1 Schoysman Infertility Management Foundation, AZ Jan Portaels, Gendarmeriestraat 65, 1800 Vilvoorde, Belgium and 2 Institut for Reproductive Medicine and Endocrinology, 6900 Bregenz, Austria
3 To whom correspondence should be addressed. e-mail: pierrevdz{at}hotmail.com
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Abstract |
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Key words: assisted hatching/cryopreservation/embryo quality/human blastocyst/vitrification
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Introduction |
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Initially, vitrification was performed after loading cleavage stage embryos (Mukaida et al., 1998) or blastocysts in 0.25 ml insemination straws (Vanderzwalmen et al., 1997
; 2002
; Yokota et al., 2000
; 2001
). In this manner, the highest cooling rates were limited to
2500°C/min.
Recently, particular attention has been paid to the cooling rate. The success rates of vitrification have been increased with the use of an ultra-rapid vitrification procedure. By highly increasing the cooling and thawing rates, the formation of ice crystals and chilling injuries are reduced. Ultra-rapid cooling procedures are based on direct contact between a reduced volume of cryoprotectant and liquid nitrogen (LN2). Mammalian blastocysts are loaded on the surface of electron microscope grids (Martino et al., 1996), in an open pulled straw (Vatja et al., 1997
), in a flexipet-denuding pipette (Liebermann et al., 2002
), or in a cryoloop (Lane et al., 1999
) before plunging them in LN2.
Successful human pregnancies were reported after carrying out high speed vitrification methods of blastocysts using the cryoloop or the electron microscope grids (Choi et al., 2000; Mukaida et al., 2001
; Cho et al., 2002
; Reed et al., 2002
; Son et al., 2003
).
In order to accomplish vitrification of blastocysts, which requires an extremely fast cooling rate while considering the amount of space required for storage, we developed the HS (Vanderzwalmen et al., 2000). Recently, Liebermann and Tucker (2002
) reported that the HS was easy to use and successful for vitrification of different stages of development (oocytes and day 3 embryos). The tip of the HS is designed to hold the blastocysts in a very small volume of vitrification solution allowing very fast cooling and warming rates as compared with those achieved by immersion of sealed 0.25 ml plastic straws into LN2.
The initial clinical application of blastocyst vitrification using the HS resulted in a pregnancy outcome that was below our expectation despite acceptable rates of re-expansion 24 h after warming. We noticed a difference in the pregnancy rates between transfers, which were performed with expanded blastocysts, or blastocysts that had hatched spontaneously. Twenty-two transfers with at least one expanded blastocyst resulted in only four pregnancies. On the other hand, 10 transfers with at least one spontaneously hatched blastocyst resulted in three pregnancies.
Considering these observations we wanted to know if the reduced ability of vitrified expanded blastocysts to further develop and implant lay in their inability to hatch out of zona pellucida (ZP).
This reduced capacity to hatch may be explained either by a hardening of the ZP or by leaving many embryos of questionable quality, and therefore of poor prognosis, for an extended period in culture.
Hardening of the ZP may occur spontaneously following fertilization, after prolonged exposure of human embryos to artificial culture conditions and also after cryopreservation (De Vos and Van Steirteghem, 2000). In the mouse, Carroll et al. (1990
) observed that the freezingthawing procedure induced changes in the ZP of oocytes which are responsible for a decrease in the rate of fertilization. To the best of our knowledge, only two reports analysed the effect of assisted hatching (AH) in conjunction with human frozenthawed embryo transfers (Tucker et al., 1991
; Check et al., 1996
). Check et al. (1996
) reported a clinical pregnancy rate of 13.7% after zona drilling on frozen day 3 embryos versus 5.3% in the control group. The clinical pregnancy rate after mechanical hatching of the ZP on day 2 embryos was 28 versus 15% in the control group, and the implantation rate was 16 and 9% respectively in the manipulated and intact ZP group (Tucker et al., 1991
). Although, the number of cases did not allow these results to reach statistical significance, a tendency toward improved outcome after artificial opening of the ZP justifies further investigation in this field.
The second factor that may interfere with the hatching process concerns the quality of the blastocyst. Although some blastocysts showed a normal morphological aspect, they originated from a group of embryos with sub-optimal morphology on days 2 and 3. Probably these blastocysts have a lower potential to be successfully cryopreserved and/or to proceed to a spontaneous hatching.
According to Balaban et al. (2000), blastocyst quality affects the success of blastocyst stage embryo transfer. The same authors reported that fresh blastocyst transfer is a possible alternative in patients who fail to produce good quality cleavage stage embryos (Balaban et al., 2001
). But what happens when blastocysts originating from poor cleavage stage embryos were vitrified? A second question concerns the usefulness of both vitrification and AH on blastocysts that originate from embryos showing dubious quality during their cleavage stage.
The purpose of this retrospective study is to present our clinical results obtained with vitrification of blastocysts using the HS method.
We wanted to determine to what extent the vitrification procedure induces a hardening of the ZP. We also evaluated the frequency of transfers with blastocysts in the hatching or hatched stage, the pregnancies and implantation rates after transfer of vitrified blastocysts in conjunction with an AH protocol or a non-AH protocol. Finally, we analysed the effectiveness of the HS technique according to the quality of the embryos during their cleavage stages.
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Materials and methods |
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Fresh embryo transfers were performed after a culture period of 3 days or following culture in sequential medium for 5 days in women ranging from 2741 years. The HS vitrification protocol was applied on supernumerary embryos that developed to the blastocyst stage after 5 days of culture.
Ovarian stimulation, oocyte retrieval, sperm preparation and embryo transfer procedures
Women undergoing assisted reproduction treatment received the combination of GnRH analogue (Suprefact, SP; Hoechst, Belgium) in association with hMG (Humegon, Organon, The Netherlands) or purified urinary FSH (Metrodin, Serono Laboratories Inc., Belgium; Menopur, Ferring, Belgium). hCG (Pregnyl, Organon; Profasi, Serono) was administered when the cohort of follicles reached a diameter of 20 mm. Luteal phase support consisted of administering 5000 IU hCG on days 4 and 8 after embryo transfer or 600 mg Utrogestan daily, vaginally, until hCG assay.
Sperm preparation was carried out using discontinuous Pure sperm (Nidacon international AB) gradient.
Oocytes were collected 3435 h after the administration of 10 000 IU of hCG and were incubated in 0.5 ml IVF 20 medium (Scandinavian Science, AB products, Sweden). When sperm exhibited normal sperm parameters according to World Health Organization (1999) (except for morphology, for which strict Kruger criteria were used) insemination was performed after a minimal delay of 34 h. In cases in which patients entered the IVF programme for male or unexplained failure of fertilization, cumulus and corona cells were removed from the oocytes 23 h after oocyte retrieval by incubation in 25 IU/ml hyaluronidase (Type VIII; Sigma Chemical Co., USA) before performing ICSI as described previously (Vanderzwalmen et al., 1996
).
In-vitro culture of embryos to the blastocyst stage and classification of day 3 and day 5 embryos
Embryo culture was carried out in 4-well multi-dishes (Nunc) containing each 500 µl of sequential media or in 40 µl microdrops under mineral oil (Cryo Bio System, France) at 37°C in a humidified atmosphere of 5% O2 in air. Injected oocytes or classical inseminated oocytes were incubated in IVF 20 medium (Scandinavian Science). Sixteen to 20 h post-insemination or ICSI, all oocytes were checked for the presence of two pronuclei. After rinsing the zygotes, they were transferred into G1-2 medium (Scandinavian Science) for an additional period of 48 h.
On day 3 (6668 h post-ICSI or insemination), the embryo cell number and the morphology were evaluated under an inverted microscope (magnification x200). They were classified and separated in wells or micro-drops of CCM medium (Scandinavian Science) according to their quality for further culture to the blastocyst stage (day 5). The good quality group consisted of grade I and II embryos. Grade I consisted of 8-cell embryos with <10% fragmentation and equal-sized blastomeres. Grade II consisted of embryos with 68 cells, with 1030% fragmentation and equal-sized blastomeres. The fair quality group consisted of embryos with 68 cells, with 3050% fragments and/or unequal-sized blastomeres. Embryos with lower morphology were not included in the study.
On day 5, the percentage of blastocyst formation was recorded and classified under an inverted microscope (magnification x200) according to the degree of expansion of the blastocoele, the quality of the inner cell mass (ICM) and the trophectoderm (Schoolcraft et al., 1999).
Vitrification: preparation of the solutions
The solutions for equilibration, vitrification and thawing were prepared using Dulbeccos phosphate-buffered saline (Sigma) plus 20% human serum albumin (Irvine) (PBSHSA).
Two solutions were prepared, one for equilibration and one for vitrification. The equilibration solution contained 10% ethylene glycol (v/v) (Fluka)10% dimethylsulphoxide (DMSO) (v/v) (Sigma) in PBSHSA. The vitrification solution was composed of 20% ethylene glycol (v/v)20% DMSO (v/v) (Fluka), 25 µmol/l (10 mg/ml) Ficoll (mol. wt 400 000; Sigma) and 0.75 mol/l sucrose (Sigma) in PBSHSA .
The solution for dilution after thawing was made of 0.5, 0.25 and 0.125 mol/l sucrose in PBSHSA.
Vitrification of the blastocysts
Before starting the vitrification procedure, in order to reduce the negative effect of the blastocoel, expanded blastocysts were collapsed by reducing artificially with a glass pipette the volume of the blastocoele (Vanderzwalmen et al., 2002; Son et al., 2003
).
Early blastocysts and shrunken blastocysts were equilibrated for 2 min at room temperature into 200 µl equilibration solution. For blastocysts, equilibration occurred at 37°C for 3 min. The blastocysts were then exposed to the vitrification solution for a maximum of 30 s. An approximate volume of 0.3 µl of cryoprotectant solution containing the blastocysts (maximum 2) was deposited using an attenuated pipette to the tip of the trough of the HS. The HS was then instantly plunged into a Dewar of LN2 and with the aid of forceps inserted in a larger pre-cooled straw (CBS; Cryo Bio System, France) before closing it (Figure 1).
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A Dewar of LN2 containing the HS was placed close to the microscope. Under LN2 and with forceps, the HS were pulled out of the larger straw and the tip of the straw holding the embryos was immediately immersed into a Petri dish containing 3 ml of 0.5 mol/l sucrose at 37°C. After 3 min, the blastocysts were transferred to 0.25 and 0.125 mol/l sucrose at intervals of 2 min at 37°C. The blastocysts were then washed several times in PBSHSA solution, then placed in CCM medium. Afterwards, the AH technique was performed on some of the embryos.
Artificial AH procedure
Three-dimensional partial zona dissection was performed as previously described by Cieslak et al. (1999). After immobilization of the blastocyst on a holding pipette, the ZP was penetrated with a micro-needle (1015 µm diameter) at the largest perivitelline space and advanced tangentially. The blastocyst was then released from the holding pipette, the micro-needle holding the embryo was gently rubbed across the holding pipette until a slice through the ZP was obtained. A large slit of approximately one-fourth of the circumference of the ZP could be obtained with the large perivitelline space. After rotating the embryo until the slit was visualized and positioned at 6 oclock, the needle was again introduced through the opening in the perivitelline space and across the ZP perpendicular to the first one. After this procedure, a T-shaped slit was obtained.
Transfers
Approximately 2024 h after embryo warming, 85 transfers were performed. Ongoing pregnancy was defined as the presence of gestational sac(s) with fetal heart activities at 30 days after blastocyst transfer.
Zona digestion by pronase
In order to assess if vitrification induces hardening of the ZP, the time for ZP lysis was recorded after enzymatic treatment of fresh or vitrified embryos. Zona digestion was applied on day 4 and day 5 embryos that were abnormally fertilized or that were not transferred. The ZP were enzymatically removed according to the method of Fong et al. (1998).
The embryos were transferred into a culture dish containing only 5 IU/ml pronase (Protease, Sigma P8811; Sigma Aldrich Co. Ltd) in CCM medium under oil. The time for complete dissolution of the zonae was recorded at x400 magnification under an inverted Hoffman modulation contrast microscope with a 37°C stage warmer.
Statistics
2-Test with Yates correction when necessary was used to determine if differences in pregnancy rate were significant between the different categories of embryos. The percentage of blastocysts that re-expanded with or without AH was also compared using the same test. Unpaired Students t-test was used to assess the statistical differences of ZP lysis times with pronase of fresh and vitrified embryos. P < 0.05 was considered as statistically significant.
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Results |
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After 24 h of culture, 72 embryos from the AH group and 96 embryos from the non-AH group developed further. As demonstrated in Table I, the overall percentages of blastocysts which hatched or were in the stage of hatching is significantly superior when the embryos had undergone AH (50%), as compared with those with non-manipulated ZP (19%) (P < 0.001). In the AH group, 23% of the blastocysts were expanded and 48% in the non-AH groups.
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If we consider only the pregnancy rate from the good quality group, similar results in favour of the AH group are observed (Table II).
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Correlation of day 3 embryo quality (Table II)
Independent of an intact or an open ZP, we observed that the percentage of blastocysts which survived the vitrification procedure and developed further after a period of 24 h culture correlated with the type of embryo quality observed during their development on day 3.
In all, 186 and 88 blastocysts from respectively the good and fair quality groups were recovered after warming. From the good quality group, 73% (n = 135: 62 from AH and 73 from no AH) survived and were suitable for embryo transfers. However, only 38% (n = 33: 10 from AH and 23 from no AH) (P < 0.001) of embryos in the fair quality group were transferable.
The percentage of blastocysts available for embryo transfer was related to previous developmental quality. In the AH group, 62 blastocysts (74%) from the good group were transferred, and from the fair quality group, only 10 (33%) were available for transfer (P < 0.001). The same trend was observed in the no-AH group where 72% of the vitrified embryos were transferred versus 40% (P < 0.001).
Consequently, an ongoing pregnancy rate per vitrification cycle of only 9% (2/22) was obtained in the fair group. Conversely, in the good quality group, an ongoing pregnancy rate of 32% (23/71) was obtained (P < 0.05).
Even though few vitrification attempts were performed, AH appears favourable in the fair group considering that out of seven vitrificationthawing assays, the only two ongoing pregnancies were obtained after AH.
Percentages of transfers with expanded or hatched blastocysts after AH versus non-AH (Tables III and IV)
Table III compares the percentage of transfers performed with at least one hatching blastocyst in conjunction with an AH protocol or a non-AH protocol.
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When at least one hatched/hatching blastocyst was transferred, no differences in the pregnancy (48% in AH group versus 38% in non-AH group) and implantation rates (25% in AH group versus 28 % in non-AH group; not significant) were observed.
Table IV compares the percentage of transfers performed with at least one expanded blastocyst in the AH and in the non-AH group.
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Higher implantation rates (43 versus 9%; P < 0.05) were observed in the AH group when expanded blastocysts were transferred, showing the beneficial effect of the AH procedure even if the embryos did not start the hatching process.
Enzymatic treatment of the zona pellucida
From a total of nine replicate experiments, the ZP of 18 morulae or blastocysts derived from abnormally fertilized zygotes or from blastocysts that were of insufficient quality for transfers were enzymatically dissolved after vitrification procedure or no procedure.
The enzymatic digestion of the ZP with pronase was significantly faster for the unfrozen embryos. In our working conditions, an average of 1 min 45 s was necessary to dissolve the ZP of unfrozen blastocysts and an average of 2 min 16 s for the vitrified one (P < 0.0001).
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Discussion |
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When compared with our classical vitrification technique inside the straw (Vanderzwalmen et al., 2002), the present study demonstrates that application of the HS in our assisted reproduction programme results in satisfactory and encouraging clinical results.
We observed from our preliminary vitrification attempts that the highest pregnancy rates were obtained after transfers of spontaneously hatched blastocysts as compared with transfers of intact expanded blastocysts. We therefore considered improving the pregnancy rate by artificially increasing the proportion of transfers with blastocysts in the hatching process through mechanical AH.
The beneficial effect of AH after vitrification is evident considering the significant increase in the percentage of blastocysts in hatching process and the percentage of transfers with at least one hatching or completely hatched blastocyst. Consequently, after creating a large opening in the ZP by mechanical means, our results indicate that zona-opened blastocysts implant at a significantly higher rate than their zona-intact counterparts.
When transfers were performed with mechanically or spontaneously hatched blastocysts, no significant difference was noted in the pregnancy rate (48 versus 38%) and implantation rate (25 versus 28%). Higher pregnancy rates were also observed after transfers of expanded blastocysts but with previously open ZP. In such a situation, they were classified as early blastocysts before vitrification and reached the expanded stage 24 h after warming. This indicates that, when mechanical opening is performed and/or when hatching occurred, satisfactory pregnancy and implantation rates are obtained.
The lower implantation rates observed after transfers of intact expanded blastocysts, suggests failed in-utero hatching, rendering implantation difficult or impossible.
Impaired hatching may result from ZP abnormalities, embryo quality, the effect of advanced maternal age, sub-optimal culture conditions, and possibly genetic abnormalities. It is impossible to predict after thawing which embryo will re-expand and hatch spontaneously and which will benefit from the artificial opening in the ZP.
Until now, the efficacy of AH was mainly evaluated on cleavage stage fresh embryos. There is no universal agreement regarding the efficacy of AH evaluated on fresh cleavage stage embryos and on blastocysts. However, we have concluded from our results that zona opening on vitrified embryos is useful. Therefore if cryopreservation can interfere in the spontaneous hatching process, we suggest that after vitrification an opening in the ZP or even a complete removal of the ZP after pronase may be beneficial for the clinical outcome. Tucker et al. (1991) and Check et al. (1996
) showed that AH using the mechanical technique (Tucker et al., 1991
) or the zona-drilling technique (Check et al., 1996
) may be beneficial for frozenthawed embryos in overcoming ZP hardening.
During the expansion of the blastocoele, the ZP becomes distended and undergoes a thinning process before rupture allowing the blastocyst to hatch. The exact nature of zona damage after freezing is not clear, the inability of cryopreserved blastocyst to hatch spontaneously may be due to changes in the structural properties of the glycoprotein membrane disturbing the elasticity and hindering the thinning process.
Different observations led us to speculate that a hardening of the ZP during vitrification may be one of the reasons that inhibit blastocysts from hatching and hence implanting. Fresh ZP are more flexible than frozen ones, leading to a difference in the resistance of the ZP when performing mechanical AH. Additionally, the resistance to enzymatic removal of the ZP by pronase increased after vitrification as compared with the unfrozen embryo. These observations suggest that hardening of the ZP in vitro may also be due to the cryopreservation process.
Another factor interfering in the hatching process, and in the capacity of the blastocyst to survive the vitrification process and to develop further, concerns its quality at the embryo stage. We observed that a proportion of morphologically normal-looking blastocysts that we vitrified, originated from cleavage embryos with sub-optimal morphology.
It is possible that impaired embryonic vitality may also contribute to reduced capacity of the embryo to survive vitrification and also to escape from the ZP after re-expansion.
After vitrification of blastocysts showing good quality during their development, the survival rate observed immediately after thawing, the percentage of blastocysts that hatch and re-expand after 24 h culture and the proportion of blastocysts available for embryo transfer is considerably higher as compared with those exhibiting poor embryo quality during their cleavage. Even if some blastocysts exhibit normal morphological aspects after 5 days in culture, the results of our study indicate variations in the percentage of embryos surviving immediately after thawing and that re-expanded after 24 h of culture. The post-thaw survival rates depend on the cleavage characteristics during the 5 days of culture. Only those developing from good quality cleavage stage embryos could be cryopreserved and thawed with good results. The percentage of embryos available for embryo transfer is 2-fold higher in the good quality group, whereas only 40% of the vitrified fair blastocysts survived the thawing and the culture period.
This underlines the need for good scoring and selection of embryos during the whole developmental stage (Alikani et al., 2000; Scott et al., 2000
; Fisch et al., 2001
). Such an approach helps to select the best embryos not only in fresh embryo transfer but also for future cryopreserved cycles. In cases of blastocysts originating from the fair group, we felt that artificial hatching could be beneficial. Until now, the number of cases has been too small to demonstrate statistical differences, but we obtained only pregnancies (2/4 transfers) after performing zona hatching.
This is in agreement with Urman et al. (2002) who recently reported that prior to fresh embryo transfer of blastocysts, enzymatic treatment of the ZP appears to be associated with increased implantation rates, especially in patients having poor quality embryos.
In the absence of uterine enzymes, hatching is entirely dependent on embryonic vitality. This embryonic vitality corresponds to cycles of expansions and contractions of the blastocyst and to the possible secretion of lytic agents (Gonzales et al., 1996). Massip et al. (1982
) suggested in the bovine species that failure of hatching may be attributed to a functional and structural immaturity or anomaly of the trophectoderm leading to impaired accumulation and retention of fluid in the blastocoele.
Whatever the cause of failure of spontaneous hatching, the presented data provide evidence that an artificial opening created in the ZP after vitrification has no detrimental effect on the embryos and may be beneficial to overcome ZP hardening.
The only suggested adverse outcome of AH could be an increased rate of monozygotic twinning (Herschlag et al., 1999). At the present time, we have not observed monozygote twins after mechanical hatching procedure. This might be due to the fact that the two intersecting slits create a sizeable opening, reducing abnormal blastocyst escape (diabolo shape).
One may avoid monozygotic twinning by removing the zona pellucida completely by enzymatic treatment (Fong et al., 1998). We observed no adverse effect of the pronase treatment on fresh and vitrified embryos. Several pregnancies have already been obtained after soft treatment of the blastocysts before fresh or frozen embryo replacements (P.Vanderzwalmen et al., unpublished data).
Some groups are reluctant to culture embryos to the blastocyst stage because the freezing of supernumerary blastocysts is problematic. Our results achieved with the HS method demonstrate that human blastocysts can be successfully vitrified. From our data we conclude that morphologically good embryos resulted in blastocysts which performed better in vitrification and thawing procedures, resulting in higher implantation rates. Therefore, a good scoring and selection of embryos during the whole developmental stage is recommended.
Cryopreservation induces hardening of the ZP, which may impair spontaneous hatching after thawing. In our experience, the artificial opening of the ZP of thawed blastocysts, with the three-dimensional partial zona dissection technique, significantly improved the implantation and pregnancy rates.
The HS vitrification technique appears to be a good alternative to other carrier systems and should generate further research for vitrification of oocytes and cleavage stage embryos.
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Acknowledgements |
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Submitted on October 28, 2002; resubmitted on February 3, 2003; accepted on April 7, 2003.