Maria Infertility Hospital, Seoul, Korea 1 To whom correspondence should be addressed at: In Vitro Fertilization Laboratory, Maria Infertility Hospital, 103-11, Sinseol-dong, Dongdaemun-gu, Seoul, Korea. e-mail: sonyoung{at}mariababy.com
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Abstract |
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Key words: artificial shrinkage/cryopreservation/electron microscopy grid/human blastocyst/vitrification
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Introduction |
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Freezing of human blastocysts has been carried out with the slow-cooling method, but clinically satisfactory results have not been obtained (Ménézo et al., 1992; Kaufman et al., 1995
). In addition, the slow-freezing method requires expensive equipment and is time-consuming. Therefore, it is essential to establish a simple, fast and reliable procedure to optimize clinical outcomes of cryopreservation at the blastocyst stage. The successes of human blastocyst vitrification procedures have been recently increased by techniques using either electron microscopy (EM) grids (Choi et al., 2000
) or cryo-loop (Yokota et al., 2000
; Mukaida et al., 2001
) that substantially increase the cooling rate.
We also established a vitrification system using EM grids and a six-step thawing method, and have reported the clinical usefulness of the system for the cryopreservation of human blastocysts (Cho et al., 2002). However, a relatively poor survival rate for the expanded blastocysts after vitrification was obtained (Cho et al., 2002
). As previously suggested (Vanderzwalmen et al., 2002
), the loss of viability after vitrification of blastocysts could be attributed to physical damage resulting from ice formation during the cooling procedure. In fact, in contrast to early blastocyst stage embryos, expanded blastocysts consist of a blastocoele, which may disturb cryopreservative potential due to ice crystal formation during the cooling step. Recently, Vanderzwalmen et al. (2002
) reported, after vitrification of blastocyst inside a 0.25 ml straw, an increase in the survival rate of the blastocyst by artificially reducing the volume of the blastocoele.
Therefore, we attempted clinical application of artificial shrinkage before vitrification on EM grids of human blastocysts at expanded stage in our cryopreservation programme.
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Materials and methods |
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Embryo culture
Culture of fertilized oocytes was the same as described in a previous study (Yoon et al., 2001). Embryos having two pronuclei were washed well and co-cultured with cumulus cells in a 10 µl YS (Yoon Sanhyun) medium supplemented with 20% human follicular fluid (hFF) (Yoon et al., 2001
) in an atmosphere of 5% CO2, 5% O2 and 90% N2. The embryos were transferred on either day 3 or day 5. The date of the embryo transfer was determined by the number of zygotes and the quality of 2 day embryos according to published criteria (Yoon et al., 2001
). After the embryo transfer, regardless of the embryo transfer date, surplus embryos were cultured until day 6, and embryos that developed to the expanded blastocyst stage (diameter
160 µm) were cryopreserved with either slow freezing or vitrification. In all, 293 blastocysts from 121 patients were vitrified on EM grids after artificial shrinkage (see below) between February 2001 and October 2001. Of these, 25 patients who had blastocysts transferred after thawing were studied.
Preparation of vitrification solution
The freezing solution for vitrification, EFS40, was prepared according to a previously described method (Kasai et al., 1990), and consisted of 40% (v/v) ethylene glycol (EG), 18% (w/v) Ficoll 70 (
70 kDa), 0.3 mol/l sucrose and 20% hFF in Dulbeccos phosphate-buffered saline (DPBS). A pretreatment solution, DPBS (EG20) containing 20% ethylene glycol and 20% hFF, was prepared.
Artificial shrinkage of expanded blastocysts and vitrification
Artificial shrinkage of expanded blastocysts was performed with two 29-gauge needles. After holding the expanded blastocyst with the flat side of a needle and placing the inner cell mass (ICM) at the 12 or 6 oclock position, a needle was pushed trough the trophectoderm cell into the blastocoele cavity until it shrank (Figure 1). Contraction of the blastocysts was then observed after 30 s to 1 min. After complete shrinkage of the blastocoele, the blastocysts were equilibrated in EG20 for 1.5 min before exposure to the vitrification solution. The blastocysts were then incubated in EFS40, loaded onto the EM grid (IGC 400; Pelco International, CA, USA) and directly plunged in liquid nitrogen within 30 s. The EM grids containing the blastocysts were sealed in a cryovial that had previously been submerged under liquid nitrogen. The cryovials were attached in canes and stored in liquid nitrogen.
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Results |
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Based on the above result, we applied the artificial shrinkage technique clinically. Figure 1 shows the morphology of human blastocysts before and after cryopreservation. Table I shows the clinical results of the human blastocyst vitrification after artificial shrinkage. A total of 90 expanded blastocysts was vitrified and warmed from 25 patients. Eighty-one blastocysts (90.0%) were re-expanded after warming. Of the 81 blastocysts that survived, 40 had hatched (49.4%) at the time of transfer. A total of 69 blastocysts was transferred into 25 patients. The implantation rate was 29.0% (20/69) and the pregnancy rate was 48.0% (12/25). Eight male and seven female infants (six sets of twins and three singletons) from nine patients were born, one cycle had a spontaneous abortion at 6 weeks of gestation, and the other two pregnancies are ongoing. Birthweights of the infants were within the range of 19503550 g, and all delivered infants had normal physical profile up to the present.
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Discussion |
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Vitrification would be a very attractive alternative to the conventional slow-freezing protocol with advantages of the lack of ice crystal formation and ease of operation. We have already reported a vitrification method for human blastocysts on EM grids and the success of vitrification procedures has been increased by use of a six-step thawing technique that substantially reduces osmotic shock (Cho et al., 2002). In the report, acceptable pregnancy and delivery were obtained after clinical application of vitrification and six-step thawing protocols, implying that vitrification on EM grids combined with a six-step thawing protocol could be applied to human blastocyst cryopreservation. However, we have observed that the survival rate of blastocysts at the expanded stage (71%) was lower than that of blastocysts at early stage (92%) with the established vitrification methods (Cho et al., 2002
). An explanation for this was that late blastocysts consist of a fluid-filled blastocoele, which may disturb cryopreservative potential due to ice crystal formation during the cooling step. Actually, we have observed that expanded human blastocysts are dehydrated and concentrated more slowly than earlier stage embryos, suggesting that intracellular ice is more likely to form.
Recently, it has been suggested that mechanical damage caused by ice crystal formation could be avoided by reducing the fluid content of the blastocoele in expanded blastocysts using a glass micro-needle (Vanderzwalmen et al., 2002). These authors also reported that the rates of pregnancy and implantation were improved after artificial shrinkage compared with the control, intact blastocyst, group.
Similarly, we applied clinically the artificial shrinkage technique in our vitrification system, using EM grids and six-step thawing procedure, and examined the effect on survival and hatching of vitrified human blastocysts. From the high survival rate (90.0%) that we obtained with this approach in our vitrification system, we can confirm that this method is a useful technique for the vitrification of expanded human blastocysts. Furthermore, the high percentage of hatching (49.4%) seen at the time of embryo transfer might be due to the effect of assisted hatching caused by the formation of a large hole in the zona pellucida produced by using a 29-gauge needle.
We achieved higher implantation and clinical pregnancy rates (29.0 and 48.0%) by application of the artificial shrinkage in our vitrification programme, and the pregnancy outcome was similar to that obtained from the transfer of fresh blastocysts in our hospital (Yoon et al., 2001). This result was compared with the report of Vanderzwalmen et al. (2002
), although the numbers in this study were too small to detect any difference. The explanation for this observation could be that even through vitrification was performed using the same artificial shrinkage technique, clinical success might depend not only on the vitrification process, but also on the apparatus which is used in the vitrification, such as EM grid, and also on the apparatus which is used for artificial shrinkage, such as a 29-gauge needle. This is probably due to the substantially increased cooling rate achieved by EM grids compared with classical vitrification using 0.25 ml straws, which were used by Vanderzwalmen et al. (2002
). Another possible explanation could be the different culture conditions used for producing the blastocysts and the quality of expanded blastocysts cryopreserved. We cryopreserved only the good quality expanded blastocysts in our hospital.
In conclusion, this study showed that vitrification of human blastocysts at the expanded blastocyst stage using EM grids and artificial shrinkage technique is a clinically useful cryopreservation method.
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References |
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Submitted on July 31, 2002; accepted on September 12, 2002