Centre for Reproductive Medicine, Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium
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Abstract |
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Key words: cryopreservation/embryo biopsy/human/survival
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Introduction |
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Patients treated for PGD may be younger. Consequently, larger numbers of oocytes may be retrieved. This is important because it has been shown that the success of a PGD cycle is dependent on the number of cumulusoocyte complexes retrieved (Vandervorst et al., 1998). The oocytes' nuclear maturity, normal fertilization, embryo quality, and embryo development are different steps that may jeopardize the number of embryos available for biopsy. After diagnosis, the limited number of transferable embryos available prevents embryo selection in a number of cycles while in others more embryos than necessary for replacement are available. Further in-vitro culture of embryos after diagnosis may allow selection of embryos that develop further for replacement and limit the number of embryos available for cryopreservation (Grifo et al., 1998
). However, for some cycles, cryopreservation of non-affected good quality embryos may still be required.
In biopsied human embryos, a large hole is made in the zona pellucida (ZP) and blastomeres are removed from the embryo. This procedure induces mechanical stress on the remaining blastomeres. There is in-vitro (Hardy et al., 1990) and in-vivo (Gianaroli et al., 1997
) evidence that the biopsy procedure does not influence further development in non-frozen biopsied embryos.
Preclinical studies using mouse embryos showed that drilling a hole in the ZP had no effect on further development after cryopreservation (Depypere et al., 1991; Garrisi et al., 1992
) and that survival and further development after blastomere removal and subsequent cryopreservation was not severely reduced (Wilton et al., 1989
; Krzyminska and O'Neill, 1991
; Liu et al., 1993
). Whether these findings are applicable to human embryos remains to be evaluated. However, the influence of cryopreservation on the survival and further development of biopsied human embryos has not yet been studied extensively (Magli et al., 1999
).
In the present study we aimed to find out whether the presence of a large hole in the zona pellucida and the removal of one or two blastomeres in human embryos influences survival and further development after cryopreservation.
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Materials and methods |
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The procedures were performed shortly after the evaluation of the embryos on day 3. The cryopreservation of the embryos was performed within 2 to 3 h. Because of the scarcity of these good quality embryos developing after abnormal fertilization, the embryos were grouped per day. Allocation to the different groups was done randomly on a daily basis. Embryos were thawed in three experiments. For each of the thawing procedures, one-third of the straws from the three different groups was selected randomly.
Recovery of embryos was defined as the embryos that were retrieved from the straws irrespective of their survival. Survival rates were expressed as embryo survival (i.e. percentage of embryos with at least 50% of the cryopreserved blastomeres intact after thawing) and as blastomere survival (i.e. the number of intact blastomeres after thawing divided by the number of blastomeres frozen). Immediately after thawing, embryos were evaluated for the number of intact blastomeres under an inverted microscope (Diaphot TMD, Nikon, Tokyo, Japan) at x200 or x400 magnification. Embryos with at least one blastomere remaining intact after freezing and thawing were cultured in S2 medium (Scandinavian IVF Science, Göteborg, Sweden). Additional evaluations for assessment of further development were performed daily for 3 days, i.e. until day 6 after insemination. Any change in the number of blastomeres, compaction, or blastocyst formation was recorded for all the embryos in culture.
Zona drilling and embryo biopsy
Zona drilling and embryo biopsy was performed under mineral oil (Sigma, St Louis, USA) in HEPES-buffered Earle's medium supplemented with 0.5% human albumin (HSA) (Belgian Red Cross, Brussels, Belgium). Acid Tyrode (pH 2.12.4) was used to perform zona drilling. The size of the hole was similar for embryos in the drilling-only group and in the biopsy group and was large enough (~3040 µm) to allow aspiration of blastomeres. The day before the biopsy procedure a separate dish containing 25 µl droplets of Ca2+Mg2+-free medium covered with mineral oil was prepared and incubated in an atmosphere of 5% CO2 in air. If embryo compaction interfered with the biopsy procedure, embryos were incubated in Ca2+Mg2+-free medium for a short period of time (510 min) in order to allow decompaction.
The embryo to be biopsied was fixed on the holding pipette in such a way that anucleate fragments or an empty area between blastomeres was at the 3 o'clock position where the hole was going to be made. The drilling pipette filled with acid was brought into the proximity of this position and acid was released. Usually this procedure took ~1015 s. To avoid too much acid solution reaching the blastomeres, the acid flow was stopped immediately by strong aspiration when the zona ruptured.
The biopsy pipette was introduced into the hole in the ZP and contact was made with a blastomere. This blastomere was then aspirated gently. When about 20% of the blastomere was aspirated into the pipette, the operator attempted to pull the cell out of the ZP. More aspiration was needed if the attempt failed. When the blastomere was removed from the embryo, it was released from the biopsy pipette. A second blastomere was removed when the embryo had 7 cells or more.
Freezing and thawing procedure
Embryos were frozen and thawed using a slow-cooling and slow-thawing procedure with dimethylsulphoxide (DMSO, Sigma) as the cryoprotectant. The cryoprotectant solution was made-up in HEPES-buffered Earle's medium supplemented with 0.5% w/v HSA (further referred to as HEPES medium). Embryos were first incubated in 300 µl HEPES medium with 0.75 mmol/l DMSO for 10 min at 22°C. The embryos were then transferred to a 150 µl droplet of HEPES medium with 1.5 M DMSO and incubated for 10 min at 22°C. The embryos were loaded into plastic ministraws [0.25 ml, Pailette Souple, Industrie de la Médecine Vétérinaire (IMV), L'aigle, Air Liquide, Machelen, Belgium]. The loading of the straws was done as follows: 25 µl of HEPES medium was aspirated into the straw and then some air was aspirated. Next, 150 µl HEPES medium with 1.5 mmol/l DMSO containing the embryos was aspirated, followed by the aspiration of another air bubble. Subsequently, HEPES medium was aspirated until the cotton plug of the straw became wet. The open end of the straw was closed with powder (IMV, Air Liquide). Up to three embryos were loaded into a straw. The loaded straws were transferred to a programmable freezer (Minicool 40 PC, Air Liquide) and placed horizontally in the freezing chamber. The controlled freezing procedure started after all the straws had been loaded. Cooling from 22°C to 7°C was done at a rate of 2°C/min. This temperature was kept for 5 min. At this point the seeding was performed by touching the straws with a liquid-nitrogen-(LN2)-cold forceps at the level of an air bubble. After another 5 min at 7°C, the temperature was lowered to 80°C at a rate of 0.3°C/min and to 100°C at 10°C/min. The straws were then plunged into LN2. Straws were stored vertically in LN2 filled containers (GT40, Air Liquide).
For thawing the straws were taken from the LN2 and transferred to the programmable freezer that had first been cooled to 100°C. After a 5 min holding period at 100°C the straws were warmed to 4°C at 4°C/min. After a 10 min holding period the straws were taken from the chamber and the content of the straws was expelled in HEPES medium containing 1 mmol/l sucrose (Sigma). After an incubation of 10 min at 22°C the embryos were transferred to 2 ml of HEPES medium without sucrose and further incubated for 10 min at 22°C. After two additional rinsing steps the embryos were put into culture.
Statistical analysis
Statistics were performed using the 2-test.
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Results |
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Fifteen embryos in the control group, 13 in the drilling-only group, and 15 in the biopsy group with at least one intact cell after thawing were put into culture. The further development of these 43 embryos is summarized in Table II. On day 4 after insemination, the number of blastomeres had increased in 15 embryos (34.9%). Six out of 15 embryos from the control group, four out of 13 embryos from the drilling-only group, and five out of 15 embryos from the biopsy group cleaved. On day 5 and day 6 after insemination, some of the embryos were able to compact and four of the compacted embryos became blastocysts. Three of the blastocysts developed in the drilling-only group and one blastocyst developed in the biopsy group.
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Discussion |
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In the present study, embryos derived from abnormal fertilization were used as a model. Human embryos obtained after abnormal fertilization may not have the same cryobiological properties and developmental potential as embryos obtained after normal fertilization. The survival rate of embryos in the control group is lower than in our current cryopreservation programme (Vitrier et al., 1998) but it is similar to the results obtained by Noto et al. who also used abnormally fertilized embryos (Noto et al., 1991
). It has also been shown that embryos that develop from 1PN oocytes may be diploid (Staessen and Van Steirteghem, 1997
) and may thus develop to the blastocyst stage. In addition, embryos developing from 3PN oocytes may also develop further, even beyond the blastocyst stage (Restagno et al., 1988
; Plachot, 1989
). Embryos derived from 1PN and 3PN oocytes may therefore serve as a model to evaluate a possible negative influence from the cryopreservation procedure on the developmental capacity of these manipulated embryos.
When comparing blastomere survival rates in the three different groups, a significantly lower number of blastomeres was intact after the thawing procedure when a relatively large hole in the ZP was present (Table I). This difference was apparent after drilling-only but was even more pronounced after blastomere removal. This lower blastomere survival rate was reflected in the number of embryos surviving with at least 50% of the frozen blastomeres intact after thawing. Although no statistical difference was found in embryo survival rates, there was an apparent trend towards a lower number of surviving embryos (Table I
) after blastomere removal compared with the two other study groups. Embryos with two blastomeres aspirated survived better as compared with embryos where only one blastomere was removed. This can be explained by a delayed cleavage and lower embryonic quality of embryos that had only five or six cells at the time of biopsy.
Preclinical studies using mouse embryos demonstrated that neither the creation of a slit or a small hole in the ZP (Depypere et al., 1991; Garrisi et al., 1992
; Thompson et al., 1995
), nor the creation of a large hole combined with blastomere removal (Wilton et al., 1989
; Krzyminska and O'Neill, 1991
; Liu et al., 1993
; Thompson et al., 1995
) jeopardizes cryosurvival. The introduction in humans of assisted fertilization techniques such as subzonal insemination and ICSI, involving the creation of a small hole in the ZP, does not result in reduced cryosurvival (Obasaju et al., 1994
; Van Steirteghem et al., 1994
; Al-Hasani et al., 1996
; Macas et al., 1998
; Kowalik et al., 1998
) and confirms the results of the preclinical studies with mouse embryos. However, the findings in our study as well as others (Magli et al., 1999
) clearly indicate that the creation of a large hole in the ZP of human embryos influences cryosurvival negatively.
After thawing, we did not observe additional zona fracture damage due to piercing of ice crystals. However, we observed that damaged cells in partially intact embryos were located close to the opening in the ZP. There may be several explanations for this observation. It has been shown in the mouse that blastomeres located close to the site where drilling has been performed divide less than blastomeres from the same embryo located on the opposite site (Van Golde et al., 1996). The acid used to create the hole may alter the membrane permeability, making these blastomeres more susceptible to damage. Another point is that the hole made in the ZP of human embryos is much larger than in mouse embryos. This may cause the ZP to be less effective during cooling or warming and blastomeres may be more easily damaged by piercing or rubbing of ice crystals formed in the vicinity of the embryo.
In this study, only two embryos from the biopsy group were incubated in Ca2+Mg2+-free medium. Whether incubation in this medium at the time of blastomere removal influences survival after cryopreservation has not yet been investigated in human embryos. Any negative influence will probably be minimal because the incubation in this Ca2+Mg2+-free medium is limited in time (maximum 10 min). It has been shown (Dumoulin et al., 1998) that in-vitro development to the blastocyst stage of human embryos is not influenced by incubation in Ca2+Mg2+-free medium.
Reduced cryosurvival in addition to removal of two blastomeres has a double negative influence on further development. Indeed, the ability of mouse embryos to develop to the blastocyst stage or to term depends on the number of cells left (Liu et al., 1993). Fewer blastocysts developed when more than two blastomeres were removed from 8-cell embryos and no living young were born when more than four blastomeres were removed. In addition, Van den Abbeel et al. demonstrated that replacement of fully intact frozen-thawed human embryos obtained after IVF, with or without ICSI, resulted in a higher clinical implantation rate than did the replacement of partially intact embryos (Van den Abbeel et al., 1997
). Together with the present data it seems that biopsied embryos require optimal cryosurvival in order to allow optimal further development after thawing. Thus, efforts must be made to improve the conditions for cryopreservation of biopsied embryos. For this, a better understanding of the cryobiological properties of human embryos is indispensable (Wood, 1997
). As long as these features of human embryos are unknown, it is possible to improve survival rates only by altering certain aspects of the biopsy procedure or by using alternative freezing procedures. Other methods of zona drilling, such as making a slit in the ZP (Thompson et al., 1995
) or making a hole by using a laser (Boada et al., 1998
), may be less traumatic to blastomeres. Protective polymers (Dumoulin et al., 1994
; Titterington and Robinson, 1996
) or host zonae (Niemann, 1991
) have been shown to improve cryosurvival rates. Ultra rapid cryopreservation procedures such as vitrification (Titterington et al., 1995
) may also increase success rates.
From the present study it can be concluded that, compared with untreated embryos, survival of human embryos after blastomere removal and subsequent cryopreservation is severely reduced. In the light of the special character of supernumerary transferable embryos after PGD, efforts have to be made to improve conditions so as to preserve these precious embryos for later use. The evaluation of further development in vitro of thawed biopsied embryos can provide important information about the potential of these frozenthawed biopsied embryos to develop to the blastocyst stage and to establish valid criteria for transferable embryos after the thawing procedure. In addition, patients undergoing PGD should be informed that, currently, their chances of becoming pregnant with frozenthawed biopsied embryos are rather low.
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Acknowledgments |
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Notes |
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References |
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Submitted on April 22, 1999; accepted on July 14, 1999.