Slow controlled-rate freezing of sequentially cultured human blastocysts: an evaluation of two freezing strategies

Etienne Van den Abbeel1, Michel Camus, Greta Verheyen, Linda Van Waesberghe, Paul Devroey and André Van Steirteghem

Centre for Reproductive Medicine, Academic Hospital, Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium

1 To whom correspondence should be addressed. E-mail: Etienne.vandenabbeel{at}az.vub.ac.be


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: To optimize blastocyst cryopreservation, the prerequisite is to develop a better understanding of factors that influence their survival and implantation potential. Therefore, the aim of the present work was to evaluate, retrospectively, the outcome of blastocyst cryopreservation in a day 2/3 fresh embryo transfer programme. METHODS: Two different freezing strategies were compared: a first strategy (strategy A: 3007 blastocysts frozen) consisted of freezing those blastocysts that had at least a cavity; a second strategy (strategy B: 3831 blastocysts frozen) consisted of freezing only more advanced stage blastocysts with a good quality inner cell mass and trophectoderm. The outcome of cryopreservation, as related to the two different freezing strategies, was analysed. In addition, after freezing and thawing, we evaluated the influence of blastocyst developmental characteristics on immediate morphological survival and further development in vitro. RESULTS: The immediate morphological survival after thawing was higher for early blastocysts as compared to advanced and hatching blastocysts. The further developmental potential in vitro of thawed blastocysts was higher for advanced and hatching blastocysts as compared to early blastocysts. As a result, the percentage of deliveries, calculated as a percentage of started thawing cycle, and the percentage of children born, calculated as a percentage of embryos transferred, was not different for strategies A and B. CONCLUSION: The results clearly indicate that culture conditions and cryopreservation procedures of blastocysts need to be further improved.

Key words: blastocysts/cryopreservation/IVF/sequential culture/slow controlled-rate freezing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
For some years, it has become accepted procedure to transfer more than one embryo to the patient in order to obtain acceptable ongoing pregnancy rates. However, the transfer of more than one embryo results in the probability of establishing multiple gestations that are at increased risk for adverse outcomes. The objective of IVF, therefore, should be to provide couples with a single, healthy baby per transfer and more than one healthy baby per treatment cycle (= delayed twin). To accomplish this goal, single embryo transfer (SET) is becoming an increasingly accepted procedure in several countries (Gerris et al., 2003Go). One benefit of SET is an increase in the number of embryos available for cryopreservation (De Neubourg et al., 2002Go), and optimized cryopreservation of embryos after SET is part of the strategy to decrease multiple pregnancies (Gerris et al., 2003Go).

There is much debate as to the developmental stage at which human embryos are best cryopreserved (Veeck, 2003Go; Ménézo, 2004Go). The disadvantage of two-pronucleate (2PN) stage embryos is that nothing is known about their developmental competence. A major complication of cleavage stage embryos on the other hand is that, after thawing, damaged blastomeres often coexist with intact ones and it has been demonstrated convincingly that the implantation potential of such embryos is much lower than that of fully intact ones (Van den Abbeel et al., 1997Go; Edgar et al., 2000Go). Blastocysts have the advantage of containing numerous small cells; thus, the loss of some cells during freezing and thawing is probably less harmful for the further development of the embryo. Furthermore, after extended culture up to the blastocyst stage, embryos with reduced viability will arrest in development and will not be cryopreserved (Alikani et al., 2000Go). Moreover, Gardner and Lane (2003)Go stated that single blastocyst transfer is the best option for some patients because of the high implantation potential of fresh blastocysts. The cryopreservation of blastocysts may be considered as a way of improving the cryopreservation programme.

Cryopreservation of human blastocyst was first reported by Cohen et al. (1985)Go. It was abandoned for years because of suboptimal culture conditions to reach the blastocyst stage. In the 1990s, co-culture on feeder cells (Vero) allowed the development of good quality blastocysts in vitro. After freezing and thawing such blastocysts, live birth rates per transferred blastocyst were ~13% (Kaufman, 1995Go). Nowadays, blastocysts are successfully cultured using sequential media formulations, and their implantation potential has been reported to be very high (Gardner et al., 2000Go). These authors also reported that blastocysts could be successfully cryopreserved, but the implantation potential of frozen–thawed blastocysts was well below that for fresh blastocysts.

In several mammalian species, the success of cryopreservation of blastocysts is known to depend on the developmental characteristics of frozen blastocysts. For mouse blastocysts, better results are obtained for early blastocysts than for expanded blastocysts (Massip et al., 1984Go), while for the ovine and the bovine better results are obtained with expanded blastocysts as compared to morulae and early blastocysts (Cocero et al., 1996Go; Hochi et al., 1996Go). Furthermore, in vitro culture conditions are known to have an important impact on the cryotolerance of mammalian embryos (Leibo and Loskutoff, 1993Go; Massip et al., 1995Go; Abe et al., 2002Go). Interestingly, Mandelbaum and Ménézo (2001)Go reported that there were differences in cryotolerance between human blastocysts obtained by co-culture or sequential culture systems (take home baby rate per frozen blastocysts transferred: 10.9% for co-cultured blastocysts versus 5% for sequentially cultured blastocysts).

To optimize blastocyst cryopreservation, the prerequisite is to develop a better understanding of factors that influence their survival and implantation potential.

Therefore, the aim of the present work was to evaluate retrospectively the outcome of blastocyst cryopreservation in a day 2/3 fresh embryo transfer programme. Two different freezing strategies were compared: a first strategy consisted of freezing those blastocysts which had at least a cavity, and a second strategy consisted of freezing more advanced stage blastocysts that had a good quality inner cell mass (ICM) and trophectoderm. The outcome of cryopreservation as related to the different freezing strategies will be analysed. Moreover, after freezing and thawing, we evaluated the influence of blastocyst developmental characteristics, such as blastocyst developmental stage and rate, on immediate morphological survival and further development in vitro.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study period and oocyte collection cycle (OCC) specifications
In a day 2/3 embryo transfer programme, the cryopreservation of supernumerary embryos frozen at the blastocyst stage on day 5 and 6 was evaluated retrospectively. The freezing and thawing outcome was evaluated over a 3 year period, from October 1, 1999 to December 31, 2002. We evaluated 4875 cycles with transfer of embryos in the OCC; 1157 were conventional IVF cycles (25.7%) and 3718 were IVF cycles in association with ICSI (74.3%). The mean age of the female patients was 33.8 years (33.9 and 33.8 for IVF and ICSI cycles respectively) and the mean number of oocytes retrieved was 10.2 (9.5 and 10.4 for IVF and ICSI cycles respectively).

Ovarian stimulation, oocyte retrieval and in vitro culture
Female patients underwent ovarian stimulation using urinary or recombinant FSH in combination with GnRH antagonist or agonist. Oocyte retrieval was carried out 36 h after HCG injection by vaginal ultrasound-guided puncture of ovarian follicles (Kolibianakis et al., 2004Go). Cumulus–oocyte complexes (COCs) were used for IVF or ICSI treatment. IVF treatment was predominantly applied for patients with tubal or idiopathic infertility indications, while ICSI was predominantly performed in male factor indications. Sperm preparations for IVF and ICSI, and the IVF and ICSI procedure were performed as described in detail by Van Landuyt et al. (2005)Go and Devroey and Van Steirteghem (2004)Go.

Oocytes and embryos were cultured in sequential culture media preparations (Cook or Vitrolife medium formulations) from the day of oocyte retrieval until day 6 of embryo culture. One single oocyte or embryo was placed in a 25 µl droplet of culture medium under equilibrated mineral oil. In vitro culture was done in incubators at 37°C in a humidified atmosphere of 6% CO2, 5% O2 and 89% N2.

Embryos for transfer and for freezing
Fertilization was checked 16–19 h post-microinjection or post-insemination. Normal fertilization was considered as the number of oocytes with two clearly visible pronuclei present. In the late afternoon of day 1, zygotes were checked a second time to assess the number of early cleaving 2-cell embryos. Embryos were further evaluated daily until day 6 of embryo culture.

Intrauterine embryo replacement was carried out on day 2/3 of the OCC. For embryo evaluation on day 2/3, the developmental stage was recorded and embryos were classified into categories A, B and C according to the percentage of fragmentation (Van Landuyt et al., 2005Go). Two or three (occasionally more than three) of the best quality available embryos were transferred during the OCC. All supernumerary embryos were transferred to blastocyst sequential culture medium (Cook or Vitrolife) on the morning of day 3 and further cultured until day 6.

On day 5 and 6, embryos were scored as compacting (compaction has started but is not yet fully completed as cell borders are still visible), compact (cell borders have disappeared completely) or blastocyst according to criteria described by Gardner et al. (2000)Go.

During a first period, from 1 October 1999 to 31 December 2000 (1942 OCC; 26.9% IVF cycles and 73.1% ICSI cycles), the strategy of freezing blastocysts (strategy A) consisted of freezing on day 5/6 early blastocysts showing some cavitation (Gardner type 1 and 2), advanced blastocysts with at least type B ICM and type B trophectoderm (Gardner type 3 and 4) and hatching blastocysts (Gardner type 5). Occasionally, some compacted or ill-defined (no ICM visible or doubtful ICM quality) embryos were also cryopreserved.

During a second period, from 1 January 2001 to 30 September 2002 (2933 OCC; 24.9% IVF cycles and 75.1% ICSI cycles), the strategy of freezing blastocysts (strategy B) consisted of freezing on day 5/6 advanced blastocysts (Gardner type 3 and 4) and hatching blastocysts (Gardner type 5) with at least type B ICM and type B trophectoderm quality.

Blastocyst cryopreservation procedure
The freezing and thawing solutions were always made up in HEPES-buffered Earle’s medium supplemented with 0.5% w/v human serum albumin (HSA; Belgian Red Cross, Brussels, Belgium), referred to hereafter as HEPES medium. Glycerol (G2025) and sucrose (S1888) were from Sigma. The freezing and thawing procedure was adapted from Ménézo et al. (1995). A schematic presentation of the freezing and thawing protocol is shown in Figures 1 and 2. Blastocysts for freezing were removed from the culture dish and placed in 0.5 ml 37°C warm HEPES medium, using a 4-well Nunc plate. When all blastocysts to be frozen were removed from the culture dishes, they were further equilibrated for 10 min at 22°C. The embryos were transferred from the HEPES medium and placed into the second well of the Nunc plates for 10 min at 22°C containing 0.5 ml HEPES medium and 5% v/v glycerol. Subsequently, the embryos were transferred to the third well of the Nunc plate for 10 min at 22°C containing 0.5 ml of HEPES medium, 9% v/v glycerol and 0.2 mol/l sucrose, which will be further referred to as freezing medium. During this equilibration step, the embryos were loaded in properly labelled 0.25 ml plastic mini-straws (IMV, paillette souple, CA006432A481; L’Air Liquide, Machelen, Belgium). The straws were loaded as follows: straws were first rinsed by aspirating freezing medium and expelling it immediately. Thereafter, using the marked sample straw as a reference, aspiration of a 3 cm freezing medium column was performed followed by a 5 mm air bubble. In turn, a 4 cm column of freezing medium also containing up to two blastocysts was aspirated followed by a 5 mm air bubble. Finally, freezing medium was aspirated until activation of the cotton plug of the straw. Up to two (occasionally three) blastocysts were loaded per straw. Straws were subsequently sealed with humidified polyvinylalcohol powder and placed into the biological freezer (minicool AS100; L’Air Liquide) previously set at 22°C. After the 10 min equilibration period, the temperature in the minicool was lowered to –7°C at 2°C/min, at which temperature a 10 min pre-seeding hold was programmed. Seeding was then performed manually by touching the straws with a liquid nitrogen (LN2) cold forceps at the level of a 5 mm air bubble. The temperature was then lowered to –37°C at 0.3°C/min, to –150°C at 50°C/min, and straws were then plunged into LN2. Straws were stored in LN2-filled containers (GT 40, L’Air Liquide).



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Figure 1. Schematic presentation of freezing protocol.

 


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Figure 2. Schematic presentation of thawing protocol.

 

For thawing, the straws were removed from LN2, kept at 22°C for 20 s and then shaken in a 30°C warm water bath for another 20 s. The contents of a straw were expelled into the first well of a 4-well Nunc plate containing 0.5 ml HEPES medium and 0.5 mol/l sucrose, and kept for 10 min at 22°C. Using a finely pulled glass pipette the blastocysts were picked up and placed consecutively into each of the three other wells for 10 min at 22°C containing either 0.5 ml HEPES medium and 0.2 mol/l sucrose, 0.5 ml HEPES medium and 0.1 mol/l sucrose, and finally 0.5 ml HEPES medium without sucrose. After this, the HEPES medium with the blastocysts was allowed to warm up gradually to 37°C for 10 min. After two further wash steps in 37°C warm HEPES medium, all thawed blastocysts were placed in blastocyst medium (Cook or Vitrolife), and further cultured before transfer.

If available, two blastocysts in excess of the number that had been decided to transfer were thawed the day before embryo replacement.

Evaluation of thawing and transfer of frozen–thawed embryo
Thawing was evaluated until 31 December 2002. Blastocysts were scored immediately after thawing and dilution of the cryoprotectant, and again after post-thaw in vitro culture.

Immediately after thawing, four types of blastocyst were recorded: fully intact blastocysts with or without blastocoelic cavity (type A); moderately damaged blastocysts with or without blastocoelic cavity (type B); severely damaged blastocysts with or without blastocoelic cavity (type C); and completely degenerated blastocysts (type D). After further culture in blastocyst medium the blastocysts were evaluated for expansion or re-expansion, and for the quality of the ICM and trophectoderm. Only blastocysts with type A or B survival immediately after thawing (= immediate morphological survival) and showing expansion or re-expansion as well as an ‘apparent’ good quality ICM and trophectoderm (Gardner type A or B quality) after post-thaw in vitro culture (= embryos suitable for transfer) were considered for transfer. If only frozen day 5 blastocysts were available for a patient, surviving blastocysts were cultured overnight and transferred if suitable for transfer. If only frozen day 6 blastocysts were available, blastocysts were thawed on the day of transfer. Surviving blastocysts were cultured for 4–8 h and transferred if suitable. If frozen day 5 as well as day 6 blastocysts were available, first day 5 embryos were thawed. Surviving day 5 embryos were then cultured overnight and transferred if suitable. If their quality was not sufficient, then day 6 embryos were thawed and transferred after 4–8 h, if suitable. For most thawing cycles, the endometrium was prepared during a natural cycle. In some patients this occurred by hormonal substitution as described by Kolibianakis et al. (2003)Go.

Embryo transfer occurred mostly asynchronously, the blastocyst for transfer being 1 day older than the endometrium.

Characterization of OCC
Cycle cryopreservation rate: number of cycles where at least one embryo was frozen/number of transfer cycles in OCC. Embryo transfer rate: number of embryos transferred in OCC/number of 2PN embryos in OCC. Blastocyst formation rate: number of blastocysts formed/number of 2PN embryos in OCC – embryos transferred in OCC. Embryo cryopreservation rate: number of embryos cryopreserved/number of 2PN embryos in OCC (Jones et al., 1997Go).

Statistical analysis
To study the relationship between two classification factors, results were analysed by {chi}2-test. If the table has two rows and three or more columns, and if there is a meaningful order in the column categories, then an exact {chi}2-test for trend was performed. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Characteristics of OCC as related to the freezing strategy (Table I)


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Table I. Characteristics of oocyte collection cycles (OCC) as related to the freezing strategy

 
There was no difference between strategies A (freezing those blastocysts which had at least a cavity) and B (freezing more advanced blastocysts that had a good quality ICM and trophectoderm) in cycle cryopreservation rate, fresh embryo transfer rate and total blastocyst formation rate. However, the embryo cryopreservation rate expressed as the number of blastocysts frozen per 2PN embryos obtained was, as expected, higher during strategy A (24.8%) as compared to strategy B (19.1%) (P < 0.0001).

Characteristics of OCC: developmental characteristics of frozen human blastocysts
On day 5 of the OCC there was no difference in the distribution of the different stages of blastocysts formed between strategies A and B. As expected, there was a difference in the distribution of the different stages of blastocysts frozen between strategies A and B. During strategy A, the percentages of early blastocysts, advanced blastocysts, hatching blastocysts, and of poorly characterized embryos frozen were 33, 48, 7 and 11% respectively. During strategy B, the percentages of advanced blastocysts and of hatching blastocysts frozen were 73 and 27% respectively (comparison between strategies A and B: P < 0.0001).

During strategy A the majority of blastocysts were frozen on day 5 of the OCC (86.5%) while during strategy B a substantial percentage of blastocysts were also frozen on day 6 of the OCC (48.5%) (P < 0.0001 between strategy A and B).

Blastocyst immediate morphological survival and further development in vitro as related to blastocyst developmental characteristics (Tables II and III)


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Table II. Thawing characteristics in strategy A: blastocyst immediate morphological survival and further development in vitro

 

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Table III. Thawing characteristics in strategy B: blastocyst immediate morphological survival and further development in vitro

 
During strategy A (Table II), a significant trend was observed in the immediate morphological survival after thawing between early blastocysts (82.6%, 381/461), advanced blastocysts (day 5 and day 6 blastocysts combined) (71.3%, 301/422), and hatching blastocysts (56.5%, 52/92) (P < 0.0001). The percentage of embryos suitable for transfer as a percentage of the immediate morphological survival, on the other hand, was higher for advanced (74.8%, 225/301 surviving embryos) as compared to hatching blastocysts (63.5%, 33/52 surviving embryos) and early blastocysts (47.0%, 179/381 surviving embryos) (P < 0.0001). Overall, the percentage of embryos suitable for transfer as a percentage of embryos thawed was higher for advanced blastocysts (53.3%, 225/422 embryos thawed) as compared to early blastocysts (38.8%, 179/461 embryos thawed) and as compared to hatching blastocysts (35.9%, 33/92 embryos thawed) (P < 0.0001).

During strategy B (Table III), the immediate morphological survival after thawing and the number of embryos suitable for transfer was not different for advanced versus hatching blastocysts. However, during strategies B and A, there was a difference depending on whether blastocysts were frozen on day 5 or day 6 of the OCC; the percentage of immediate morphological survival and the percentage of embryos suitable for transfer were higher when the blastocysts were frozen on day 5 as compared to day 6 of the OCC (P < 0.0001).

Outcome of frozen embryo transfer (Table IV)


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Table IV. Outcome of frozen embryo transfer

 
The percentage of frozen embryo transfer and of deliveries as a percentage of thawing cycles was not significantly different for strategies A and B. Furthermore, the percentage of children born, calculated as a percentage of blastocysts transferred, was not different for strategies A and B.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The aim of the present work was to evaluate retrospectively the outcome of human blastocyst cryopreservation in a day 2/3 fresh embryo transfer programme. In total, and divided over two different freezing strategies, we analysed the freezing of 6838 blastocysts from which 2480 were thawed. After thawing, 1095 blastocysts were transferred in 622 transfer cycles. To the best of our knowledge, this represents the broadest study of human blastocyst cryopreservation.

Characteristics of OCC
The blastocyst cryopreservation rate was higher during strategy A (24.8%) as compared to strategy B (19.1%). This was simply because, during strategy B, only blastocysts with a clear visible ICM were frozen, while during strategy A early blastocysts were also frozen; in other words, there was a stronger selection of the blastocysts for freezing during strategy B as compared to strategy A.

The blastocyst formation rate was ~50% and was not different for strategies A and B. This total blastocyst formation rate is comparable to that described in the literature (Gardner et al., 2000Go).

The overall blastocyst cryopreservation rate (strategy A and B combined) was 21.3%. This value is significantly lower than that obtained for day 2/3 freezing (Van den Abbeel, 2000Go), and is indicative of the better selection of embryos for freezing on day 5/6 (Blake et al., 2004Go). The embryo cryopreservation rate is important because this allows for a comparison between centres (Jones et al., 1997Go). In most papers on the cryopreservation of human embryos, this information is lacking; as a result, the data cannot be correctly interpreted. It is evident that centres with strong selection criteria for embryo freezing (low embryo cryopreservation rate) must have better cryopreservation outcomes than centres that are less restrictive in the embryos for freezing (high embryo cryopreservation rate). The number of embryos frozen is an important consideration when assessing the effectiveness of a treatment, because it offers couples an additional opportunity to achieve a pregnancy from the same OCC. So a disadvantage of fewer embryos being cryopreserved as a result of stronger selection must be weighed against a substantial improvement in the cryopreservation results (Mandelbaum and Ménézo, 2001Go; Blake et al., 2004Go).

Influence of developmental characteristics of blastocysts on immediate morphological survival after thawing and on further development in vitro
The results after thawing clearly demonstrated that developmental characteristics of blastocysts influenced the freezing–thawing outcome. The immediate morphological survival after thawing was highest for early blastocysts. The further developmental capacity in vitro, however, was higher for advanced and hatching blastocysts. Furthermore, immediate morphological survival and further development in vitro were dependent on the day of freezing in the OCC. The results were significantly better for blastocysts frozen on day 5 in the OCC as compared to day 6.

From these results it can be concluded that the size of the blastocoelic cavity influences the immediate morphological survival of human blastocysts. This is in accordance with observations in mouse blastocysts (Massip et al., 1984Go), but is in conflict with observations in bovine and ovine (Cocero et al., 1995; Hochi et al., 1996Go). Interestingly, Vander Zwalmen et al. (2002)Go mentioned that an artificial shrinkage of the blastocoelic cavity significantly improves the vitrification results of human advanced blastocysts. Our own observations as well as those by Van der Zwalmen et al. (2002)Go may indicate that the risk of detrimental ice crystal formation in human blastocysts is higher for fully expanded blastocysts than for early cavitating blastocysts. The evaluation of blastocyst immediate morphological survival is difficult and highly subjective. Some centres describe blastocyst survival as they do for cleavage stage embryos (>50% of cells surviving). In our experience, it is impossible to quantify immediate morphological survival and predict developmental competence. Therefore, more blastocysts than had been decided to transfer were thawed, and surviving day 5 blastocyst were cultured overnight so as to achieve a better appreciation of their quality before transfer (Guérif et al., 2003Go). Interestingly, Veiga et al. (2003)Go showed that the synchrony between embryo and endometrium is not affected if blastocysts are thawed the day before the predicted time of transfer. Day 6 surviving blastocysts, however, were cultured for only 4–8 h after thawing to avoid culture in vitro up to day 7 of the OCC. Consequently, some day 6 blastocysts remained collapsed, thus making it difficult to evaluate their developmental competence.

After further culture, the advantage of a better immediate morphological survival of early blastocysts as compared to advanced blastocysts was completely lost due to the lower developmental capacity of frozen–thawed early blastocysts. In early blastocysts there is no pre-freezing indication of a subsequent formation of an ICM or healthy trophectoderm. It may be hypothesized that some early blastocysts frozen during strategy A would have been excluded for freezing in strategy B because of suboptimal development of the ICM and/or the trophectoderm. Furthermore, it might be argued that early blastocysts on day 5 of the OCC are somewhat retarded as compared to advanced blastocysts on day 5 (Gardner et al., 2000Go). Retarded embryos are known to have a suboptimal viability (Shoukir et al., 1998Go); therefore the capacity of retarded blastocysts to sustain the freezing–thawing stress might be inferior as compared to normally developing blastocysts. This argument is sustained by observations by Shapiro et al. (2001)Go, who concluded that day 5 transfer of expanded blastocysts is more successful than transfer (on day 6) of blastocysts for which expansion has been delayed until day 6. Our results and the observations by Shapiro et al. (2001)Go, however, contradict observations by Veeck et al. (2004)Go, who found no implantation differences, whether blastocysts were frozen on day 5 or day 6 after retrieval. A possible explanation for this difference might be that our culture conditions are not fully optimal for sustained growth of embryos up to day 5 and day 6, leading to a higher susceptibility of cultured blastocyts to freezing–thawing damage. Our results clearly indicate that advanced or hatching blastocysts obtained on day 5 of the OCC have the highest chance of surviving cryopreservation and developing further in vitro. Gardner et al. (2000)Go reported that in a day 5 embryo transfer programme and under optimal culture conditions (group culture, several culture medium renewal steps), >90% of good quality blastocysts were obtained on day 5 of the OCC; in the present study and during strategy B, this percentage is only 52%. Suboptimal culture conditions might have influenced blastocyst susceptibility to the freezing and thawing process.

Further research should be conducted to obtain better quality and cryotolerant human blastocysts, to develop optimized slow-controlled rate cryopreservation procedures (Gardner et al., 2003Go), and to investigate whether vitrification is a feasible option for the cryopreservation of blastocysts in order to obtain better survival and implantation rates (Mukaida et al., 2003Go).

Outcome of frozen blastocyst transfer
Overall, the outcome of frozen–thawed blastocyst transfer could be summarized as follows: we obtained a delivery rate of 11.0% per thawing cycle; of 15.1% per frozen transfer; and the implantation potential expressed as the number of children born per frozen–thawed blastocyst transferred was 10.3%.

In selected patient populations, and using sequential culture systems, several authors reported high implantation rates of fresh or frozen blastocysts. However, the implantation potential of frozen–thawed blastocysts varied considerably between centres, and was mostly lower than the implantation potential of fresh blastocysts (Gardner et al., 2000Go; Langley et al., 2001Go; Mandelbaum and Ménézo, 2001Go; Pantos, 2001Go; Behr et al., 2002Go; Anderson, 2003Go; Virant-Klun et al., 2003Go; Guérif et al., 2003Go; Ding, 2004Go; Veeck et al., 2004Go). Typically, delivery rates (or ongoing pregnancy rates) varied between 12.8 and 50.2%, and the implantation potential (children or sacs per blastocyst transferred) varied between 5.3 and 38.6%. It is difficult to compare between centres, because essential information on OCC characteristics is often lacking.

When comparing day 5/6 freezing results (this paper) with previously published results from our centre on the cryopreservation of day 2/3 embryos (Van den Abbeel et al., 2000Go), it is clear that despite a better selection of the embryos frozen in a day 5/6 freezing programme, the outcome after thawing and transfer is not substantially enhanced.

In conclusion, despite important differences between strategies A and B in the distribution of the different stages of blastocysts frozen, and despite important differences in cryotolerance between these blastocysts, the outcome expressed as delivery rate per started thawing cycle and the number of children born per frozen–thawed blastocyst transferred were not different for strategies A and B. The benefit of a better morphological survival for early blastocysts during strategy A was lost as a result of their low developmental capacity. The benefit of freezing advanced blastocysts with clearly visible ICM and good quality trophectoderm was lost because a substantial number of blastocysts demonstrated retardation in development as many advanced blastocysts were only obtained and frozen on day 6 of the OCC.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors acknowledge the expertise of clinical embryologists and laboratory technologists of the Centre for Reproductive Medicine, AZ-VUB (Brussels, Belgium). We also wish to thank Hubert Joris and Walter Meul for assistance with data collection and analysis. The authors also thank Michael Withburn of the Language Education Centre (Dutch-speaking Brussels Free University) for correcting the manuscript. This work was supported by Grants from the ‘Fonds voor Wetenschappelijk Onderzoek – Vlaanderen’ (G.0470.99 and G.0375.03).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on April 5, 2005; resubmitted on May 4, 2005; accepted on May 12, 2005.