 30 °C for 30–40 s. In the dilution procedure, the embryos were serially transferred into 1.0 mol/l PROH with 0.2 mol/l sucrose, 0.5 mol/l PROH with 0.2 mol/l sucrose and 0.2 mol/l sucrose for 5 min each, followed by sucrose-free medium at room temperature for 10 min.

Modified freezing method. The modified freezing method of Jericho et al. (2003)Go was used: briefly the concentration of PROH in the freezing solutions remained at 1.5 mol/l, but the sucrose concentration was doubled to 0.2 mol/l. The concentration of sucrose was also increased up to 0.3 mol/l during the first thawing steps. Thawed embryos were rehydrated by sequential transfer to 0.75 mol/l PROH + 0.3 mol/l sucrose (5 min), 0.3 mol/l sucrose (5 min) and 0.2 mol/l sucrose (10 min). Rehydration was completed by transfer to sucrose-free HEPES-buffered HTF medium (10 min).

Modified thawing method. Based on the standard freezing method, modification was made only in the thawing procedure as follows: the sucrose concentration of the warming media was increased to 1.0 mol/l, and the solution was kept heated (37 °C) during the warming procedure. The straw was held vertically, just above the surface of the warming medium after being taken out from the liquid nitrogen, and the two ends were cut off. The ice content of the straw would slip in seconds into the 37 °C medium with 1.0 mol/l sucrose. The embryos were kept in the warm media for 30 s altogether and were serially transferred into 0.5, 0.4, 0.3, 0.2, 0.1 and 0 mol/l sucrose at room temperature (5 min each).

Vitrification. Biopsied embryos were pre-equilibrated for 3 min in 10% ethylene glycol (EG; Sigma) at room temperature. Embryos were then placed for the final equilibration in 30% EG with 0.5 mol/l sucrose for 30 s. In the meantime, one to three embryos were mounted on the tip of a Cryotop (Kitazato Ltd, Tokyo, Japan) and excessive medium was sucked away using a fine pipette. The Cryotops containing embryos were immediately plunged into liquid nitrogen, and a Cryocap was used to cover the tip of the Cryotop. For the thawing process, after removing the Cryocap, the Cryotop was quickly immersed in 1 mol/l sucrose at 37 °C for 10–20 s. Then the embryos were transferred to 1.0 mol/l sucrose for 1 min at room temperature and subsequently to 0.5, 0.4, 0.3, 0.2, 0.1 and 0 mol/l of sucrose at intervals of 5 min at room temperature.

Assessment of the viability of frozen–thawed embryos
After thawing, embryos were placed in 10% SSS HTF-cleavage medium (SAGE, BioPharma, USA) under mineral oil at 37 °C in a humidified atmosphere of 5% CO2 in air. After 2 h incubation, embryos were assessed to determine their integrity and the number of surviving blastomeres. Embryos with half or more blastomeres intact after thawing were defined as surviving embryos and those with all blastomeres fully intact were defined as intact embryos. Therefore, the survival rate was the percentage of embryos that have ≥50% intact blastomeres after thawing. Surviving embryos were incubated further for 3 days to assess the blastulation potential.

Statistical analysis
The {chi}2 test was used to compare results for embryo survival, total blastomere survival and blastulation among five groups. Statistical significance was defined as a P-value <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Table I shows the proportion of embryos with >50% of the original blastomeres that survived, and the total blastomere survival rate of biopsied embryos was significantly higher (P<0.001) with vitrification than all the other three methods. Therefore, vitrification produced the highest survival rates (94%), intact embryos (80%) and total blastomere survival rate (90%) for biopsied embryos. Both modified freezing (75 and 63%) and modified thawing (76 and 55%) methods also had significantly (P<0.001) higher embryo survival and total blastomere survival rates than the standard method (16 and 25%). Both survival rate and total blastomere survival rate of biopsied embryos with the modified freezing and thawing methods were very similar to non-biopsied embryos with the standard method (85 and 71%). There was no statistically significant difference in the total blastomere survival rate between the modified freezing and modified thawing methods (P>0.05).


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  		Table I. Survival rate of embryos post-freezing–thawing with different methods

 
The results for blastulation of those biopsied and non-biopsied embryos that survived the freezing–thawing process are summarized in Table II. There was no statistically significant difference (P>0.05) in the percentage of blastocysts present in biopsied embryos compared with the non-biopsied group (24%), standard freezing group (13%), modified freezing group (31%), modified thawing group (18%) and vitrification group (20%).


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  		Table II. Results of blastulation of surviving embryos post-freezing–thawing with different methods

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
It has become clear from a number of studies that blastomere loss in frozen–thawed early cleavage stage embryos is associated with a reduction in implantation potential. Frozen embryo transfer (FET) cycles in which all embryos transferred remain fully intact at thawing achieve a better outcome than those in which at least one partially damaged embryo is transferred (Edgar, 2000Go; Guerif et al., 2002Go; El-Toukhy et al., 2003Go). The present study shows clearly that standard slow freezing methods are not optimal for biopsied human embryos. Non-transferable (poor or abnormal) embryos derived from clinical IVF/ICSI are useful for evaluation of optimal procedures for cryopreservation of biopsied embryos. The data in this study showed that vitrification produced the highest (95% embryos with >50% blastomere survival and 80% intact embryos) survival of biopsied embryos of all the methods investigated (standard, modified freezing and modified thawing). However, blastulation rates of surviving biopsied embryos were very similar for all four methods. This result suggests that different freezing methods mainly affect the outcome of survival of embryos and not the development potential of surviving embryos (Edgar et al., 2001Go). In the literature, others have reported that vitrification is the most effective and simple method for freezing human oocytes, embryos and blastocysts (Kasai, 1997Go; Mukaida et al., 2003Go; Kasai and Mukaida, 2004Go).

In this study, we also confirmed further that the modified freezing method of Jericho et al. (2003)Go with an increased sucrose concentration to 0.2 mol/l significantly enhances the survival of biopsied embryos compared with standard freezing methods. The survival rate of biopsied embryos with modified freezing methods (75%) was very similar to the survival rate of non-biopsied embryos with standard methods (85%). Similarly, a modified thawing method with 1.0 mol/l sucrose added to the warming media also significantly increased the survival rate of biopsied embryos (76%). The latter result suggests that the thawing procedures are also important for recovery of frozen biopsied embryos.

It is obvious that standard cryopreservation with slow programmed procedures is very different from vitrification. Vitrification could be obtained by combining the high concentration cryoprotectant with high cooling and warming rates. During vitrification, there will not have been cell damage caused by ice. EG is a common cryoprotectant used for vitrification of mammalian oocytes and embryos because of its low toxicity and fast permeation of the cell compared with other cryoprotectants (Ali, 1993Go; Newton, 1998Go).

Increasing sucrose concentrations in the modified method of Jericho et al. (2003)Go appear to be beneficial to survival of frozen–thawed biopsied embryos and the further development of blastocysts of surviving embryos. Others have also reported that increasing the concentration of sucrose to 0.2 mol/l improves survival of frozen–thawed human oocytes (Fabbri et al., 2001Go; Chen et al., 2004Go; Stachecki and Cohen, 2004Go). For slow freezing programmes for embryos, the cryoprotectant generally used is PROH (membrane-permeating cryoprotectant) and sucrose (non-membrane-permeating cryoprotectant). Sucrose does not enter the cell and it mainly induces cellular dehydration through changes in osmotic pressure. Therefore, varying the sucrose concentration can change the rate of cellular dehydration to draw water out of the cells sufficiently during freezing and change the speed of cellular rehydration during warming.

Biopsied embryos have a hole on the zona pellucida, and cryoprotectant and frozen medium can freely enter the previtelline space. It is suggested that the increased susceptibility of biopsied embryos could be a consequence of both zona drilling and blastomere removal (Joris et al., 1999Go). However, others found that micromanipulation does not appear to be a major factor for reducing the cryosurvival of human embryos since embryos derived from ICSI survive cryopreservation at similar rates to those derived from conventional IVF (Ludwig et al., 1999Go). The hole on the zona pellucida after ICSI is obviously much smaller than that after biopsy and the small hole tends to close. We noted that blastomeres near to the hole of the zona pellucida were more likely to lyse after the warming of standard slow cryopreservation. It is possibly because water velocity is different among the blastomeres located close to and further away from the hole in the zona pellucida during the warming procedure. It is proposed that the zona pellucida acts as a partial water barrier to prevent blastomeres from rupturing by excessive high speed rehydration during the warming procedure. To verify the hypothesis, the cryopreservation protocols were revised accordingly. The common component of these successful modified protocols is that the concentration of sucrose of the warming media was increased to slow down the rehydration velocity. Also the cells shrink more after freezing with higher osmolality sucrose media. These factors help the cells to remain intact during the warming process.

There are limited normal spare human embryos available for methodological research to test cryopreservation methods for biopsied embryos, therefore using non-transferable embryos generated from clinical IVF/ICSI is a good alternative. Although mouse embryos have been widely used for evaluation of optimal conditions of culture medium and for frozen oocyte and embryo methods, species difference is still a major concern of validation of the methods for human embryos. Non-transferable (poor or abnormal) embryos derived from clinical IVF/ICSI still survived well with the modified protocols after biopsy.

In summary, the preliminary results of this study suggest that vitrification may be a better method for cryopreservation of biopsied human embryos. Also the modified method is better than the standard method for biopsied embryos. This preliminary finding is worthy of further investigation using normal human embryos.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The authors thank W.L.Peng, Q.Wang and Y.P.Zhong for their expert advice for this study.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Ali J and Shelton JN (1993) Design of vitrification solutions for the cryopreservation of embryos. Reprod Fertil 99, 471–477.

Chen ZJ, Li M, Li Y, Zhao LX, Tang R, Sheng Y, Gao X, Chang CH and Feng HL (2004) Effects of sucrose concentration on the developmental potential of human frozen-thawed oocytes at different stages of maturity. Hum Reprod 19, 2345–2349.[Abstract/Free Full Text]

Ciotti PM, Lagalla C, Ricco AS, Fabbri R, Forabosco A and Porcu E (2000) Micromanipulation of cryopreserved embryos and cryopreservation of micromanipulated embryos in PGD. Mol Cell Endocrinol 169, 63–67.[CrossRef][Web of Science][Medline]

Edgar DH, Bourne H, Speirs AL and McBain JC (2000) A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 15, 175–179.[Abstract/Free Full Text]

Edgar DH, Jericho H, Bourne H and McBain JC (2001) The influence of prefreeze growth rate and blastomere number on cryosurvival and subsequent implantation of human embryos. Assist Reprod Genet 18, 135–138.

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Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S and Flamigni C (2001) Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 16, 411–416.[Abstract/Free Full Text]

Geraedts J, Handyside A, Harper J, Liebaers I, Sermon K, Staessen C, Thornhill A, Viville S and Wilton L (2000) ESHRE preimplantation genetic diagnosis (PGD) consortium: data collection II (May 2000). Hum Reprod 15, 2673–2683.[Abstract/Free Full Text]

Guerif F, Bidault R, Cadoret V, Couet ML, Lansac J and Royere D (2002) Parameters guiding selection of the best embryos for transfer after cryopreservation: a reappraisal. Hum Reprod 17, 1321–1326.[Abstract/Free Full Text]

Jericho H, Wilton L, Gook DA and Edgar DH (2003) A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos. Hum Reprod 18, 568–571.[Abstract/Free Full Text]

Joris H, Van den Abbeel E, Vos AD and Van Steirteghem A (1999) Reduced survival after human embryo biopsy and subsequent cryopreservation. Hum Reprod 14, 2833–2837.[Abstract/Free Full Text]

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Kasai M (1997) Crypreservation of mammalian embryos. Mol Biotechol 7, 173–179.

Kasai M and Mukaida T (2004) Cryopreservation of animal and human embryos by vitrification. Reprod Biomed Online 9, 164–170.[Medline]

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Ludwig M, Al-Hasani S, Felberbaum R and Diedrich K (1999) New aspects of cryopreservation of oocytes and embryos in assisted reproduction and future perspectives. Hum Reprod 14 (Suppl), 162–185.[Abstract/Free Full Text]

Magli MC, Gianaroli L, Fortini D, Ferraretti AP and Munne S (1999) Impact of blastomere biopsy and cryopreservation techniques on human embryo viability. Hum Reprod 14, 770–773.[Abstract/Free Full Text]

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Submitted on November 9, 2004; resubmitted on January 6, 2005; accepted on January 19, 2005.


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