Hum. Reprod. Advance Access originally published online on August 26, 2006
Human Reproduction 2006 21(12):3246-3252; doi:10.1093/humrep/del285
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Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts
1 Hiroshima HART Clinic, Naka-ku, Hiroshima and 2 Tokyo HART Clinic, Shibuya-ku, Tokyo, Japan
3 To whom correspondence should be addressed at: Hiroshima HART Clinic, 5-7-10 Ohtemchi, Naka-ku, Hiroshima, 730-0051, Japan. E-mail: info{at}hiroshima-hart.jp
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Abstract |
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BACKGROUND: Since we reported the first successful birth from a blastocyst vitrified using a cryoloop technique, our results showed that the survival rate of vitrified blastocysts was negatively correlated with the expansion of the blastocoele. We speculated that a large blastocoele may disturb the efficacy of vitrification. Therefore, we evaluated the effectiveness of artificial shrinkage (AS) of blastocoeles before vitrification, on increasing the survival rate of vitrified blastocysts. METHODS: Supernumerary expanded blastocysts on day 5 were vitrified after AS, which was performed by puncturing the blastocoele with a micro-needle, or by making a hole in the blastocoele with a laser pulse. After warming, viable blastocysts (confirmed by re-expansion of the blastocoele) were transferred to patients with hormone replacement cycle. We compared these data with those of our previous report where AS was not carried out. RESULTS: The survival rate was significantly higher (97.2%, 488/502) in this study than that of the previous report (86%). After 266 transferable cycles, 160 patients became pregnant (60.2%), which was significantly higher than our previous results (34.1%, 29/85). The implantation rate was 46.7% (209/448). CONCLUSIONS: Our results revealed that the survival rate and the pregnancy rate of vitrified expanded and hatching blastocysts can be improved by using AS to collapse the blastocele before vitrification.
Key words: artificial shrinkage/blastocoele/cryoloop/human blastocyst/vitrification
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Introduction |
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Recent advances in culture systems with sequential media have made it possible to develop human IVF embryos into blastocysts quite easily. Because the blastocyst is better suited to the uterine environment and blastocyst formation is a form of selection for more viable embryos, blastocyst transfer has become a promising option to raise the pregnancy rate (Gardner et al., 1998
; Cruz et al., 1999). Accordingly, the need to cryopreserve human blastocysts has increased.
Human blastocysts were successfully vitrified in straws (Yokota et al., 2001). However, our attempts to vitrify human blastocysts using straws resulted in disappointing survival rates (unpublished data), and others (Vanderzwalmen et al., 1999) also reported a low pregnancy rate with this method. This is probably because human blastocysts are much less permeable to cryoprotectant and water, as we have observed that they shrink more slowly than mouse and bovine blastocysts in the cryoprotectant solution. This in turn suggests that human blastocysts are more likely to be injured by intracellular ice.
Increased rates of cooling and warming can help circumvent the problem of intracellular ice formation in less-permeable embryos. Faster rates of cooling and warming can be achieved by minimizing the volume of the solution with which embryos are vitrified, that is, by using minute tools such as electron microscopic grids (Martino et al., 1996), open pulled straws (Vajta et al., 1998), cryoloops (Lane et al., 1999a), Hemi-straw (Vanderzwalmen et al., 2003) or cryotop (Kuwayama et al., 2005) (for a review, see Kasai, 2002). It was shown that the transfer of human blastocysts vitrified with cryoloops leads to successful births (Mukaida et al., 2001). After this report, we summarized the results obtained with 223 warming cycles and confirmed the effectiveness of the cryoloop technique in the cryopreservation of human blastocysts (Mukaida et al., 2003a). However, our previous report revealed the survival rates were dependent on the developmental stage of blastocysts and were negatively correlated with the expansion of the blastocoele (Mukaida et al., 2003a). The survival rates of early blastocysts with smaller blastoceolic cavities, which were scored 1 and 2 according to Gardner’s criteria (Gardner and Schoolcraft, 1999), were 87% (48/55) and 97% (62/64), respectively. Also, full blastocysts lacking an expanded blastoceolic cavity, which were scored 3, had a survival rate of 89% (99/111). The total survival rate of blastocysts scored 1–3 together was 91% (209/230). However, the survival rate of both expanded and hatching blastocysts, scored 4 and 5 respectively, was 85.0% (288/339), which was significantly lower than that of the score 1–3 group (p<0.05). We therefore postulated that a large blastocoele might lessen cryopreservative potential because of ice crystal formation during the rapid cooling of the vitrification process. To overcome this problem, we thought shrinkage of the blastoceole to be the appropriate approach. Several studies reported an increase in the survival rate of blastocysts when the volume of the blastocoele was artificially reduced with a glass micro-needle (Vanderzwalmen et al., 2002), a 29-gauge needle (Son et al., 2003) and a micropipetting with a hand-drawn Pasteur pipette (Hiraoka et al., 2004).
In this study, we evaluate the improvement in the survival rate and clinical outcome of vitrified blastocyst transfer programmes when artificial shrinkage (AS), through puncturing the blastocoele with a micro-needle or a laser pulse, is used before vitrification using a cryoloop. We believe our clinical results to confirm the safety of this procedure.
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Materials and methods |
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Patients and IVF
We perform blastocyst transfer programmes on patients who have had previous multiple failures of conventional day 2 or day 3 embryo transfer and who have agreed to use of the cryoloop vitrification method to cryopreserve their supernumerary blastocysts obtained 5 or 6 days after oocyte retrieval. This study was carried out in both Hiroshima and Tokyo HART Clinics between May 2003 and July 2005, and only patients who had expanded and/or hatching blastocysts vitrified on day 5 in the same cryoloop and warmed subsequently for transfer were included.
Two hundred and forty-five patients were entered in the study. The mean age of the women was 35.6 (range 25–41) years. Women were treated with GnRH agonist (GnRHa) and hMG using either a long or a short treatment protocol. The injection of hCG was given when dominant follicles reached a diameter of 18mm. Oocytes were collected 36h after hCG administration using the vaginal ultrasound-guided procedure. The oocytes were inseminated either by conventional IVF or by ICSI and were incubated in human tubal fluid (HTF) medium containing 5mg/ml of human serum albumin (HSA) or Blast Assist Medium 1 (Medicult, Jyllinge, Denmark) respectively, in a four-well multi-dish under mineral oil in a CO2 incubator at 37°C.
Previous results of studies on vitrified expanded blastocysts without AS, which were carried out between 1999 and 2002, were referred to as a control. We fully understood that the control group did not consist of randomly selected blastocysts from the same experimental period but rather from an earlier experimental period. Accordingly, time-, experience- and technical-related biases may influence the comparison. However, we believe that these two human studies represent acceptably close correlations. Patient characteristics are summarized in Table I.
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Embryo culture and grading of blastocysts
Fertilization was assessed 15–18h after insemination by the presence of two pronuclei. Zygotes were washed well and cultured in groups of two or three in Blast Assist Medium 1 (Medicult) for 48h and then in Blast Assist Medium 2 (Medicult) for another 48–72h. All culturing of embryos was performed in a CO2:O2:N2 (6:5:89%) environment. In conventional cases, one or two 4- to 8-cell embryos were transferred to the patient after 24–48h of culture, and only supernumerary embryos were extended to culture in Blast Assist Medium 2 (Medicult).
On day 5 after the oocyte retrieval, blastocyst development was examined. At this point, each embryo developed to the blastocyst stage was scored depending on the developmental stage and graded according to quality criteria (Gardner and Schoolcraft, 1999; Gardner et al., 2000) with slight modifications (Mukaida et al., 2003a).
Briefly, blastocysts were first given a score from 1 to 6 according to their degree of development. Second, the blastocysts were graded in three ranks based on morphological appearance. For example, the inner cell mass (ICM) was graded as A (many tightly packed cells), B (several loosely grouped cells) or C (few cells), and the trophectoderm was graded as A (many cells forming a cohesive epithelium), B (fewer cells forming a loose epithelium) or C (very few large cells).
On day 5, if at least one supernumerary blastocyst was graded as A or B, all the blastocysts of the patient were vitrified regardless of the developmental stage and the grading. If all the blastocysts of the patient were graded C in both ICM and trophectoderm grading, they were not cryopreserved.
AS of expanded blastocyst
The technique of shrinkage using micro-needle puncture has been described previously (Vanderzwamen et al., 2002). Briefly, about 10min before the vitrification, the expanded blastocysts were placed in 50-µl drops of pre-equilibrated Blast Assist Medium 2 (Medicult). The expanded blastocyst was held with a holding pipette connected to the micromanipulator, and the ICM was placed at the 6 or 12 o’clock position, and a glass micro-needle was pushed through the cellular junction of the trophectoderm into the blastocoele cavity until it shrank (Figure 1). After removing the micro-needle, we observed the contraction of the blastocoele within a few minutes. After complete shrinkage of the blastocoele, we vitrified the blastocyst and stored in the liquid nitrogen tank.
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Since September of 2004, a laser pulse generated by laser system ZILOS-tkTM (Hamilton Thorn Bioscience Inc., Beverly, MA, USA) has been introduced to perform AS, instead of micro-needle puncture. The ICM should be located away from the targeted point of the laser pulse. One single laser pulse (200ms) targeted at the cellular junction of the trophectoderm creates a hole to induce collapsing of the blastocoelic cavity. The blastocoele of the expanded blastocyst shrinks immediately (Figure 2). Using this laser system, it is not necessary to hold and locate the expanded blastocyst with a holding pipette connected to a micromanipulator. The laser technique makes the procedure simple and convenient.
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Vitrification of blastocysts
The protocol for the cryoloop vitrification of blastocysts was adopted from a previous report (Lane et al., 1999a
,b), albeit with slight modifications, and has been described previously (Mukaida et al., 2001, 2003a,b). The cryoloop consists of a nylon loop (20µm wide; 0.5–0.7mm in diameter) mounted on a stainless steel pipe inserted into the lid of a cryovial (Hampton Research, Laguna Niguel, CA, USA). A metal insert on the lid enables the use of a stainless steel handling rod with a small magnet (Crystalwand, Hampton Research) for the manipulation of the loop at low temperature (Mukaida et al., 2003b). One to three blastocysts were vitrified in a cryoloop after a two-step procedure to load the blastocysts with cryoprotectants at 37°C. As the base medium, HEPES-buffered-modified HTF medium containing 5mg/ml of HSA was used. Initially, blastocysts were placed in the base medium containing 7.5% (v/v) dimethylsulphoxide (DMSO) and 7.5% (v/v) ethylene glycol (EG) (Cryoprotectant solution I). After 2min, the blastocysts were transferred into cryoprotectant solution II, which is the base medium containing 15% (v/v) DMSO, 15% (v/v) EG, 10mg/ml of Ficoll 70 (average molecular weight 70,000; Pharmacia Biotech, Uppsala, Sweden) and 0.65mol/l sucrose. Both cryoprotectant solutions had been warmed briefly in an incubator at 37°C, and blastocysts were handled on the stage warmer of a dissecting microscope at 37°C.
While the blastocysts were in cryoprotectant solution II, a cryoloop was dipped into cryoprotectant solution II to create a thin, filmy layer of solution, by surface tension, on the nylon loop. The blastocysts were then washed quickly in solution II and transferred onto the filmy layer on the nylon loop using a micropipette. Immediately after the loading of blastocysts, the cryoloop was plunged into liquid nitrogen. The time blastocysts were exposed in solution II before cooling was limited to 25–30s. Using the stainless steel rod, the loop containing the blastocysts was sealed in a cryovial, which was previously submerged in liquid nitrogen. The vials were attached in standard canes and stored in liquid nitrogen. The entire procedure was completed within 5min. Vitrified blastocysts were kept in the liquid nitrogen tank from 1 month to 5 years depending on the patient requirements.
Warming of blastocysts and assessment of survival
In a four-well multi-dish, 1ml of base medium containing 0.33mol/l sucrose in well number 1, base medium containing 0.2mol/l sucrose in well number 2 and base medium in well number 3 were warmed briefly in an incubator at 37°C and then placed on the stage warmer of a dissecting microscope. With the cryovial submerged in liquid nitrogen, the vial was opened with the stainless steel rod, and the loop containing the blastocysts was removed from the liquid nitrogen and placed directly and quickly into the 0.33 mol/l sucrose solution in well number 1. Blastocysts immediately fell from the loop into the solution. Thus, blastocysts were warmed and diluted instantly at 
37°C, the temperature being controlled by the stage warmer. After 2min, the blastocysts were transferred to the 0.2mol/l sucrose solution in well number 2. After an additional 3min, blastocysts were washed and kept in the base medium in well number 3 for 5min. During this 5min, assisted zona hatching was performed with acidic tyrode as previously described (Cohen et al., 1992; Obruca et al., 1994) on the warmed blastocysts. Then the blastocysts were returned to Blast Assist Medium 2 (Medicult) for further culture until transfer.
About 2–3h after warming, the blastocysts were examined on an inverted microscope at x400 magnification, and survival was assessed based on the morphological integrity of the blastomeres, ICM and trophectoderm and re-expansion of the blastocoele. The surviving blastocysts were scored as to developmental stage and were graded according to quality as described above.
Transfer of blastocysts and assessment of pregnancy
All women received transdermal estradiol (Estrana 0.4mg/day, Hisamitsu, Tokyo, Japan) with GnRHa for the preparation of the endometrium. The administration of progesterone (50mg in oil, daily) was initiated when endometrial thickness exceeded 10mm. On day 5 after the initiation of progesterone treatment, the blastocysts were warmed and the surviving blastocysts were transferred into the patient’s uterus. Most patients received one or two blastocysts; occasionally, three blastocysts were transferred depending on the patient’s background [i.e. multiple failures of assisted reproduction techniques (ART)]. Pregnancy was assessed by serum hCG 14 days after administration of progesterone, and then implantation was confirmed by the presence of a gestational sac. Clinical pregnancy was confirmed by the presence of fetal heart activity.
Statistics
The data were examined for differences by 
2 analysis, unless the expected frequency was <5, in which case Fisher’s exact probability test was used.
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Results |
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Table I summarizes patient characteristics and the survival and pregnancy rates of vitrified human blastocysts with AS (study group) or without AS (control group). Two hundred and forty-five patients were entered in the study, and 270 cycles that had only expanded and/or hatching blastocysts vitrified were attempted. The average age of the patients was 35.6 (27–41). Two hundred and sixty-six cycles had vitrified blastocyst transfer with AS. In four cycles, no blastocysts survived and embryo transfer was cancelled. Five hundred and two vitrified blastocysts were warmed for transfer and 488 survived. Survival rate was 97.2%. Four hundred and forty-eight vitrified blastocysts were transferred, and the mean number of blastocysts transferred per cycle was 1.7. Of 266 transfers, 160 resulted in clinical pregnancy; the pregnancy rate was 59.3% per warming cycle (160/270) and 60.2% per transfer (160/266).
The results of vitrified expanded and hatching blastocysts in our previous study, which we reported in 2003, served as a control group. The survival rate of both expanded and hatching blastocysts, scored 4 and 5 respectively, was 85.0% (288/339). A statistical difference was noted between the study and control groups (P<0.05). When the pregnancy rate of the study group was compared with the control group, a statistically significant improvement was noticed in the AS group (60.2 versus 34.1%; P<0.01). The difference in samples between the two groups has already been discussed, and, again, we believe two such similar human studies should provide acceptably similar groupings.
Table II summarizes the clinical outcome of vitrified blastocyst transfer in the study. Two hundred and sixty-six cycles of 270 attempted cycles achieved vitrified blastocyst transfer, and 448 vitrified blastocysts were transferred. Of 266 transfers, 160 resulted in clinical pregnancy; the pregnancy rate was 59.3% per warming cycle (160/270) and 60.2% per transfer (160/266). Although 35 of 160 pregnancy cycles (28%) ended in miscarriage, of the 448 transferred embryos, 209 (46.7%) were diagnosed as implanted, by confirmed presence of gestational sacs, about 21 days after transfer. Seventy-seven healthy babies were born in 57 deliveries, and 68 pregnancies are ongoing. No bias in the sex ration was observed because 41 babies were boys and 36 were girls.
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Table III summarizes the comparison between the results when using the micro-needle and the laser pulse for blastocoele shrinkage. AS using a micro-needle was performed on 240 cycles with 462 blastocysts and AS using a laser pulse was on 26 cycles with 40 blastocysts. Survival rate achieved with the two methods was similar (the micro-needle: 97.2% versus the laser pulse: 97.5%). The mean number of survived blastocysts transferred was also similar. The clinical pregnancy rate, implantation rate and miscarriage rate were also similar. No statistical difference was observed in the results achieved with the two methods.
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Discussion |
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The aim of this study was to improve the survival rate of the cryoloop technique using AS before vitrification for expanded and hatching blastocysts and to use our large clinical data to evaluate the safety of AS. In a total of 266 warmed vitrified blastocyst transfer cycles, high rates of survival (97.2%) and pregnancy (60.2%) were obtained using AS before vitrification. This result demonstrates that AS of human expanded and hatching blastocysts is a useful approach to improve clinical outcomes.
Other reports showing the effectiveness of blastocoele shrinkage contained 25 cycles (Son et al.), 29 cycles (Hiraoka et al.) and 49 cycles of transfers (Vanderzwalmen et al., 2002). However, in our study, 270 cycles of transfer with AS were available to support our findings.
Since 1999, we have adopted vitrification using the cryoloop technique for human blastocyst cryopreservation, and we reported its usefulness (Mukaida et al., 2001, 2003a). We also previously reported perinatal outcomes of blastocyst vitrification, including no increase of major and minor malformation (Takahashi et al., 2005).
Achievement of pregnancy is related to many different factors. One of the most important factors to achieve pregnancy is embryo viability. More developed blastocysts at day 5 should have better developmental competency and higher viability. Theoretically, the more developed blastocysts should have a better pregnancy rate than that of early stage blastocysts. However, we have observed that the survival rate of expanded (86%) and hatching (82%) blastocysts was significantly lower than that of early stage (97%) even after we had established the vitrification steps. This is in accordance with the observation of Vanderzwalmen et al. (1999, 2002) that the efficiency of vitrification of human blastocysts was negatively correlated with the expansion of the blastocoele. Increase of survival rate might contribute to the improvement of the viability of survived blastocysts in the AS group and could facilitate implantation.
A possible explanation for the lower survival rate is that the large blastocoele of more developed blastocysts may disturb the efficacy of vitrification due to inappropriate dehydration and the permeation of cryoprotectant, which might cause ice crystal formation in the rapid cooling and warming steps during vitrification.
A study on mouse blastocysts also reported that survival rates of vitrified blastocysts after a one-step exposure to EFS40 decreased as the volume of the blastocoelic cavity increased (Miyake et al., 1993). In mouse blastocysts, a two-step loading of cryoprotectant was effective in preventing the decrease in post-warming survival of fully expanded blastocysts (Zhu et al., 1993). Bovine blastocysts were also successfully vitrified after a two-step loading of cryoprotectant (Mahmoudzadeh et al., 1995). However, human blastocysts appear to be different (Vanderzwalmen et al., 2002) and are thought to have specific characteristics related to the lower cryoresistance. Such characteristics would be attributable to the permeability of the membrane, which would decrease as the blastocyst develops. Therefore, it would be preferable to cryopreserve human blastocysts on day 5 before the blastocoele fully develops, or earlier on day 4.
Recently, it has been suggested that mechanical damage caused by ice crystal formation could be avoided by reducing the fluid content of the blastocoele of more developed stage blastocysts (Vanderzwalemen et al., 2002). One of the strategies reported was reducing the size of the blastocoele mechanically. Vanderzwalmen et al. (2002) reported making a small hole in the trophectoderm with a needle and thus causing the blastocoele to shrink. They vitrified shrunken blastocysts in straws in EFS40 and reported that this AS dramatically raised the post-warming survival rate, which resulted in eight pregnancies from 35 transfers. Son et al. (2003) reported using a 29-gauge needle for AS of the blastocoele. Hiraoka et al. (2004) reported collapsing the blastocoele by micropipetteing without puncturing the zona and trophectoderm.
An alternative method is to increase the concentration of and exposure to cryotectant. The use of a high concentration and the increase in the duration of exposure of cryoprotectant might be effective for preventing injury from intracellular ice while allowing sufficient dehydration. However, the risk of injury from the chemical toxicity of the cryoprotectant would increase as the concentration and the duration of exposure increase. For these reasons, reducing the fluid content of the blastocoele of more developed stage blastocysts is preferred to increasing the concentration of cryoprotectant.
Thus, these authors reported that the rates of survival were improved after AS of the blastocoele. Similarly, we applied the AS technique in our vitrification using the cryoloop for human blastocyst cryopreservation.
Initially, we collapsed the blastocoele by puncturing with a fine needle using micromanipulation procedures. Occasionally, we came across cases where collapsing of the blastocoele occurred partially or incompletely, because the elastic plasma membrane of trophectoderm cells adhered to the puncture opening. In those cases, puncturing was performed again at a different site of the trophectoderm until complete collapsing of the blastocele was achieved. However, since September 2004 we have used a laser pulse for AS, and incomplete collapse of the blastocoele has seldom been observed. We speculated that the single laser pulse created an instant heat effect that caused cellular degeneration of the trophectoderm and allowed a large enough hole to open. Also, collapsing the blastocoele by laser pulse did not require preparation before the procedure, such as setting the needle. It was also not necessary to hold the blastocyst at the manipulation stage. It is only necessary to locate the peripheral junction of the trophectoderm cells of an expanded blastocyst and administer a single laser pulse. The safety of laser application to human embryos and blastocysts such as biopsy has already been reported (Veiga et al., 1997; Ebner et al., 2005).
We compared the various techniques of AS in other reports and observed that blastocoele collapse by micropipetting (Hiraoka et al., 2004) and 29-gauge needle (Son et al., 2003) do not require micromanipulation. Both techniques can be performed under the dissecting microscope. Micropipetting was much the same as removal of the cumulus cells of the oocyte for ICSI. However, the size of human oocytes is quite regular compared with the variation in the size of blastocoeles depending on the developmental stage of the blastocysts. Thus, for micropipetting, it is necessary to prepare several sizes of hand-drawn Pasture pipettes according to the expanded size of the blastocoele. Also, shrinkage by 29-gauge needle requires a very precise hand movement, because the edge of the 29-gauge needle is quite huge compared with a glass micro-needle. Among these techniques, laser pulse is the simplest and most convenient for AS. The only drawback is the expense of the equipment. In this comparison between the results of AS using either a micro-needle or a laser pulse, no statistical difference was observed. This result indicated that the methodology used to collapse blastocoeles did not affect the clinical outcome, as long as collapsing of blastocoeles was confirmed.
According to our observations, the average time taken for re-expansion of survived blastocysts in the AS group tends to be shorter than that in the control group. This also might be related to the viability of survived blastocysts.
In conclusion, this study proved that AS of human expanded and hatching blastocysts before vitrification statistically improves the survival and pregnancy rate, and delivery of healthy babies confirms the safety of this technique.
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Footnotes |
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This work was undertaken at Hiroshima HART Clinic and Tokyo HART Clinic. Both clinics are private fertility centres. We did not have Institutional Review Board (IRB) when we started this work in 1999. Drs K. Takahashi, T. Mukaida, T. Goto and C. Oka are members of Japan Society of Obstetrics and Gynecology (JSOG), Japan Society of Fertility and Sterility (JSFS) and Japan Society of Fertilization and Implantation (JSFI). Both clinics have been registered as certified fertility centres by JSOG. Our clinical practices and research works have been performed following the guidelines of JSOG. Our clinical research works have also been performed according to the ethical principles for medicine, that is, the Declaration of Helsinki.
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Submitted on March 30, 2006; resubmitted on June 13, 2006; accepted on June 23, 2006.
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