1 Hiroshima HART Clinic, 5-7-10 Ohtemchi, Naka-ku, Hiroshima, 2 Osaka HART Clinic, Snowcrystal 10F 2-6-20, Umeda, Kita-ku, Osaka, 3 Tokyo HART Clinic, 1-22-2, Higashi, Shibuya-ku, Tokyo and 4 Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi, Japan
5 To whom correspondence should be addressed at: Hiroshima HART Clinic, 5-7-10 Ohtemchi, Naka-ku, Hiroshima, 730-0051 Japan. e-mail: hart{at}enjoy.ne.jp
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Abstract |
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Key words: blastocyst/cryoloop vitrification/cryopreservation/embryo/human
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Introduction |
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In 1985, the first report was made of an innovative approach called vitrification, in which injuries related to ice are minimized by using very high concentrations of cryoprotectant (Rall and Fahy, 1985). This approach simplifies the cooling process, because embryos can be cooled directly in liquid nitrogen. Although embryos subjected to vitrification are liable to be injured by the toxicity of the high concentration of cryoprotectant, the method has been refined and proven effective for the cryopreservation of embryos at various stages of development in laboratory and domestic species. In 1998, it was shown that vitrification using an EG-based vitrification solution (EFS40) (Kasai et al., 1990
) with conventional cryo-straws was effective for human embryos at the 4- to 8-cell stage (Mukaida et al., 1998
). The effectiveness of vitrification was confirmed for human embryos at the 8- to 16-cell stage (Saito et al., 2000
) and the morula stage (Yokota et al., 2001b
), also using EG-based solutions.
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 because 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 is increasing. One group (Ménézo et al., 2000
) cryopreserved human blastocysts which were developed in a co-culture system using the slow-freezing method with glycerol and obtained reasonable clinical results (27% pregnancy rate, 17% implantation rate). However, results reported by other clinics have not been consistent (Troup et al., 1990
; Nakayama et al., 1995
; Ludwig et al., 1999
). It has also been speculated that the outcome of cryopreservation is influenced by the culture conditions (Ménézo et al., 2000
).
Although a recent report described the successful vitrification of human blastocysts in straws (Yokota et al., 2001a), our own attempts to repeat this approach resulted in only 45% survival (39/86, unpublished data). Others (Vanderzwalmen et al., 1999
) also reported a low pregnancy rate with human blastocysts vitrified in straws. Human blastocysts are much less permeable to cryoprotectant and water, and have been observed to shrink more slowly than mouse and bovine blastocysts when placed in the cryoprotectant solution; this suggests that they 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, i.e. by using minute tools such as electron microscopic grids (Martino et al., 1996), open pulled straws (Vajta et al., 1998
) or cryoloops (Lane et al., 1999a
) (for a review, see Kasai, 2002
). It was shown that the transfer of human blastocysts vitrified with cryoloops leads to the successful birth of a baby (Mukaida et al., 2001
), and this method of vitrification has been used in the present authors group of three clinics to cryopreserve blastocysts on days 5 and 6. The present report summarizes the results obtained with 223 warming cycles, and confirms the effectiveness of the cryoloop technique in the cryopreservation of human blastocysts.
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Materials and methods |
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Women were treated with GnRH agonist and hMG using either a long- or a short-treatment protocol. They were administered hCG when dominant follicles reached a diameter of 18 mm. Oocytes were collected 36 h after hCG administration using a vaginal ultrasound-guided procedure. The oocytes were inseminated using either conventional IVF or ICSI, and incubated in human tubal fluid (hTF) medium containing 5 mg/ml human serum albumin (hSA) or G1.2 medium (Vitrolife, Gothenburg, Sweden) in a 4-well multi-dish under mineral oil in a CO2 incubator at 37°C in an atmosphere of 6% CO2, 5% O2 and 89% N2.
Embryo culture and grading of blastocysts
Fertilization was assessed at 1518 h after insemination by the presence of two pronuclei. One-cell embryos were washed well and cultured in groups of two to three in 4-well dishes under oil in the CO2 incubator. Embryos were cultured in G1.2 medium or Blast Assist Medium 1 (Medicult, Jyllinge, Denmark) for 48 h, and then placed in G2.2 medium (Vitrolife) or Blast Assist Medium 2 (Medicult) for 4872 h. In some cases, when 4- to 8-cell embryos were removed for transfer to the patient after 2448 h of culture in the first medium, only supernumerary embryos were cultured in the second medium.
After 4854 h of culture in the second medium, 5 days after oocyte retrieval, embryos were examined for development into blastocysts. If all the embryos were at the morula stage or earlier, they were cultured until day 6 and then examined for development. On day 5 or 6, each embryo which had developed to the blastocyst stage was scored depending on the developmental stage, and graded according to quality using published criteria (Gardner and Schoolcraft, 1999; Gardner et al., 2000
) with slight modifications.
First, blastocysts were given a numerical score from 1 to 6 on the basis of their degree of development, as follows: 1, early blastocysts with a blastocoele <50% of the embryo volume; 2, early blastocysts with a blastocoele 50% of the embryo volume; 3, full blastocysts with a blastocoele completely filling the embryo but not expanded; 4, expanded blastocysts with a blastocoele volume larger than that of the early embryo, with a thinning zona; 5, hatching blastocysts with the trophectoderm starting to herniate through the zona; and 6, hatched blastocysts, in which the blastocyst has completely escaped from the zona.
Second, the blastocysts were graded in three ranks by quality. Early blastocysts (scored as 12) were assessed based on morphological appearance as A (many equal-shaped cells), B (many unequal-shaped cells) or C (few cells and degeneration). More advanced blastocysts (scored as 36) were classified on both the quality of the inner cell mass (A = many tightly packed cells; B = several loosely grouped cells; C = few cells), and the quality of the trophectoderm (A = many cells forming a cohesive epithelium; B = fewer cells forming a loose epithelium; C = very few large cells).
When the patient had their fresh embryos transferred on day 23, all the remaining embryos were cultured to allow those which developed into blastocysts to be vitrified. Patients who received transfers of fresh blastocysts had all their remaining supernumerary blastocysts vitrified. 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. In a few cases, compacted morulae were also vitrified with the blastocysts. If all the blastocysts of the patient were graded C, they were not cryopreserved. On day 6, if at least one blastocyst had a large blastocoele (i.e. scored as 36) and was graded as A or B, all the developed blastocysts scored as 36 were vitrified.
Vitrification of blastocysts
The protocol for the cryoloop vitrification of blastocysts was adopted from previous reports (Lane et al., 1999a,b), albeit with slight modifications, and had been described previously (Mukaida et al., 2001
). The cryoloop consisted of a nylon loop (20 µm wide; 0.50.7 mm diameter) mounted on a stainless steel pipe that was inserted into the lid of a cryovial (Hampton Research, Laguna Niguel, CA, USA). A metal insert on the lid enabled the use of a stainless steel handling rod with a small magnet (Crystalwand, Hampton Research) for manipulation of the loop at low temperature.
One to three blastocysts were vitrified in each cryoloop after a two-step procedure to load the blastocysts with cryoprotectants at 35°C. As the base medium, HEPES-buffered modified hTF medium containing 5 mg/ml hSA was used. Initially, blastocysts were placed in the base medium containing 7.5% (v/v) DMSO and 7.5% (v/v) EG (cryoprotectant solution I). After 2 min, the blastocysts were suspended in the base medium containing 15% (v/v) DMSO, 15% (v/v) EG, 10 mg/ml Ficoll 70 (average molecular weight 70 000; Pharmacia Biotech, Uppsala, Sweden) and 0.65 mol/l sucrose (cryoprotectant solution II). 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 embryos were suspended in cryoprotectant solution I, a cryoloop was dipped into cryoprotectant solution II in order 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 that blastocysts were exposed in solution II before cooling was limited to 2530 s. 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 5 min.
Warming of blastocysts and assessment of survival
In a 4-well multi-dish, 1 ml of base medium containing 0.33 mol/l sucrose, base medium containing 0.2 mol/l sucrose, and base medium 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 aid of the stainless steel rod, and the loop containing blastocysts was removed from the liquid nitrogen and placed directly and quickly into a well of the 0.33 mol/l sucrose solution. Blastocysts immediately fell from the loop into the solution. Thus blastocysts were warmed and diluted instantly at
35°C. After 2 min, blastocysts were transferred to the 0.2 mol/l sucrose solution. After an additional 3 min, blastocysts were washed and kept in the base medium for 5 min, and then returned to G2.2 medium for further culture until transfer.
At 2 h after warming, the appearance of the blastocysts was examined on an inverted microscope at x400 magnification, and survival was assessed based on the morphological integrity of the blastomeres, inner cell mass and trophectoderm, and re-expansion of the blastocoele. The surviving blastocysts were scored as to developmental stage and graded for quality as described above.
Transfer of blastocysts and assessment of pregnancy
All women received transdermal estradiol (Estrana, 0.4 mg/day; Hisamitsu, Tokyo, Japan) with GnRH agonists for preparation of the endometrium. Administration of progesterone (50 mg in oil, daily) was initiated when the endometrial thickness was >10 mm. At 5 days (in a few cases, 6 days) after the initiation of progesterone treatment, blastocysts were warmed and surviving blastocysts were transferred into the patients uterus. Most patients received one to three blastocysts; occasionally, four to five blastocysts were transferred, depending on the patients background (i.e. multiple failures of assisted reproduction). Chemical pregnancy was assessed based on serum hCG levels at 910 days after blastocyst transfer, after which implantation and clinical pregnancy was confirmed by the presence of fetal heart activity or a gestational sac at 30 days after blastocyst transfer.
Statistical analysis
The data obtained were examined for differences using 2 analysis, unless the expected frequency was <5, in which case Fishers exact probability test was used.
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Results |
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It was not possible to follow the survival and developmental potential of each blastocyst, because one to three blastocysts were vitrified on a cryoloop, embryos of each patient were cultured in a group after warming, and one or more embryos were transferred to each woman. Thus, a group of blastocysts for each attempted transfer was classified by the most developed stage (i.e. the highest score number) of the group of blastocysts for retrospective analysis: that is, as group I (with a stage 1 embryo); group II (with a stage 2 embryo); group III (with a stage 3 embryo); group IV (with a stage 4 embryo); and group V (with a stage 56 embryo). For example, if three blastocysts of a patient were scored as 4, 3 and 2, all the blastocysts in this group were classified as group IV. The survival rates of vitrified blastocysts classified by this criterion before vitrification are shown in Table II. Average proportions of score 1, 2, 3, 4 and 56 embryos included in group I, II, III, IV and V embryos were 80, 45, 52, 53 and 41% respectively. The survival rate of embryos in group II or III, which included unexpanded blastocysts (9097%), was statistically significantly higher than the rates of blastocysts in group IVV, including those at more developed stages (6879%) (P < 0.05). Accordingly, pregnancy rates with earlier blastocysts in groups IIII (44%; 37/85 in total) tended to be higher than those with more developed blastocysts in groups IVV (32%; 39/122 in total), although the difference was not statistically significant (Table III). By contrast, when the pregnancy rate was evaluated according to the most developed stage of the group of embryos after warming and a short period of culture (Table IV), higher pregnancy rates (4049%) were obtained with embryos in groups IIIV, which regained a larger blastocoele, than with those in group I, which remained at less developmental stages. Average proportions of score 1, 2, 3, 4 and 56 embryos included in group I, II, III, IV and V embryos were 81, 59, 52, 55 and 51% respectively.
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Discussion |
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The present authors have adopted blastocyst transfer since sequential culture systems first became available. Initially, to cryopreserve blastocysts, straw vitrification was attempted using EFS40 (which contains 40% EG), but post-warming survival was quite poor (45%, unpublished data). It has been speculated that blastocysts are much less permeable to water and cryoprotectant than embryos at early cleavage stages, as blastocysts respond to hypertonic solutions much more slowly than earlier embryos (Mukaida et al., 2001). This suggests that intracellular ice is more likely to form in the blastocyst during cryopreservation.
An attempt has been made to vitrify human morulae and blastocysts in straws (Vanderzwalmen et al., 2002), but the efficiency of vitrification was shown to decrease with expansion of the blastocoele. The subsequent strategy of these authors to cryopreserve human blastocysts was to make a small hole in the trophectoderm with a needle, thereby causing the embryo to shrink. The artificially shrunken blastocysts were vitrified in straws in EFS40, and this resulted in a dramatic increase in post-warming survival rate such that eight pregnancies resulted from 35 transfers. By contrast, others (Yokota et al., 2001a
) vitrified intact blastocysts in straws and reported six pregnancies from 18 transfers. Considering that these authors used a vitrification solution containing 25% EG + 25% DMSO, the use of a high concentration (50%) of cryoprotectant might be effective in preventing injury caused by intracellular ice. However, the risk of injury caused by chemical toxicity of the cryoprotectant would increase as the concentration increased. An alternative strategy to cryopreserve human blastocysts would be to adopt ultrarapid vitrification, in which embryos are vitrified extremely rapidly using a minimal amount of vitrification solution, as this reduces the chance of intracellular ice forming (Kasai, 2002
). Using this approach, one group vitrified human blastocysts using electron microscopic grids, and reported five pregnancies from 20 transfers (Choi et al., 2000
).
The cryoloop is a sophisticated tool which enables handling of the loop with a magnet-attached stainless steel rod and preservation of embryos in a cryovial (Lane et al., 1999a,b). Preliminary results of the cryoloop vitrification of human blastocysts have been reported in which six pregnancies with one delivery were obtained (Mukaida et al., 2001
). The present data with 207 transfers resulting in 76 pregnancies demonstrate the effectiveness of the cryoloop technique for the cryopreservation of human blastocysts (Mukaida et al., 2001
). It has also been reported that cryoloop vitrification yields a superior survival of Rhesus monkey blastocysts (Yeoman et al., 2001
). These authors showed that while a vitrification solution containing 25% EG and 25% glycerol was effective, an alternative solution containing 20% EG and 20% DMSO was ineffective. This shows that permeation by the cryoprotectant and concentration by the embryo are important factors, even in the cryoloop vitrification of primate blastocysts. The vitrification solution used in the present study contained a lower concentration of permeating cryoprotectant (15% EG and 15% DMSO), which might be less effective in preventing the formation of intracellular ice. In order to promote dehydration and permeation of the cryoprotectant, however, blastocysts were loaded with cryoprotectants at a higher temperature (
35°C). Successful vitrification with a lower concentration of cryoprotectant suggests that this approach is actually effective at preventing the formation of intracellular ice in human blastocysts. The use of a lower concentration of cryoprotectant must be more effective in minimizing the risk of toxic injury.
Although the overall survival rate (80%, 583/725) with vitrified blastocysts was high, the rate was affected by embryonic factors, such as the age (day) and the developmental stage of blastocysts. Most embryos (78%) developed to the blastocyst stage on day 5 and were vitrified on that day, whereas retarded embryos were vitrified on day 6. The overall survival rate was higher for blastocysts vitrified on day 5 than day 6 (87 versus 55%; Table I). More precisely, however, the distribution of embryos in terms of developmental stage was different among the blastocysts of different ages. If the survival rates of blastocysts on day 5 and day 6 are compared in more homogeneous groups, i.e. only for blastocysts in the same groups with embryos scored as 35 (groups IIIV) from the data shown in Table II, the survival rate of day 5 blastocysts was 86% (387/450), whereas that of day 6 blastocysts was 55% (86/156), and with a statistically significant difference (P < 0.01) between these subgroups.
The effect of developmental rate in vitrification outcome of blastocysts parallels that obtained with day 23 human embryos, in that more of the morphologically normal on-time embryos (86%) survive than retarded embryos (58%) (Mukaida et al., 1998). It is strange that both rates are quite similar to the survival rates for day 5 and day 6 blastocysts shown above, but it indicates that normally developing human embryos are more resistant to cryopreservation than retarded ones, not only in embryos at early cleavage stages but also in blastocysts, when the survival was assessed by the appearance. Vitrified human blastocysts could be damaged as a result of the chemical toxicity of the cryoprotectant, intracellular ice formation, or osmotic stress during the removal of the cryoprotectant. Because toxic injury is not reflected in the morphological survival of blastomeres (Kasai et al., 2002
), possible mechanisms for the difference in cryoresistance among normally developed and retarded blastocysts would be intracellular ice formation or osmotic stress. Both injuries are closely related to the lower permeability of the membrane.
The implantation rates of the surviving blastocysts after transfer to the recipient were similar for both day 5 and day 6 blastocysts (21 and 18%; Table I). Although the pregnancy rate for day 6 blastocysts (29%) was lower than that of day 5 blastocysts (39%), the difference was not statistically significant. It should be noted that while day 5 blastocysts include early blastocysts, which tends to result in higher pregnancy rates (Table II), the day 6 blastocysts did not. In addition, the number of embryos per transfer was larger for day 5 blastocysts (2.5 in average) than day 6 blastocysts (1.8 in average), because the number of transferable embryos tended to be small on day 6; the present results also show that the pregnancy rate increases as the number of transferred embryos per recipient increases, as expected. Considering these factors, it seems that once they have survived vitrification, blastocysts on day 5 and 6 have comparable potential to develop further.
As one or more embryos of each patient were vitrified and cultured together, it was not possible to assess post-warming survival and development for each blastocyst. In an attempt to assess the effect of the developmental stage briefly, a group of blastocysts of each patient was classified retrospectively based on the most developed stage of the blastocysts. Hence, embryos classified as early blastocysts (groups III) did not include developed blastocysts, whereas embryos classified as developed blastocysts (groups IIIV) included early blastocysts. Thus, this classification was not accurate. Nevertheless, analyses on the classification suggested that earlier blastocysts were more resistant to vitrification than more developed blastocysts (Tables II and III). This finding was in accord with the observation of others (Vanderzwalmen et al., 1999, 2002)that the efficiency of vitrification of human blastocysts was negatively correlated with the expansion of the blastocoele.
One reason for the lower survival rate in more developed blastocysts could be the presence of a large amount of blastocoelic fluid, in which ice may form. 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, 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 seem to be different (unpublished results; Vanderzwalmen et al., 2002
), and thus must have specific characteristics related to the lower cryoresistance. The characteristics could 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 is fully developed.
The pregnancy rate in human assisted reproduction is affected not only by embryonic factors but also by factors related to patients. It is well known that the age of the recipient has significant effects (Wang et al., 2001). In the present study, the pregnancy rate was lower in older women (33%) than in younger women (40%), although the difference was not statistically significant (Table VI). The cause of infertility affected the pregnancy rate significantly; when infertility was related to male factors, the rate was high (Table VII), probably because recipient women had no problems. However, the morphological survival of vitrified blastocysts was not affected either by the age of the woman (Table VI) or the cause of infertility (Table VII). This shows that these factors do not affect the quality of embryos, at least in terms of cryoresistance.
While 28% (21/76) of pregnancies ended in miscarriages, this outcome cannot be attributable to cryopreservation, because 22% (27/126) of pregnancies established from fresh blastocyst transfers in the authors clinics during the same period also resulted in miscarriage. There was no statistically significant difference among the two miscarriage frequencies, though others (Ménézo et al., 2000) reported that the overall miscarriage rate with frozen blastocysts was 20%.
In conclusion, the clinical outcome with 725 vitrified embryos and 207 transfers confirmed that vitrification using the cryoloop technique is effective and practical for the cryopreservation of human blastocysts. Early blastocysts on day 5 seem to be the most suitable in terms of stage and age for cryopreservation, but more developed blastocysts and even retarded ones on day 6 can also be cryopreserved, which would lead to the best use of valuable embryos.
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FOOTNOTES |
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Submitted on August 27, 2002; accepted on October 8, 2002.