Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts

Kenichiro Hiraoka1, Kaori Hiraoka, Masayuki Kinutani and Kazuo Kinutani

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Manual puncture of the trophectoderm of human blastocysts with a needle before vitrification increases their survival rate, but the embryos take a long time to re-expand. This study examined whether causing human blastocysts to collapse by manual pipetting before vitrification would allow more rapid re-expansion and improve pregnancy rates. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were placed in cryoprotectant and then artificially shrunk by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. The shrunken embryos were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. The blastocysts were thawed by warming and then dilution in 1 mol/l sucrose. RESULTS: Of 49 expanded vitrified blastocysts, 48 (98%) re-expanded within 3 h after warming. Following transfer (48 blastocysts in 28 cycles), 14 women (50%) became clinically pregnant, and the implantation rate was 33% (16/48). Eight healthy babies have been born in six deliveries, and the other eight pregnancies are ongoing. To date, there have been no spontaneous abortions. CONCLUSIONS: The results suggest that artificial shrinkage with pipetting is a simple and effective technique to assist successful cryopreservation of expanded blastocysts by vitrification.

Key words: artificial shrinkage/cryopreservation/cryotop/human expanded blastocyst/vitrification


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since the first pregnancy after vitrification of a human blastocyst was reported using cryostraws (Yokota et al., 2000Go), attention has focused mainly on using very small volumes of cryoprotectant. This greatly increases the cooling and warming rate, while reducing chilling injuries and ice crystal formation. The efficacy of vitrification in small volumes is demonstrated by good survival rates of early human blastocysts using the cryotop (Kuwayama and Kato, 2000Go), the cryoloop (Mukaida et al., 2001Go, 2003Go; Reed et al., 2002Go), electron microscope grids (Choi et al., 2000Go; Son et al., 2003Go) and the hemi-straw (Vanderzwalmen et al., 2003Go). However, expanded blastocysts exhibit relatively poor survival rates after vitrification (Cho et al., 2002Go). Expanded blastocysts have more blastocoelic fluid than early blastocysts, in which ice crystals may form during cooling. Recently, Vanderzwalmen et al. (2002)Go and Son et al. (2003)Go reported an increase in the survival rate of blastocysts when the volume of the blastocoele was artificially reduced with a needle. However, this method is invasive because it makes a hole in the zona and trophectoderm. On the other hand, Motoishi (2000)Go reported that the blastocoele of expanded blastocysts could be artificially collapsed by mechanical pipetting with a glass pipette slightly smaller in diameter than the blastocyst. By using this artificial shrinkage method before vitrification of human expanded blastocysts, the author reported a survival rate of 91% after warming, a clinical pregnancy rate of 65% and an implantation rate of 61% after the transfer of vitrified blastocysts. This artificial shrinkage method is thought to be non-invasive and useful for vitrification of expanded blastocysts, but the usefulness of this method has not been well documented.

Therefore, we report here on our clinical results for human expanded blastocysts subjected to artificial shrinkage by pipetting before vitrification.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and IVF
All patients who entered this vitrified blastocyst transfer programme had had previous multiple failures of conventional fresh day 2 or day 3 embryo transfers. The day of oocyte retrieval was considered as day 0. The patients' mean age was 34.3±4.1 (range, 27–45) years.

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 5–7 h after oocyte retrieval at a concentration of 100 000 to 200 000 motile sperm per ml for 5–10 oocytes, or microinjected with a single spermatozoon. Fertilization was confirmed at 15–18 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., 2003Go). We had conducted consecutive embryo transfer for 75 cycles of 70 patients who had 4.3±3.8 (range, 2–29) 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, 26–45) 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., 1992Go) 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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Figure 1. The cryotop: (a) laminate film and cover straw: (b) the cryotop covered with cover straw.

 
Preparation of vitrification solutions
The expanded blastocysts were vitrified by the method developed by Kuwayama (2001)Go using a cryotop that consists of polyethylene laminate film (20 mm x 0.7 mm x 0.1 mm, L x W x T; Kitazato Supply, Inc., Japan) (Figure 1).

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 (~100–120 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., 1998Go) and with the aid of forceps covered in a pre-cooled cover straw (Figure 1a) before closing it (Figure 1b).



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Figure 2. Cryopreserving and warming of human expanded blastocysts after artificial shrinkage. Human expanded blastocysts (a) before artificial shrinkage, (b) during artificial shrinkage by pipetting with a glass pipette ~140 µm in diameter, (c) during artificial shrinkage by pipetting with a glass pipette ~120 µm in diameter, (d) after artificial shrinkage, (e) loaded onto cryotop with cryoprotectant solution, and (f) 3 h after warming. Scale bars (a, b, c, d and f)= 100 µm. Scale bar (e)=700 µm.

 
Warming of blastocysts
Before warming blastocysts, 1.0 mol/l sucrose solution, 0.5 mol/l sucrose solution, and the base medium 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. The warming procedure was done as follows. The cryotop tip with the blastocysts was plunged directly into 1.0 mol/l sucrose solution for 1 min. The blastocysts were then transferred to 0.5 mol/l sucrose solution for 3 min and washed twice in the base medium for 5 min. All steps were completed on the stage warmer of a dissecting microscope at 38°C. All of the warmed blastocysts were still within their zonae and then cultured in Blastocyst medium (Irvine) containing 10% SSS (Irvine) for further culture until transfer. The post-warming survival of blastocysts was observed 3 h after warming under a microscope, and re-expanded blastocysts were judged to have survived (Figure 2f). Embryo transfer was scheduled on day 5 after ovulation in the spontaneous cycles irrespective of whether they had been vitrified on day 5 or day 6. The time from warming to transfer ranged from 3 to 5 h. One to three surviving blastocysts were transferred into the patient's uterus. Pregnancy was first assessed by urinary hCG 9 days after blastocyst transfer, and then clinical pregnancy was confirmed by the presence of fetal heart activity 30 days after blastocyst transfer.

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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In all, 10 expanded blastocysts were cryopreserved by a slow freezing method without performing artificial shrinkage and thawed for three patients in four thawing cycles. Six blastocysts (60%) re-expanded after warming. A total of 10 blastocysts was transferred to three patients in four cycles. The implantation rate was 10% (1/10) and the pregnancy rate was 25% (1/4).

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)Go. 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, 3–18) 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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Table I. Results of human expanded blastocyst vitrification on day 5 or day 6 after artificial shrinkage

 

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Table II. Birth outcome for pregnancies following human expanded blastocyst vitrification on day 5 or day 6 after artificial shrinkage

 
The effect of the day of vitrification day is shown in Table I. Of 49 vitrified blastocysts, 24 (49%) were vitrified on day 5, and 25 (51%) on day 6. Although the average time of contraction with day 5 blastocysts (7.4±4.5 min) was shorter than that with day 6 blastocysts (8.9±4.2 min), the difference was not statistically significant. The re-expansion, implantation and pregnancy rates of day 5 blastocysts (100%, 24/24; 29%, 7/24; and 54%, 7/13) were similar compared with those of day 6 blastocysts (96%, 24/25; 38%, 9/24; and 47%, 7/15).

The average contraction time of the blastocysts transferred to pregnant women (8.0±4.0 min, range 3–18) was similar compared with that of the embryos transferred to women who failed to become pregnant (8.3±4.9 min, range 3–18). 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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study demonstrates that the artificial shrinkage of the blastocoele by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts.

Unlike the method of Vanderzwalmen et al. (2002)Go and Son et al. (2003)Go 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 (0.5–2 min) of Vanderzwalmen et al. (2002)Go and Son et al. (2003)Go. 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)Go and Son et al. (2003)Go. 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., 2003Go), 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)Go and Son et al. (2003)Go 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)Go. 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., 2003Go). 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., 2004Go). 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.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cho HJ, Son WY, Yoon SH, Lee SW and Lim JH (2002) An improved protocol for dilution of cryoprotectants from vitrified human blastocysts. Hum Reprod 17, 2419–2422.[Abstract/Free Full Text]

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Goto S, Takebayashi K, Shiotani M, Fujiwara M, Hirose M and Noda Y (2003) Effectiveness of 2-step (consecutive) embryo transfer. Comparison with cleavage-stage transfer. J Reprod Med 48, 370–374.[ISI][Medline]

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Kuwayama M (2001) Vitrification of human oocytes and embryos. In Suzuki S (ed). IVF Update [Japanese]. Medical View Co, Tokyo, Japan, pp. 230–234.

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Vajta G, Lewis IM, Kuwayama M, Greve T and Callesen H (1998) Sterile application of the open pulled straw (OPS) vitrification method. Cryo Lett 19, 389–392.[ISI]

Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, van Roosendaal E, Vandervorst M, Bollen N, Zech H, Mukaida T, Takahashi K and Schoysman R (2002) Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 17, 744–751.[Abstract/Free Full Text]

Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, Bollen N, van Roosendaal E, Vandervorst M, Schoysman R and Zech N (2003) Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum Reprod 18, 1504–1511.[Abstract/Free Full Text]

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Submitted on April 26, 2004; accepted on August 12, 2004.





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