Zona pellucida damage to human embryos after cryopreservation and the consequences for their blastomere survival and in-vitro viability

Etienne Van den Abbeel1 and André Van Steirteghem

Centre for Reproductive Medicine, University Hospital and Medical School, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Brussels, Belgium


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study objective was to quantify zona pellucida (ZP) damage in cryopreserved human embryos. The influence of two different freezing containers was investigated, and the influence of freezing damage on the survival and viability of the embryos evaluated. ZP damage did not differ according to whether embryos originated from in-vitro fertilization (IVF) cycles or from IVF cycles in association with intracytoplasmic sperm injection (ICSI). The freezing container, however, significantly influenced the occurrence of ZP damage after cryopreservation. More damage was observed when the embryos were frozen–thawed using plastic cryovials than using plastic mini-straws (16.6% versus 2.3%; P < 0.0001). A clear association was found between blastomere survival and ZP intactness. Consequently, the percentage of embryos with 100% blastomere survival was higher when embryos were frozen–thawed using plastic mini-straws. The further cleavage of frozen–thawed embryos suitable for transfer was not different whether there was ZP damage or not; however, it was higher when there was 100% blastomere survival as compared with when some blastomeres were damaged (79.0% versus 43.7%; P < 0.0001). Consequently, more embryos suitable for transfer cleaved further when they were frozen–thawed using plastic mini-straws. In conclusion, the aim of a cryopreservation programme should be to have as many fully intact embryos as possible after thawing. Increased ZP damage might indicate a suboptimal cryopreservation procedure.

Key words: cryopreservation/damage/in-vitro viability/survival/zona pellucida


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cryopreservation of supernumerary embryos is nowadays a well-accepted procedure in human assisted reproduction programmes. However, it has been shown that not all human embryos survive the cryopreservation procedure (Wood, 1997Go). Cryopreservation damage to mammalian embryos can be induced by several factors, including intracellular ice formation, solution effects, osmotic effects, or physical damage by growing ice crystals at the advancing ice front (Leibo et al., 1978Go; Rall et al., 1984Go; Ashwood-Smith et al., 1988Go). When cryopreserved mammalian embryos are recovered from liquid nitrogen, therefore, embryos with damaged blastomeres and/or with a cracked zona pellucida (ZP) are found (Lehn-Jensen and Rall, 1983Go; Rall et al., 1984Go).

Under certain freezing and thawing conditions, more than 50% of mammalian embryos may have ZP damage (Lehn-Jensen and Rall, 1983Go; Rall and Meyer, 1989Go; Schiewe et al., 1991Go). These conditions depend on the speed of cooling and warming, the type of storage container, and the cryoprotectant used. Careful observations of ZP damage to embryos may therefore help us to understand the reasons for its occurrence.

Zona pellucida damage has been quantified in different mammalian species, and several studies have reported on the influence of such damage on further in-vitro and in-vivo development (Whittingham and Adams, 1976Go; Willadsen et al., 1976Go; Lehn-Jensen and Rall, 1983Go; Bielanski et al., 1986Go; Rall and Meyer, 1989Go; Todorow et al., 1989Go; Schiewe et al., 1991Go; Kasai et al., 1996Go).

Previously, it had been reported (Freeman et al., 1986Go; Van Steirteghem et al., 1987Go) that human cryopreserved multicellular embryos with damaged ZP are better not transferred. However, the incidence of ZP damage and the relationship between such damage and blastomere damage is poorly described in human embryo cryopreservation programmes. To the best of our knowledge, ZP damage after freezing–thawing was carefully quantified on a limited number of whole human embryos and isolated zonae in four publications (Cohen et al., 1986Go, 1988aGo; Hartshorne et al., 1991Go; Dumoulin et al., 1994Go), two of which indicated that blastomere survival and embryo viability were much lower in embryos with ZP damage (Cohen et al., 1986Go, 1988aGo).

The aim of the present study was to quantify ZP damage in human cryopreserved embryos. We investigated the influence of two different freezing containers, and the influence of such damage on blastomere survival and viability of the embryos was also evaluated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Embryos studied
The embryos evaluated for thawing outcome were frozen between January 1995 and December 1997. Culture conditions were kept constant. Further cleaving multicellular embryos were selected for transfer 24 h after thawing and further in-vitro culture. This allowed us also to evaluate further cleavage in vitro of the frozen–thawed embryos (Van der Elst et al., 1997Go).

Embryos for freezing were supernumerary embryos obtained after in-vitro fertilization (IVF) with or without intracytoplasmic sperm injection (ICSI). After selection of two to three excellent to good quality embryos for transfer in the oocyte-collection cycle, the remaining embryos were frozen (Staessen et al., 1993Go; Van Steirteghem et al., 1993Go). Two types of embryos were frozen: excellent quality embryos containing regular or irregular blastomeres, and good quality embryos containing regular or irregular blastomeres and also containing up to 20% of their volume filled with anucleate fragments. The embryos were frozen irrespective of their developmental stage.

Embryos were frozen in polypropylene cryovials up to October 1996 and in plastic mini-straws between November 1996 and December 1997. Thawing results were evaluated up to January 1998. Blastomere damage and ZP damage were carefully evaluated under an inverted microscope at x400 magnification.

Freezing and thawing
Freezing and thawing in polypropylene cryovials
For freezing, embryos were put into HEPES-buffered Earle's medium supplemented with 0.5% w/v human serum albumin (HSA, CAF-Belgian Red Cross, de Tyraslaan 109, Brussels, Belgium), henceforward referred to as HEPES-medium. The embryos were then placed into 0.3 ml of HEPES-medium containing 0.75 mol/l dimethylsulphoxide (DMSO; Sigma D2650, Bornem, Belgium) for 10 min at 22°C and transferred to a properly labelled polypropylene cryovial (Nunc, 1.2 ml; Simplon, VEL, Leuven, Belgium) also containing 0.3 ml of HEPES-medium and 1.5 mol/l DMSO for 10 min at 22°C. Each vial was loaded with up to three embryos. The vial was then transferred to a programmable freezer (KRYO 10, series III, Planer, VEL) and cooling was initiated to –7°C at 2°C/min. At –7°C a 5 min holding period was built in and manual seeding was performed by touching the vial with liquid nitrogen-cold forceps at the level of the fluid meniscus. After another 5 min post-seeding holding period at –7°C, the temperature was lowered to –80°C at 0.3°C/min and to –110°C at 10°C/min. The vials were then plunged into liquid nitrogen. Vials were kept in liquid nitrogen-filled containers (GT40, Air Liquide, Machelen, Belgium).

For thawing, the vials were taken from liquid nitrogen and put in a programmable freezer (KRYO 10 series III) previously set at –100°C. After a 5 min holding period at –100°C the embryos were thawed at 4°C/min to 22°C. When all ice crystals had melted, the contents of the vial were emptied into a Petri dish. The embryos were picked up using a finely pulled Pasteur pipette and placed in a Petri dish containing HEPES-medium and 1 mol/l sucrose (Analar Grade, British Drug Houses, Pasture, Ghent, Belgium) for 10 min at 22°C. Thereafter, the embryos were washed several times in HEPES-medium and put into culture.

Freezing and thawing in plastic mini-straws
After addition of the cryoprotectant DMSO as for polypropylene cryovials, the embryos were loaded in properly labelled 0.25 ml plastic mini-straws (pailette souple, CA 006430ZA481; L'air Liquide, Brussels, Belgium). The loading of the straws was carried out as follows: 0.02 ml of HEPES-medium and 1.5 mol/l DMSO were aspirated, an air bubble was made, 0.15 ml of HEPES-medium containing 1.5 mol/l DMSO and up to three embryos was aspirated, an air bubble was made, and finally HEPES-medium with 1.5 mol/l DMSO was aspirated until the cotton plug became wet. The straw was then sealed with PVA sealing powder and transferred horizontally to a programmable freezing unit (Minicool, AS100, L'Air Liquide). Cooling was then initiated to –7°C at 2°C/min. After a 5 min holding period at –7°C, the straw was seeded by touching it with a liquid nitrogen-cooled metal device where an air bubble was located. After a further holding period for 5 min at –7°C the straw was cooled to –80°C at 0.3°C/min and from –80°C to –110°C at 10°C/min. The straws were then plunged horizontally into liquid nitrogen. The straws were stored vertically in liquid nitrogen-filled containers.

For thawing, straws were taken from liquid nitrogen and put in a programmable freezer (Minicool, AS 100, L'air Liquide) previously set at –100°C. After a 5 min holding period at –100°C, the straws were warmed to 22°C at 4°C/min. Thawed straws were taken from the programmable freezer and the contents of the straw were expelled into HEPES-medium containing 1 mol/l sucrose. The embryos were then processed further for dilution of the cryoprotectants as described for the polypropylene cryovials.

Evaluation of damage after thawing
Immediately after thawing and dilution of the cryoprotectants, as well a few hours later, the embryos were inspected for morphological survival under an inverted microscope (x200). Two types of damage were then evaluated, namely of the blastomere and the ZP.

Blastomeres were considered to be damaged if the cytoplasm appeared granular and degenerative, when they remained contracted after dilution of the cryoprotectant, or if they had smooth membranes (Cohen et al., 1988bGo). Several types of ZP damage (Rall and Meyer, 1989Go) were also observed. To be recorded as damaged, the ZP had to be cracked on at least one spot. Whether there was one crack or more cracks, or whether there was complete absence of the ZP, was not separately recorded. Minor ZP damage (dark ZP, a thinner but not completely cracked ZP at local spots) was not recorded.

After freezing and thawing, embryos were suitable for transfer if they contained at least 50% of the initial number of blastomeres intact. Embryos suitable for transfer were put into culture for 24 h. They were then inspected for further cleavage, which was defined as the number of embryos suitable for transfer where after 24 h the number of blastomeres had increased.

In-vitro culture
Embryos were cultured individually in 0.025 ml droplets of B2 Ménézo medium under a mineral oil (Sigma, M8240) overlay in a humidified incubator (5% O2, 5% CO2, 90% N2) at 37°C.

Statistical analysis
Chi-square analysis was used for comparison of the numbers of embryos between different groups. P-values less than 0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of 774 thawing cycles in which 3884 embryos were thawed were analysed. The quality of the embryos thawed was analysed before freezing according to the presence of anucleate fragments and to the developmental stage. Grade A embryos contained no anucleate fragments (11.4% of thawed embryos), and grade B embryos contained up to 20% anucleate fragments (88.6% of thawed embryos). The evaluation of damage to embryos after thawing revealed that damage to the ZP was visible immediately after thawing, while blastomere damage was visible either immediately, or after dilution of the cryoprotectant or after a few hours in culture.

The morphological survival of frozen–thawed embryos and their further cleavage as related to the assisted reproductive procedure in the oocyte-collection cycle is reported in Table IGo. The incidence of ZP damage and the survival rate of blastomeres in frozen–thawed embryos did not differ between the two procedures. Embryos suitable for transfer were put into in-vitro culture. After 24 h, the percentage of embryos that cleaved further did not differ between the two different assisted reproduction procedures.


View this table:
[in this window]
[in a new window]
 
Table I. Morphological survival and further cleavage of frozen–thawed embryos as related to the assisted reproduction procedure in the oocyte collection cycle
 
The morphological survival of frozen–thawed embryos and their further cleavage as related to the storage container is reported in Table IIGo. The incidence of ZP damage was different according to whether the embryos were frozen–thawed in polypropylene cryovials or in plastic mini-straws (16.6% versus 2.3%; P <0.0001). The percentage of embryos with 100% blastomere survival was higher using plastic mini-straws (59.8% versus 34.9%; P <0.0001). Subsequently the percentage of embryos suitable for transfer that cleaved further after a 24 h culture period was higher when using plastic mini-straws (81.0% versus 61.0%; P <0.0001).


View this table:
[in this window]
[in a new window]
 
Table II. Morphological survival and further cleavage of frozen–thawed embryos as related to the freezing procedure
 
The distribution of the different embryonic qualities of thawed embryos did not differ between the two assisted reproduction procedures or whether the freezing–thawing was done in cryovials or plastic mini-straws (data not shown). Also, the incidence of ZP damage was not influenced by the quality of the embryos thawed (data not shown).

Among 556 embryos showing ZP damage after thawing, only 5.9% showed 100% blastomere survival, 32% showed >=50% blastomere survival, and 62.1% showed <50% survival. Among 3328 embryos showing no ZP damage after thawing these percentages were significantly different; 44.8% of embryos showed 100% blastomere survival, 25.4% showed >=50% survival, and 30.3% showed <50% blastomere survival (P <0.001, P <0.001 and P <0.001 respectively). This indicates that there was a clear relationship between the incidence of ZP damage and the blastomere survival rate.

The further cleavage of thawed embryos as related to blastomere survival and ZP damage is reported in Table IIIGo. The further cleavage of frozen–thawed embryos suitable for transfer depended on the blastomere survival rate. More embryos cleaved further when there was 100% blastomere survival (79.0% versus 43.7%; P <0.0001). The incidence of ZP damage in thawed embryos suitable for transfer did not influence their further cleavage. The distribution of the different embryonic qualities was not significantly different whether there was 100% blastomere survival or >=50% blastomere survival.


View this table:
[in this window]
[in a new window]
 
Table III. Further cleavage of frozen–thawed embryos as related to blastomere survival and zona pellucida (ZP) intactness
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, the incidence of ZP damage to human cryopreserved embryos and the relationship between ZP damage, blastomere damage and the ability of the embryos suitable for transfer to cleave further in vitro were analysed.

Our results indicate that human embryos obtained after IVF in association with ICSI survive the cryopreservation procedure equally well as do embryos resulting from IVF alone. The hyaluronidase exposure followed by mechanical removal of cumulus cells by aspirating the oocyte–cumulus complexes in and out of a finely pulled pipette and the mechanical piercing of the ZP and oolemma in ICSI do not make the embryo more susceptible to blastomere damage and do not alter their capacity to cleave further. These results are in agreement with previously published reports (Van Steirteghem et al., 1994Go; Al-Hasani et al., 1996Go; Hoover et al., 1997Go; Kowalik et al., 1998Go; Mandelbaum et al., 1998Go). Our study adds that ICSI does not make the embryo more susceptible to ZP damage. These observations allowed us to pool the data for the subsequent analysis on the influence of the storage containers.

Due to increasing storage problems in our centre we introduced the use of plastic mini-straws from November 1996 onwards instead of cryovials to store human cryopreserved embryos. In the present retrospective analysis we found that the incidence of ZP damage was significantly higher when a freezing procedure with cryovials as storage containers was used, indicating that the material in which the human embryos are stored has important consequences for the outcome after thawing. Cracks in the ZP are considered to be caused by mechanical stress produced from non-uniform volume changes of the freezing medium during phase changes, and are termed fracture damage (Rall et al., 1984Go; Rall and Meyer, 1989Go). These authors concluded that the differences observed between plastic mini-straws and glass or plastic cryovials were associated with thermally induced fracturing of the freezing suspensions during rapid changes of temperature. Although the biological freezer used was different for the two types of container we compared, cooling rates and thawing rates used for both were identical. Furthermore, the biological freezers were regularly calibrated for temperature. Nevertheless, it should be recognized that, besides the biological freezer, other factors might also have influenced the results, e.g. change of operator and inter-batch variation in the chemicals used. Any of these factors which changed during the two different periods analysed could explain the difference seen in ZP damage, although we speculate that it was the type of storage container which was responsible. However, since the study lacks control data, the results presented in this paper should be confirmed in a properly designed, controlled study.

In this study, the percentage of thawed embryos with 100% blastomere survival was 39%, and 26% of thawed embryos had more than 50% but less than 100% of the initial number of blastomeres surviving. These overall figures are comparable with those described in the literature when freezing–thawing multicellular human embryos (Mandelbaum et al., 1987Go). Our study indicated that the percentage of embryos with 100% blastomere survival was significantly higher when a freezing procedure using plastic mini-straws was used, and that a clear relationship was found between the incidence of ZP and blastomere damage. Zona pellucida damage was correlated with low blastomere survival. The same observations were made in another analysis (Cohen et al., 1988aGo). It has long been considered that the ZP may play a role in the cryopreservation of embryos (Willadsen et al., 1976Go), by acting either as a barrier to the diffusion of water and cryoprotectants or as a physical barrier to the growth of extracellular ice (Kanagawa et al., 1979Go). Blastomere damage has been described as the result of intracellular ice crystal formation, osmotic stress during addition and removal of concentrated solutions of cryoprotective agents, gas bubble formation, or fracture planes which sometimes pass through the cytoplasm (Leibo et al., 1978Go; Ashwood-Smith et al., 1988Go; Oda et al., 1992Go). Compared with embryos without ZP damage, our study indicates that in thawed embryos with ZP damage after cryopreservation, blastomere damage is probably also the result of fracture planes passing through the cytoplasm (data not shown).

We were unable to relate the type of ZP anomalies and the blastomere damage seen. In a prospective study, it would be interesting to investigate whether less severe ZP damage is correlated with higher blastomere survival and improved in-vitro viability.

One study (Cohen et al., 1986Go) concluded that human embryos with ZP damage after cryopreservation were less likely to cleave further in vitro. We also studied the consequence of cryopreservation damage to human embryos in terms of their capacity to cleave further in vitro. We found that ZP damage had no effect on the further cleavage in vitro of the embryo. Blastomere damage, however, significantly reduced the capacity of the frozen–thawed embryo to cleave further in vitro. The damage to the embryos was not related to the quality of the embryos before freezing, indicating that the blastomere damage in itself was responsible for their impaired further cleavage.

Previous reports with regard to mice (Depypere et al., 1991Go; Garrisi et al., 1992Go; Grossmann et al., 1994Go) also reported that small gaps in the ZP of early mouse embryos did not disturb further cleavage, and that the viability of zona-free embryos which survived the freezing–thawing process was similar to that of zona-intact embryos cultured in vitro up to the morula stage. Other studies described significantly lower developmental rates when a slit in the ZP of early mouse embryos was made, and that the size of the hole in the ZP affected dramatically the further development of cryopreserved ZP-damaged embryos (Nichols and Gardner, 1989Go; Kuo-Kuang Lee et al., 1997Go).

In a mouse model, damage to blastomeres was described as being negatively correlated with further culture in vitro, and that restoration of partially damaged embryos improves their viability (Alikani et al., 1993Go; Rülicke and Autenried, 1995Go). For human embryos, it was concluded that after a post-thaw culture period, a cleaved embryo group had a significantly increased number of intact blastomeres (Ziebe et al., 1998Go), while in a previous study at our centre we reported that partially damaged embryos implanted less well than fully intact ones (Van den Abbeel et al., 1997Go). We postulated that damaged blastomeres exerted `toxic' effects on intact blastomeres. The present study seems to confirm the hypothesis that damaged blastomeres have some toxic effect on the intact ones.

Zona pellucida damage and (or) blastomere damage after cryopreservation might be signs of suboptimal cryopreservation procedures (Lehn-Jensen and Rall, 1983Go). The introduction of plastic mini-straws as storage containers in our practice resulted in a clear improvement of the cryopreservation outcome. Further improvements might be expected by using polymeric substances in the freezing media or by using vitrification procedures (Ali et al., 1995Go; Titterington et al., 1995Go; Shaw et al., 1997Go).

In conclusion, this study indicated that the morphological survival and further cleavage in vitro of human cryopreserved embryos obtained after IVF treatment in association with ICSI are no different from those obtained after IVF only. Zona pellucida damage and blastomere damage after cryopreservation can have dramatic consequences for the viability of the human embryo in vitro. The occurrence of ZP and blastomere damage can be carefully controlled using an optimized freezing procedure. Such a procedure includes the use of plastic mini-straws as storage containers and slow-cooling, slow-thawing rates with 1.5 mol/l DMSO as the cryoprotectant. The general aim of a cryopreservation programme should be to have as many fully intact embryos as possible after thawing.


    Acknowledgments
 
We wish to thank Mrs Sabrina Vitrier for excellent technical assistance. Mr F.Winter of the Language Education Centre is kindly acknowledged for correction of the English text. This study was supported by grants from the Belgian National Fund for Medical Research.


    Notes
 
1 To whom correspondence should be addressed at: Centre for Reproductive Medicine, Laarbeeklaan 101, 1090 Brussels, Belgium

Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Al-Hasani, S., Ludwig, M., Gagsteiger, F. et al. (1996) Comparison of cryopreservation of supernumerary pronuclear human oocytes obtained after intracytoplasmic sperm injection (ICSI) and after conventional in-vitro fertilisation. Hum. Reprod., 11, 604–607.[Abstract]

Ali, J., Bongso, A. and Ratnam, S. (1995) Chromosomal analysis of day-2 human embryos vitrified with VS14. Med. Sci. Res., 23, 539–540.[ISI]

Alikani, M., Olivennes, F. and Cohen, J. (1993) Microsurgical correction of partially degenerate mouse embryos promotes hatching and restores their viability. Hum. Reprod., 8, 1723–1728.[Abstract]

Ashwood-Smith, M., Morris, G., Fowler, R. et al. (1988) Physical factors are involved in the destruction of embryos and oocytes during freezing and thawing. Hum. Reprod., 3, 795–802.[Abstract]

Bielanski, A., Schneider, U., Pawlyshyn, V. et al. (1986) Factors affecting survival of deep frozen bovine embryos in vitro: the effect of freezing container and method of removing cryoprotectant. Theriogenology, 25, 429–437.[ISI]

Cohen, J., Simons, R., Fehilly, C. et al. (1986) Factors affecting survival and implantation of cryopreserved human embryos. J. Assist. Reprod Dev., 3, 46–52.

Cohen, J., DeVane, G., Elsner, C. et al. (1988a) Cryopreservation of zygotes and early cleaved human embryos. Fertil. Steril., 49, 283–289.[ISI][Medline]

Cohen, J., Wiemer, K. and Wright, G. (1988b) Prognostic value of morphological characteristics of cryopreserved embryos: a study using videocinematography. Fertil. Steril. 49, 827–834.[ISI][Medline]

Depypere, H., Carroll, J., Vandekerckove, D. et al. (1991) Normal survival and in-vitro development after cryopreservation of zona-drilled embryos in mice. Hum. Reprod., 6, 432–435.[Abstract]

Dumoulin, J., Bergers-Janssens, J., Pieters, M. et al. (1994) The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertil. Steril., 62, 793–798.[ISI][Medline]

Fehilly, C., Cohen, J., Simons, R. et al. (1985) Cryopreservation of cleaving embryos and expanded blastocysts in the human: a comparative study. Fertil. Steril., 44, 638–644.[ISI][Medline]

Freeman, L., Trounson, A. and Kirby, C. (1986) Cryopreservation of human embryos: progress on the clinical use of the technique in human in vitro fertilisation. J. Assist. Reprod. Genet., 3, 53–61.

Garrisi, G., Talansky, B., Sapira, V. et al. (1992) An intact zona-pellucida is not necessary for successful mouse embryo cryopreservation. Fertil. Steril., 57, 677–681.[ISI][Medline]

Grossmann, M., Egozcue, J. and Santalo, J. (1994) Cryopreservation of zona-free mouse embryos. Cryo-Letters, 15, 103–112.[ISI]

Hartshorne, G., Elder, K., Crow, J. et al. (1991) The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts. Hum. Reprod., 6, 136–141.[Abstract]

Hoover, L., Baker, A., Check, J. et al. (1997) Clinical outcome of cryopreserved human pronuclear stage embryos resulting from intracytoplasmic sperm injection. Fertil. Steril., 67, 621–624.[ISI][Medline]

Kanagawa, H., Frim, J. and Kruuv, J. (1979) The effect of puncturing the zona pellucida on freeze thaw survival of bovine embryos. Can. J. Anim. Sci., 59, 623–626.[ISI]

Kasai, M., Zhu, S., Pedro, P. et al. (1996) Fracture damage of embryos and its prevention during vitrification and warming. Cryobiology, 33, 459–464.[ISI][Medline]

Kowalik, A., Palermo, G., Barmat, L. et al. (1998) Comparison of clinical outcome after cryopreservation of embryos obtained from intracytoplasmic sperm injection and in-vitro fertilization. Hum. Reprod., 13, 2848–2851.[Abstract/Free Full Text]

Kuo-Kuang Lee, R., Jin-Tsung, S., Yu-Wen, C. et al. (1997) A comparison of the effects of different degrees of zona-pellucida damage followed by cryopreservation on the postthaw development of mouse embryos. J. Assist. Reprod. Genet., 14, 170–173.[ISI][Medline]

Lehn-Jensen, H. and Rall, W. (1983) Cryomicroscopic observations of cattle embryos during freezing and thawing. Theriogenology, 19, 263–277.[ISI]

Leibo, S., McGrath, J. and Cravalho, E. (1978) Microscopic observation of intracellular ice formation in unfertilised mouse ova as a function of cooling rate. Cryobiology, 15, 257–271.[ISI][Medline]

Mandelbaum, J., Junca, A., Plachot, M. et al. (1987) Human embryo cryopreservation, extrinsic and intrinsic parameters of success. Hum. Reprod., 2, 709–715.[Abstract]

Mandelbaum, J., Belaisch-Allart, J., Junca, A. et al. (1998) Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum. Reprod. 13, (Suppl. 3), 161–177.[Abstract]

Nichols, J. and Gardner, R. (1989) Effect of damage to the zona-pellucida on development of preimplantation embryos in the mouse. Hum. Reprod., 4, 180–187.[Abstract]

Oda, K., Gibbons, W. and Leibo, S. (1992) Osmotic shock of fertilised mouse ova. J. Reprod. Fertil., 95, 737–747.[Abstract]

Rall, W. and Meyer, T. (1989) Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos. Theriogenology, 31, 683–692.[ISI]

Rall, W., Reid, D. and Polge, C. (1984) Analysis of slow-warming injury of mouse embryos by cryomicroscopical and physiochemical methods. Cryobiology, 21, 106–121.[ISI][Medline]

Rülicke, T. and Autenried, P. (1995) Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation. Lab. Anim., 29, 320–326.[ISI][Medline]

Schiewe, M., Rall, W., Stuart, L. et al. (1991) Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos. Theriogenology, 36, 279–293.[ISI]

Shaw, J., Kulishova, L., MacFarlane, D. et al. (1997) Vitrification properties of solutions of ethylene glycol in saline containing PVP, ficoll, or dextran. Cryobiology, 35, 219–229.[ISI][Medline]

Staessen, C., Janssenswillen, C., Van den Abbeel, E. et al. (1993) Avoidance of triplet pregnancies by elective transfer of two good-quality embryos. Hum. Reprod., 8, 1650–1653.[Abstract]

Titterington, J., Robinson, J., Killick, S. et al. (1995) Synthetic and biological macromolecules: protection of mouse embryos during cryopreservation by vitrification. Hum. Reprod., 10, 649–653.[Abstract]

Todorow, S., Siebzehnrüble, E., Koch, R. et al. (1989) Comparative results on survival of human and animal eggs using different cryoprotectants and freeze–thawing regimens. I. Mouse and hamster. Hum. Reprod., 4, 805–811.[Abstract]

Van den Abbeel, E., Camus, M., Van Waesberghe, L. et al. (1997) Viability of partially damaged human embryos after cryopreservation. Hum. Reprod., 12, 2006–2010.[Abstract]

Van der Elst, J., Van den Abbeel, E., Vitrier, S. et al. (1997) Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates. Hum. Reprod., 12, 1513–1521.[Abstract]

Van Steirteghem, A., Van den Abbeel, E., Camus, M. et al. (1987) Cryopreservation of human embryos obtained after gamete intra-Fallopian transfer and/or in-vitro fertilisation. Hum. Reprod., 2, 593–598.[Abstract]

Van Steirteghem, A., Nagy, P., Joris, H. et al. (1993) High fertilisation and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 1061–1066.[Abstract]

Van Steirteghem, A., Van der Elst, J., Van den Abbeel, E. et al. (1994) Cryopreservation of supernumerary multicellular human embryos obtained after intracytoplasmic sperm injection. Fertil. Steril., 62, 775–780.[ISI][Medline]

Whittingham, D. and Adams, C. (1976) Low temperature preservation of rabbit embryos. J. Reprod. Fertil., 47, 269–274.[Abstract]

Willadsen, S., Polge, C. and Moor, R. (1976) Deep freezing of sheep embryos. J. Reprod. Fertil., 46, 151–154.[Abstract]

Wood, M. (1997) Embryo freezing: is it safe? Hum. Reprod., 12 (Suppl., 2), 32–37.[ISI]

Ziebe, S., Bech, B., Petersen, K. et al. (1998) Resumption of mitosis during post-thaw culture: a key parameter in selecting the right embryos for transfer. Hum. Reprod., 13, 178–181.[Abstract]

Submitted on January 5, 1999; accepted on June 21, 1999.