Human Reproduction, Vol. 17, No. 9, 2419-2422,
September 2002
© 2002 European Society of Human Reproduction and Embryology
An improved protocol for dilution of cryoprotectants from vitrified human blastocysts*
In vitro Fertilization Laboratory, Maria Infertility Hospital, 103-11, Sinseol-dong, Dongdaemun-gu, Seoul, Korea
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Abstract |
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BACKGROUND: The purposes of this study were to compare the survival rate of human blastocysts thawed by two different methods after vitrification using electron microscopic (EM) grids, and to report the successful pregnancies and live births resulting from the transfer of blastocysts that had survived the established thawing method. METHODS: The blastocysts produced from three pronuclei zygotes in IVF cycles were vitrified on an EM grid. After dilution of cryoprotectant by either a 6- or 2-step method, the survival rate of the blastocysts was compared. RESULTS: The survival rate of blastocysts thawed using the 6-step method (82.6%, 100/121) was significantly higher than that of the 2-step method (50.6%, 87/172; P < 0.01). Therefore, we applied the 6-step method in our blastocyst cryopreservation programme. Overall, 34.1% (14/41) of clinical pregnancies and 11 live births were achieved. CONCLUSIONS: The present results indicated that a 6-step thawing method was more effective than a 2-step thawing method for thawing vitrified human blastocysts.
Key words: EM grid/human blastocyst/live birth/survival rate/vitrification
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cryopreservation of human embryos has become a routine procedure to increase cumulative pregnancy rates, to help avoid the risk of multiple pregnancies after the transfer of many embryos and to avoid unnecessary additional stimulation procedures.
Recently, blastocyst transfer based on an improved culture system has been proven effective for increasing the pregnancy rate in assisted reproductive technology (Gardner et al., 1998
; Yoon et al., 2001). Therefore, a reliable procedure for the cryopreservation of supernumerary blastocysts is needed because only a small number of blastocysts after transfer are likely to be available for cryopreservation. 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). Therefore, it is essential to establish a simple, fast and reliable procedure to optimize clinical outcomes of cryopreservation at the blastocyst stage. Several investigators have reported the clinical usefulness of vitrification for the cryopreservation of human embryos (Mukaida et al., 1998; Yokota et al., 2000; Vanderzwalmen et al., 2002).
Martino and colleagues observed a higher percentage of blastocyst formation from oocytes surviving vitrification on electron microscopic (EM) grids to obtain more rapid cooling and warming rates (Martino et al., 1996). Since then, several researchers have applied EM grids successfully in the vitrification of human oocytes and blastocysts (Choi et al., 2000; Yoon et al., 2000). In addition, we reported previously that a higher survival rate of bovine blastocysts derived from in-vitro maturation (IVM) could be obtained by a simple 2-step vitrification method using EM grids and freezing solution (Park et al., 1999). Several successful cases of human blastocyst vitrification have been reported (Choi et al., 2000; Yokota et al., 2000; Mukaida et al., 2001). EM grids (Choi et al., 2000) or cryo-loop (Mukaida et al., 2001) have been used to substantially increase the cooling rate. In this study, we compared a new thawing protocol with a protocol used for bovine blastocysts (Park et al., 1999) on the survival of vitrified human blastocysts. In addition, we attempted clinical application of the new thawing protocol of vitrified blastocysts.
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Materials and methods |
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Approval for the study was obtained from the Institutional Review Board of the Maria Infertility Hospital.
IVF of oocytes
Women were treated with GnRH agonist and hMG in either a long or short treatment protocol. When more than two follicles reached 18 mm in diameter, 10 000 IU hCG (IVF-C; LG Chemical, Korea) was administered. Oocytes were retrieved transvaginally 36–38 h after hCG injection and the oocytes were inseminated by either conventional IVF or ICSI. Fertilization was examined 17–19 h after insemination for the presence and number of pronuclei (PN).
Embryo culture
The procedure used to culture fertilized oocytes was the same as described in a previous study (Yoon et al., 2001). All zygotes were co-cultured with cumulus cells in a 10 µl YS (Yoon Sanhyun) medium supplemented with 10% hFF for 5 or 6 days (Yoon et al., 2001). The hFF was prepared using a previously reported method (Chi et al., 1998). The cumulus cells for co-culture were prepared using the method reported by Yoon et al. (Yoon et al., 2001). The developed blastocysts were classified according to their degree of expansion as follows (Figure 1): Early blastocyst (ErB), the blastocoele is less than half the volume of the embryo and diameter <140 µm; early expanding blastocyst (EEB), the blastocoele is greater than, or equal to, half of the volume of the embryo and diameter 140–160 µm; middle expanding blastocyst (MEB), the blastocoele completely fills the embryo and diameter 160–180 µm; expanded blastocyst (EdB), the blastocoele volume is larger than that of the early embryo with thinning zona and diameter >180 µm.
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Vitrification of blastocysts using an EM grid
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; Sigma Chemical Co., St Louis, USA), 18% (w/v) Ficoll (Ficoll 70, average MW 70 000 Da; Pharmacia Biotech, Uppsala, Sweden), 0.3 mol/l sucrose and 20% hFF in modified Dulbecco’s phosphate-buffered saline (m-DPBS). A pretreatment solution, m-DPBS (EG20) containing 20% EG and 10% hFF, was prepared. The blastocysts were classified according to developmental stage and vitrified on an EM grid. The blastocysts were equilibrated in EG20 for 1.5 min before exposure to the vitrification solution at room temperature. The blastocysts were then incubated in EFS40 at room temperature, loaded onto the EM grid (IGC 400; Pelco International, CA, USA), had excess cryoprotectant removed using sterilized filter paper, and were directly plunged in LN2 within 30 s. The EM grids containing the blastocysts were sealed in a cryovial that had previously been submerged under LN2. The cryovials were attached in canes and stored in LN2.
Thawing of blastocysts derived from 3PN zygotes
For thawing of vitrified blastocysts, EM grids containing blastocysts at the same developmental stage were divided randomly into two groups. For one group a 2-step cryoprotectant dilution method was used in which the EM grids stored in LN2 were directly transferred into a 100 µl drop of 0.3 mol/l sucrose solution (prepared in DPBS containing 20% hFF) as soon as possible, and then quickly transferred into fresh 0.3 mol/l sucrose and incubated for 1.5 min at room temperature. Recovered blastocysts were transferred into DPBS containing 20% hFF at room temperature. After 1.5 min, the embryos were washed three times in culture medium and co-cultured with cumulus cells in 10 µl YS medium containing 10% hFF.
For the other group a 6-step cryoprotectant dilution method was used in which the EM grids containing blastocysts were transferred to a 100 µl drop of 0.5 mol/l sucrose. After 3 min, the blastocysts were transferred sequentially to 100 µl drops containing 10% hFF in DPBS supplemented with 0.4, 0.3, 0.2, 0.1 and 0 mol/l sucrose at intervals of 1.5 min at room temperature. The blastocysts were then washed three times in culture medium and co-cultured with cumulus cells in 10 µl YS medium containing 10% hFF. The post-thawing survival of blastocysts was observed ~18–20 h after warming under a microscope, and blastocysts with a morphologically intact inner cell mass, trophectoderm and re-expanding blastocoele were judged to have survived.
Thawing of blastocysts derived from 2PN zygotes (clinical use)
A total of 806 blastocysts from 258 patients were cryopreserved by vitrification. The patients consented to have their supernumerary embryos vitrified after fresh embryo transfer. The 6-step cryoprotectant dilution method and blastocyst transfer after thawing was conducted in 41 patients between September 1999 and July 2000. The blastocysts had been stored for 3–12 months in LN2. The age and duration of infertility of the 41 patients (mean ± SD) were 32.6 ± 3.7 and 3.1 ± 2.7 years respectively. The causes of infertility were male factor in 11, tubal factor in 19, unexplained infertility in two, anovulation in three and mixed causes in six patients. Vitrified day 5 and 6 blastocysts were thawed in the afternoon of the day before embryo transfer. Embryo transfer was scheduled on days 4–5 after ovulation in the spontaneous cycles of 38 patients, or on days 19–20 in artificial cycles prepared with exogenous estrogen and progesterone for three anovulatory patients. One to three surviving blastocysts were transferred into the patient’s uterus. Pregnancy was first assessed by serum ß-hCG 9 days after blastocyst transfer, and then clinical pregnancy was determined by the presence of fetal heart activity 30 days after blastocyst transfer.
Statistical analysis
Differences between treatments groups in each experiment were compared with the 
2-test using the Statistical Analysis System (SAS Institute, Cary, NC, USA) software package.
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Results |
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In the preliminary experiment, 293 out of 930 3PN zygotes developed to blastocyst stage (31.5%) and were cryopreserved by vitrification. Blastocysts were thawed with either the 2- or 6-step cryoprotectant dilution method. Figure 2
shows the morphology of human blastocysts before and after cryopreservation. Table I shows the survival rate of blastocysts after thawing. As shown in Table I, the survival rate of blastocysts following the 6-step dilution method was significantly higher than that of the 2-step dilution method (P < 0.01). There was a tendency towards higher survival in the early blastocyst stages (ErB, EEB) than in the late (MEB, EdB) in both thawing methods, but the difference was not significant.
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Based on the above result, we applied the 6-step thawing method clinically. Table II
shows the clinical results. A total of 41 patients and 120 blastocysts were included in this study. There were 90 day 5 and 30 day 6 frozen blastocysts. There was no difference in survival rates between day 5 (84.4%, 76/90) and day 6 (83.3%, 25/30) frozen blastocysts. After thawing of day 5 frozen blastocysts, 90.9% of ErB (20/22), 91.9% of EEB (34/37), 72.2% of MEB (13/18) and 69.2% of EdB (9/13) became re-expanded. After thawing of day 6 frozen blastocysts, 100% of ErB (6/6), 90% of EEB (9/10), 66.7% of MEB (4/6), and 75% of EdB (6/8) became re-expanded. Out of 101 survived blastocysts, 15 (14.6%) hatched at the time of embryo transfer. Before transfer, all embryos for each patient were pooled and selected for transfer. All 41 patients underwent embryo transfer. After transfer, 14 clinical pregnancies (34.1%) were achieved. Nine male and six female infants (four sets of twins and seven singletons) from 11 patients were born and three others ended in miscarriage. Birth weights of the infants were within the range of 1850–3750 g, and all delivered infants had normal physical profiles up to the present.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, for the first time, we examined the survival rate of vitrified human blastocysts after different cryoprotectant dilution methods. Acceptable pregnancy and delivery were obtained after clinical use of vitrification and 6-step cryoprotectant dilution protocols. This study indicates that vitrification on EM grids and 6-step cryoprotectant dilution can be applied to human blastocyst cryopreservation.
Cryopreserved blastocyst survival depends in part on the freezing–thawing procedure. Previously, Park et al. reported that higher survival and hatching rates of vitrified–thawed bovine blastocysts could be obtained using EM grids and a 2-step cryoprotectant dilution (Park et al., 1999). In our hospital, a few investigations into various concentrations and times of cryoprotectant dilution after thawing to improve the efficiency of human blastocysts vitrified on EM grids resulted in disappointing survival rates (data not shown). However, we had obtained a good survival rate by changing the first concentration of sucrose to 0.5 mol/l for 5 min in cryoprotectant dilution after thawing. Thus, we compared a new thawing protocol of 6-step dilution with a 2-step dilution used for bovine blastocysts (Park et al., 1999) on the survival of vitrified human blastocysts derived from 3PN. We obtained a low survival rate (50.6%) of vitrified–thawed human blastocysts using a 2-step cryoprotectant dilution. In contrast, a higher survival rate (82.6%) with a 6-step thawing method was obtained. When we observed microscopically the blastocysts during the thawing process of a 2-step cryoprotectant dilution, thawed human blastocysts were morphologically normal in first 0.3 mol/l sucrose solution, but degenerated abruptly in second 0 mol/l sucrose solution.
Thus, the low percentage of blastocysts that survived after thawing in a 2-step dilution method might be due to dramatic osmotic shock. So, it could be speculated that the higher survival of human blastocysts in a 6-step cryoprotectant dilution may result from decreasing osmotic shock. Another author (Kobayashi et al., 1998) also demonstrated that stepwise dilution of the cryoprotectant after warming appears to reduce or perhaps eliminate osmotic injury to cells of vitrified porcine embryos.
It was observed that the survival rate of blastocysts at late stage (MEB, EdB) was lower than that of blastocysts at early stage (ErB, EEB) in both 2- and 6-step dilution methods, although the numbers in this study were too small to tell any possible significant difference. The explanation for this observation could be that late blastocysts consist of a full-filled blastocoele, which may disturb cryopreservative potential due to ice crystal formation caused by inadequate permeation of the cryoprotectants during the cooling step. Actually, we have observed that late human blastocysts are less permeable than earlier stage embryos, suggesting that intracellular ice is more likely to form as has been previously asserted (Vanderzwalmen et al., 2002). Therefore, further study is necessary to avoid mechanical damage, which may be caused by ice crystal formation, and this possibility is under investigation.
We applied the 6-step thawing method clinically in vitrified human blastocysts in our cryopreservation programme. After thawing of the vitrified blastocysts by the 6-step method, 84.2% of thawed blastocysts survived. Several investigators have reported high survival and pregnancy rates by vitrification of human blastocysts (Mukaida et al., 1998; Yokota et al., 2000). Mukaida et al. showed 63% survival and a 32% clinical pregnancy rate in human blastocysts vitrified on nylon loops (Mukaida et al., 2001). Yokota et al. reported 80% survival and a 33.3% clinical pregnancy rate after vitrification that used a modified vitrification solution and straw (Yokota et al., 2000). These studies are similar to our results. In contrast, Choi and colleagues observed that 51.6% of human blastocysts survived after thawing (Choi et al., 2000). It could be implied that even in vitrification performed using the same EM grids as this study, survival of blastocysts after thawing might depend on the concentrations and times they are exposed to cryoprotectant solutions. Another possible explanation could be the different culture conditions used for producing the blastocysts. We obtained a reasonable clinical pregnancy (34.1%) rate following transfer, but this pregnancy outcome was lower than that obtained from the transfer of fresh blastocysts in our hospital (Yoon et al., 2001). Therefore, further studies are necessary to determine the optimal method for vitrification of human blastocysts in order to improve viability after thawing.
A disadvantage of vitrification on EM grids or loops is the risk of contamination for pathogens such as virusus, prions and bacteria caused when the vitrification solution comes into direct contact with LN2 during cooling or storage (Bielanski et al., 2000). Although there are no cases of contamination occurring via LN2 in our vitrification system until now, the development of safety strategies for reducing the risk of contamination by larger pathogens is necessary.
In this report, we have shown that a 6-step thawing method was more effective than a 2-step method after vitrification of human blastocysts on EM grids. In addition, we achieved acceptable pregnancy rates clinically by combining vitrification on an EM grid and 6-step thawing. Therefore, vitrification of human embryos at the blastocyst stage will be a reliable approach in the near future.
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Notes |
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1 To whom correspondence should be addressed. E-mail: sonyoung{at}mariababy.com
* Presented at the 56th annual meeting of the American Society for Reproductive Medicine, San Diego, California, USA, Oct. 21–26, 2000
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Submitted on January 4, 2002; resubmitted on March 18, 2002; accepted on May 9, 2002.
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