Kinutani Women's Clinic, 2-1-4-3F, Ohtemachi, Naka-ku, Hiroshima 730-0051, Japan
1 To whom correspondence should be addressed. Email: hiraoka{at}chive.ocn.ne.jp
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Abstract |
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Key words: artificial shrinkage/cryopreservation/cryotop/human expanded blastocyst/vitrification
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Introduction |
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Therefore, we report here on our clinical results for human expanded blastocysts subjected to artificial shrinkage by pipetting before vitrification.
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Materials and methods |
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Women were treated with GnRH analogue buserelin acetate (Mochida, Japan) from either the preceding mid-luteal phase in a long treatment protocol or from the second day of the cycle in a short treatment protocol. Controlled ovarian stimulation was carried out with hMG (Nikken, Japan) or urinary FSH (Fertinorm; Serono, Japan). Follicular development was monitored with serial vaginal ultrasound examinations and serum E2 measurements. hCG (Teizo, Japan) was administered when dominant follicles reached a diameter of 18 mm. Oocytes were collected 35 h after hCG (Teizo) administration using a vaginal ultrasound-guided procedure and were incubated in HTF medium (Irvine Scientific, USA) containing 10% (v/v) serum substitute supplement (SSS; Irvine) at 37°C in an atmosphere of 6% CO2, 5% O2 and 89% N2. Sperm preparation was carried out using discontinuous IsolateTM (Irvine) gradient. Mature oocytes were either inseminated with sperm 57 h after oocyte retrieval at a concentration of 100 000 to 200 000 motile sperm per ml for 510 oocytes, or microinjected with a single spermatozoon. Fertilization was confirmed at 1518 h after insemination by the presence of two pronuclei.
Embryo culture
Fertilized oocytes were washed well and cultured in P-1TM Medium (Irvine) or Blast Assist Medium 1 (Medicult, Denmark) until day 3, and then placed in Blastocyst medium (Irvine) or Blast Assist Medium 2 (Medicult) until day 6. In all cases, consecutive embryo transfer was performed, in which one or two cleaved embryos were transferred on day 2 or day 3 (first step of embryo transfer) and one or two blastocysts were transferred on day 5 or day 6 (second step of embryo transfer) (Goto et al., 2003). We had conducted consecutive embryo transfer for 75 cycles of 70 patients who had 4.3±3.8 (range, 229) prior failures of conventional fresh day 2 or day 3 embryo transfers between October 2001 and April 2004. The mean patients' age was 35.4±4.2 (range, 2645) with an average of 11.0±8.0 matured oocytes retrieved per cycle. The fertilization rate was 70.7% (584/826) with a mean of 7.8±4.2 fertilized embryos per retrieval. An average of 1.5±0.5 embryo was transferred on day 2 or day 3 and 1.1±0.4 blastocyst on day 5 or day 6. The clinical pregnancy rate was 48.0% (36/75) per retrieval, with a 21.2% (41/193) implantation rate and a 39.0% (185/474) expanded blastocyst development rate on day 5, 57.0% (270/474; days 5 and 6) on day 6. During this period, two blastocyst culture media systems were used in order to evaluate and compare the results of consecutive embryo transfer between two sequential media at the same gas phase (37°C in an atmosphere of 6% CO2, 5% O2 and 89% N2) in each case. However, blastocyst culture media systems had no effect on the percentage of expanded blastocysts on days 5 and 6, or on the pregnancy and implantation rates. After a consecutive embryo transfer, surplus embryos that developed to the expanded blastocyst stage (diameter
160 µm) (Figure 1a) were cryopreserved on day 5 or day 6. Eighteen blastocysts from four patients were cryopreserved with a slow cooling method (Ménézo et al., 1992
) without performing artificial shrinkage between October 2001 and May 2002. In all, 161 blastocysts from 63 patients were vitrified between July 2002 and April 2004 after artificial shrinkage by a pipetting method (see below). Data for 49 blastocysts that had a zona at the start of cryopreservation from 29 cycles of warming (28 patients) are presented. Of these 28 patients, 26 women had never carried an IVF pregnancy to term and had failed to become pregnant after consecutive embryo transfer. One woman had conceived and delivered by consecutive embryo transfer after six previous failures (two fresh day 2, one frozen day 2, two fresh day 3, one frozen day 3 embryo transfers). One woman had conceived and delivered following fresh day 2 embryo transfer after two previous failures (two fresh day 2 embryo transfers); since then the woman failed to conceive after two transfers (one fresh day 2, one consecutive).
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As the base medium, Dulbecco's phosphate-buffered saline solution (PBS 1x; Irvine) plus 20% (v/v) SSS (Irvine) was used. The equilibration solution contained 7.5% (v/v) ethylene glycol (EG) (Sigma Chemical Co., USA) and 7.5% (v/v) dimethylsulphoxide (DMSO) (Kanto Chemical Co., Japan). The vitrification solution was composed of 15% (v/v) EG, 15% (v/v) DMSO and 0.5 mol/l of sucrose (Nacalai Tesque, Inc., Japan). Both cryoprotectant solutions were warmed 10 min in a heated incubator at 37°C in an atmosphere of 100% air, which was long enough for the temperature to reach
35°C, and blastocysts were handled on the stage warmer of a dissecting microscope at 38°C.
Artificial shrinkage of expanded blastocysts and vitrification
Before starting the vitrification procedure, artificial shrinkage was performed as soon as the expanded blastocysts were placed in the equilibration solution. First, pipetting of the expanded blastocyst was conducted with a glass pipette slightly smaller in diameter (140 µm) than the expanded blastocyst (Figure 2b). The pipettes used for artificial shrinkage were manually made from a Pasteur pipette hand-drawn using a spirit lamp. The size of the pipette was assessed using stage micrometer (Nikon Co., Japan). The pipette opening was not flamed. After confirmation of slight shrinkage of the blastocoele, pipetting was performed with a pipette slightly smaller in diameter than the first one (
100120 µm) (Figure 2c). This procedure was repeated two or three times until the blastocoele collapsed completely (Figure 2d). Although the shape of the zona and blastocyst was distorted by being aspirated into the pipette, the zona remained intact and all embryos remained within their zona after the artificial shrinkage. After blastocoele contraction, the blastocysts were equilibrated in the equilibration solution for another 2 min before exposure to the vitrification solution. The blastocysts were then incubated in the vitrification solution and loaded onto the tip of the cryotop within 45 s with
1 µl of cryoprotectant solution (Figure 2e). Then the cryotop was immediately plunged into liquid nitrogen which had been filter-sterilized through a 0.22 µm filter (Millipore, Ireland) (Vajta et al., 1998
) and with the aid of forceps covered in a pre-cooled cover straw (Figure 1a) before closing it (Figure 1b).
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Statistical analysis
The data obtained were examined for differences using Student's group t-tests and Fisher's exact probability test as appropriate. Results are expressed as mean±SD.
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Results |
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In a preliminary experiment, we evaluated the effect of artificial shrinkage on blastocyst survival after warming of poor quality vitrified day 6 expanded blastocysts, which had been donated by consenting patients. The rate of survival (90%, 9/10) in the artificial shrinkage group was significantly higher than a group (40%, 4/10) which was vitrified after only a short (2 min) equilibration step using a protocol adopted from Lane et al. (1999). Usefulness of the procedure was noted.
Based on the above result, we applied the artificial shrinkage technique clinically. Figure 1 shows the morphology of human expanded blastocysts before and after cryopreservation. An average of 8.2±4.4 (range, 318) min was necessary for the blastocoele of expanded blastocysts to collapse completely before vitrification using artificial shrinkage by the pipetting method. Since the blastocysts were moved to the vitrification solution 2 min after collapsing, the embryos were in the equilibration solution between 5 and 20 min. The results of human expanded blastocyst vitrification after artificial shrinkage are summarized in Table I. A total of 49 expanded blastocysts were vitrified and warmed from 28 patients in 29 warming cycles. Forty-eight blastocysts (98%) re-expanded after warming. In one cycle for one patient, no blastocyst survived and embryo transfer was not conducted. A total of 48 blastocysts was transferred to 27 patients in 28 cycles. The implantation rate was 33% (16/48) and the pregnancy rate was 50% (14/28). Three male and five female infants (one set of triplets and five singletons) from six patients have been born (Table II), and all of the other eight pregnancies are ongoing and diagnosed as singletons. The triplets following transfer of three day 6 blastocysts were diagnosed as non-identical by ultrasound at 7 weeks of gestation and resulted in delivery of one male and two female infants. To date, there have been no spontaneous abortions, and all delivered infants have had normal physical profiles up to the present.
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The average contraction time of the blastocysts transferred to pregnant women (8.0±4.0 min, range 318) was similar compared with that of the embryos transferred to women who failed to become pregnant (8.3±4.9 min, range 318). One woman became pregnant following the transfer of two vitrified blastocysts that took 16 and 18 min to collapse (i.e. 18 and 20 min equilibration time) respectively.
During this study, two blastocyst culture media systems were used, but there is no difference in survival, implantation or pregnancy rates after warming in relation to the medium used.
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Discussion |
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Unlike the method of Vanderzwalmen et al. (2002) and Son et al. (2003)
using a needle, we collapsed the blastocoele by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. This artificial shrinkage method was simple because the pipetting procedure was much the same as that of removal of cumulus cells of mature oocytes before ICSI.
The mechanism of collapsing the blastocoele by mechanical pipetting is unclear. The pipetting procedure may cause some damage and rupture of the trophectoderm, possibly at a point of weakness, for example in the region of dividing cells where the intercellular contacts may be less strong than normal. Hence, it is suggested that blastocoelic fluid escapes via some rupture of trophectoderm cells by the pressure of the pipetting procedure on the blastocyst. However, there were two observations that lead us to suggest that the damage of artificial shrinkage by the pipetting method is less compared with that of the method using a needle.
First, the average time of blastocyst contraction (8.2±4.4 min) using artificial shrinkage by the pipetting method was longer than the method (0.52 min) of Vanderzwalmen et al. (2002) and Son et al. (2003)
. If the time taken to collapse is determined by the size of the hole, then pipetting makes smaller holes than needles. The blastocysts vitrified in this study were collapsed artificially in the equilibration solution. In addition, the blastocysts were equilibrated in the equilibration solution for another 2 min after blastocoele contraction, and so might be damaged as a result of the chemical toxicity of the cryoprotectant. However, the equilibration solution used in our clinic contained a lower concentration of permeating cryoprotectant (7.5% EG and 7.5% DMSO) than that of the reports (20% EG) of Vanderzwalmen et al. (2002)
and Son et al. (2003)
. The lower concentration of the equilibration solution used in the present study might be effective in minimizing the risk of injury caused by chemical toxicity.
Secondly, warmed re-expanded blastocysts, shrunk artificially by the pipetting method, formed a large blastocoelic cavity 3 h after warming (Figure 1f). The re-expansion time after warming of vitrified blastocysts in the present study (
3 h) was not so different from that of vitrified blastocysts without performing artificial shrinkage (
2 h) (Mukaida et al., 2003
), thus confirming rapid repair of the trophectoderm. Therefore, the viability of the post-warming expanded blastocysts could be assessed 3 h after warming. On the other hand, Vanderzwalmen et al. (2002)
and Son et al. (2003)
estimated the blastocyst survival post-warming
20 h after warming. The blastocysts shrunk artificially by a needle might take a long time to re-expand because of the hole made in the trophectoderm.
The survival rate (100%) of day 5 blastocysts that were vitrified in the present study was not different from that of the report (87%) of Mukaida et al. (2003). However, the survival rate (96%) of day 6 blastocysts in the present study was significantly higher than that of the report (55%). Since both groups use the same equilibration solution, the higher survival in our study may reflect our longer exposure time and the artificial shrinkage. The day 6 blastocysts tend to be at the expanded stage and may be less permeable to cryoprotectants (Mukaida et al., 2003
). Therefore, the day 6 blastocysts may be more vulnerable to damage by ice formation or osmotic stress compared to the day 5 blastocysts. However, both injuries of vitrified day 6 blastocysts can be avoided by the artificial shrinkage. We have previously reported a successful pregnancy after vitrification of human day 6 hatched blastocyst using the same artificial shrinkage method (Hiraoka et al., 2004
). The woman has now delivered a healthy girl (3128 g) at 38 weeks of gestation (unpublished data). No obvious anomalies were detected. Consequently, we can confirm that the artificial shrinkage method is a useful technique for the vitrification of both day 6 blastocysts and even zona-free hatched blastocysts.
In conclusion, this study showed that vitrification of human blastocysts at the expanded stage using artificial shrinkage by pipetting is a clinically useful and simple cryopreservation method.
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References |
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Submitted on April 26, 2004; accepted on August 12, 2004.
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