1 Division of Infertility Clinic, Lee Women's Hospital, 2 Institute of Biochemistry, Chung-Shan Medical University and 5 Department of Obstetrics and Gynecology, Chung-Shan Medical University Hospital, Taichung, 3 Department of Biotechnology, Chungtai Institute of Health Science and Technology, 4 Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, Taipe, and 6 Department of Medicine, China Medical University, Taiwan
7 To whom correspondence should be addressed at: Lee Women's Hospital, 263 Pei-Tun Road, Taichung 406, Taiwan. Email: msleephd{at}giga.net.tw
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Abstract |
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Key words: blastocyst cryopreservation/super-cooling,ultra-rapid vitrification
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Introduction |
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The slow cooling cryopreservation method for cleaved embryos has been widely used since the first human pregnancy resulted from cryopreserved 4- to 8-cell embryos, as reported in 1983 (Trounson and Mohr, 1983). Subsequent to these initial developments, however, the time-consuming and laborious process of slow cooling has made vitrification an attractive alternative embryo cooling technique. The first successful human cleavage-stage embryo vitrification followed by a successful delivery was reported in 1990 (Gordts et al., 1990
). Unfortunately, however, to the best of our knowledge, the clinical results from human blastocyst cryostorage by slow freezing or vitrification are not entirely consistent (Hartshorne et al., 1991
; Menezo et al., 1992
; Kaufman et al., 1995
; Choi et al., 2000
).
Vitrification is performed by suspending the embryo(s) in a solution containing a high concentration (58 mol/l) of cryoprotectants and then directly plunging the embryo(s) into liquid nitrogen (LN) (196°C) (Rall and Fahy, 1985). The advantage of this technique is the prevention of ice crystal formation within the embryo tissue or outside the cytoplasm. However, the osmotic stress and toxic effect from high cryoprotectant concentrations may constitute an obstacle to using this method. Several new techniques and applications were developed recently that improve the survival rate for human blastocysts following vitrification. These procedures include the use of an electron microscope (EM) grid (Martino et al., 1996
; Cho et al., 2002
), cryoloops (Lane et al., 1999a
,b
; Mukaida et al., 2001
) and an open pulled straw (OPS) (Vajta et al., 1998
; Chen et al., 2000
). These techniques attempt to accelerate the blastocyst cooling rate and decrease the cryosolution volume needed for this procedure.
A new device (the Vit-MasterTM, IMT, Nes Ziona, Israel) (Figure 1A) was designed and manufactured to reduce the LN temperature as low as 205 to 210°C by applying negative pressure. The super-cooled LN facilitates heat transmission between LN and the cryosolution interface (Arav et al., 2002). This characteristic has proven to be efficient for bovine gamete and blastocyst cryopreservation (Arav et al., 2002
). Whether embryos from other species can survive such a cooling process and maintain full development potential remains to be determined. The efficiency of such a freezing technique in clinical applications for human blastocysts warrants further investigation.
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Materials and methods |
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The B6CBF1 females were injected with 5 IU of pregnant mare serum gonadotrophin (PMSG; Sigma Chemical Co., St Louis, MO) followed 48 h later by injection with 5 IU of HCG (Pregnyl; Serono, Anbunne, Switzerland). The female mice were caged with fertile B6CBF1 males. The female mice were sacrificed by cervical dislocation 96 h after impregnation. To facilitate blastocyst collection, the blastocysts were flushed from the excised uterus and rinsed three times with sterile phosphate-buffered saline (PBS; Sigma) solution (37°C). All mouse blastocysts were cultured in G2 medium (Vitrolife) in a humidified atmosphere; 95% air and 5% CO2 at 37°C until cooled (after 11.5 h of culture).
Human blastocyst collection
The patients participating in this study underwent a general medical work-up for infertility and were enrolled into the in-house IVF programme. Initially, participating women took the GnRH agonist leuprolide acetate (Lupron, Takeda Chemical Industries Ltd, Osaka, Japan) commencing from the mid-luteal phase. A serum estradiol (E2) level <50 pg/ml was used to confirm pituitary suppression using a recombinant FSH (rFSH; Gonal-F, Serono) treatment. The participant's ovarian response was monitored using serial serum E2 levels and ultrasound examination. When the leading follicles reached 18 mm in diameter and the appropriate serum E2 level was determined, 10 000 IU of HCG (Profasi, Serono) was administered. Transvaginal oocyte retrieval was performed 3436 h later.
The retrieved oocytes were fertilized in vitro and collected for further in vitro embryo culture in the microdrops of G2.1/G2.2 (Vitrolife) medium. Embryo development was observed by microscope after 72 h subsequent to fertilization. The in vitro culture was continued if the number of good-quality embryos exceeded three. Two or three of the best quality blastocysts were selected for embryo transfer. A fraction of the remaining blastocysts were randomly cryopreserved with slow or super-cooling vitrification using a random number table.
Human blastocysts criteria
Human blastocysts for cryopreservation must have an intact inner cell mass (ICM) and trophectoderm: grade A, where the blastocoels of early blastocysts were less than half the volume of the embryo; grade B, where the blastocoels were greater than half the embryo volume; grade C, where the blastocoel completely filled the embryo; and grade D, where the blastocysts were expanded or expanding with a distinct trophectoderm and eccentrically located ICM.
Super-cooling vitrification protocol
The vitrification procedure was adapted from Lane et al. (1999a,b)
. A two-step cryoprotectant loading process was used. The 100% vitrification solution (VS) was composed of 20% (3.6 mol/l) ethylene glycol (EG), 20% (2.4 mol/l) dimethylsulphoxide (DMSO) and 0.5 mol/l sucrose in human tubular fluid (HTF) medium with 20% human serum albumin (HSA). The VS was pre-warmed in 37°C incubators for balance. The blastocysts were then exposed to 50 and 100% VS at 37°C for 2 min and 30 s, respectively.
Two to three blastocysts treated with 100% VS were transferred onto a thin layer formed by coating 100% VS (0.5 µl) onto the nylon loop of a cryoloop (20 µm wide; 0.50.7 mm in diameter; Hampton Research, Laguna Niguel, CA). Before cryoloops containing blastocysts were placed into the super-cooled LN, cryovials (Hampton Research) with 1.8 ml capacity and LN were placed into the Vit-MasterTM apparatus. Negative pressure (0.9 bar) was then applied and, as a consequence, the LN temperature decreased from 196 to 206°C (Figure 1B). After the cryoloops containing blastocysts were plunged directly into the super-cooled LN, they were mounted on a stainless-steel tube fixed to the inside of the cryovials and screwed into place using a magnetic holding rod (Figure 1C).
Warming process of vitrification
All vitrified blastocysts were warmed using a two-step sucrose dilution treatment. After the cryovials with vitrified embryos were opened and removed from the liquid nitrogen, the loops with blastocysts were plunged directly into 0.25 mol/l sucrose solution. The blastocysts were treated in 0.25 and 0.125 mol/l sucrose [HTF medium with 20% fetal bovine serum (FBS)] solution for 2 and 3 min, respectively. The warmed blastocysts were then returned and cultured into G2.2 (Vitrolife, Gothenburg, Sweden) medium in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The human blastocysts were observed and transferred after 16 h.
The morphology of surviving blastocysts after warming was defined with an expanding, intact trophectoderm and ICM; however, dehydration of embryos was found during treatment with cryoprotectants and warming solutions. The different degree of dehydration was observed and recorded.
Slow cooling and warming protocol
We modified the blastocyst freezing protocol reported by Cohen et al. (1985). The supernumerary blastocysts were exposed to 5% glycerol and 9% glycerol with 0.2 mol/l sucrose (in G2 medium with 20% albumin) for 10 min at room temperature. After treatment, the blastocysts were cooled using slow cooling (the cooling rates were as follows: 2°C/min from room temperature to 7°C), seeding at 7°C and holding for 15 min. The temperature was then decreased from 7 to 30°C (0.3°C/min) followed by temperature reduction from 30 to 180°C (50°C/min). The frozen blastocysts were then stored in LN.
When appropriate, the human blastocysts were removed from cryovials for warming and then assessed for their viability. The blastocysts were warmed by placing them in a solution (37°C) containing 0.5 mol/l sucrose for 10 min. After this, they were transferred into 0.2 mol/l sucrose for an additional 10 min. The warmed blastocysts were then cultured in G2.2 medium overnight to observe and evaluate their survival status.
Experiment I: mouse blastocysts with super-cooling vitrification
All mouse blastocysts were randomly assigned into three groups: group I was a super-cooled vitrification group. The embryos were treated with vitrification solution and vitrified using cryoloops combined with a Vit-MasterTM apparatus (n=108). Group II was a cryosolution effect (toxic) group. The embryos were treated with vitrification solution but without any enforced super-cooling temperature change with LN (n=88). Group III was a control group. These embryos were treated with neither vitrification solution nor super-cooled LN (n=112). All warmed mouse blastocysts were cultured for 24 h and the survival and development rates were observed and recorded.
Experiment II: donated human blastocyst with super-cooling vitrification
In order to test whether the new protocol is successful for human blastocysts, 153 blastocysts were donated by 38 patients who participated in our hospitals' IVF programme. The blastocysts were cyropreserved using slow freezing (n=72, 15 patients) or vitrification with cryoloops combined with a super-cooled LN procedure (n=81, 23 patients). All remaining blastocysts were collected and cultured from day 3 or day 5 embryo transfer. The remaining embryos after transfer and early or expanded blastocysts were all included in this study. Blastocysts were divided randomly into slow cooling or super-cooling groups. Because all remaining embryos on day 3 were in poor condition, there were no blastocysts good enough for transfer in the next cycle. The warmed blastocyst survival rate in the slow-cooled and vitrified blastocysts was compared after 16 h.
Experiment III: clinical super-cooled vitrification application
The supernumerary blastocysts available after in vitro culture were cryopreserved for future use. Informed consent was requested and obtained from all participating couples prior to using their blastocysts for the vitrification procedure.
If the patient elected to have her cryopreserved blastocysts subsequently thawed with a view to her becoming pregnant, serial serum E2 levels were ascertained and ultrasound examinations were performed to determine the patient's specific ovulation day. The timing of embryo thawing and transfer was determined by synchronizing the cryopreservation time with the period corresponding to the implantation window. We treated women in artificial cycles using a GnRH hormone agonist (Sathanandan et al., 1991). Only those blastocysts determined to be viable subsequent to warming were selected for embryo transfer (ET). Serum HCG levels were determined 12 days after ET for pregnancy status diagnosis. Ultrasound was performed twice, 1 and 3 weeks following ET to determine the presence of a viable fetal heart beat and overall pregnancy status.
Survival and development potential assessment
The warmed mouse and human blastocysts were transferred into G2.2 culture medium for a period of 24 and 16 h for further observation. Blastocysts were determined to have survived the freezing/thawing process if they presented an ICM, trophoectoderm and a re-expanding blastocoel cavity. Hatching blastocysts were determined to constitute surviving embryos.
Statistical analysis
The likelihood of blastocyst survival subsequent to treatment and the associated hatching rate were compared by use of the 2 test. A confidence level of P<0.05 was considered to constitute the limit of statistical significance for comparison.
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Results |
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Experiment III: clinical super-cooled vitrification application
Ninety-six blastocysts from 13 super-cooling vitrification cycles were warmed before transfer (on experiment II). Seventeen embryos were lost during the warming process (17.7%, 17 out of 96) and these embryos were lost from the loops into LN rather than from loop loading. The 79 remaining warmed embryos were cultured and the survival rate was 77% (74 out of 96) because five warmed embryos degenerated before transfer (5.2%). Sixty surviving blastocysts (74) were viable and transferred into 13 patients, resulting in seven successful pregnancies (53.8%, Table II). There were four patients with eight live births and two spontaneous abortions among the seven pregnancies. The one remaining pregnancy is going smoothly.
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Discussion |
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It has been determined previously that the factors necessary for successful vitrification include a faster cooling rate, a higher viscosity cryoprotectant solution and a smaller cryosolution volume for the blastocysts to decrease ice crystal formation inside and outside the cytoplasm (Rall, 1987; Arav et al., 2002
; Kasai et al., 2002
; Liebermann et al., 2002
). An ultra-rapid cooling rate offers two advantages over a slower rate. First, the cryoprotectant concentration used can be decreased with a consequent decrease in the associated potential toxicity. Secondly, a more rapid passage through the dangerous temperature zone results in reduced chilling injury (Vajta et al., 1998
). The cooling rate is
2500°C/min in conventional vitrification procedures (Rall and Fahy, 1985
). An ultra-rapid cooling rate, which is a special characteristic of super-cooling vitrification, further increases the cooling rate up to 24 000°C/min using an EM grid (Steponkus et al., 1990
), 20 000°C/min when using the OPS technique (Vajta et al., 1998
) and 20000°C/min using a cyroloop (Mukaida et al., 2001
). The achievable cooling rate with super-cooled LN (
100 000°C/min according to the manufacturer's instructions) is
5 times that achieved using only a cryoloop. Our study indicates that mouse and human blastocysts are able to traverse the dangerous temperature zone safely during this ultra-fast cooling rate and the post-thawed embryos maintained their developmental capability.
In a conventional vitrification process, embryos are plunged into high cryoprotectant solution concentrations (58 mol/l) (Rall and Fahy, 1985). Yeoman et al. (2001)
compared two different cryoprotectant levels in the vitrification of Rhesus monkey blastocysts in combination with a cryoloop (Yeoman et al., 2001
). One cryosolution contained 25% (4.5 mol/l) EG and 25% (3.4 mol/l) glycerol and proved to be effective, constituting an appropriate freezing medium. However, the other cryosolution, which contained 20% (3.6 mol/l) EG, 20% (2.4 mol/l) DMSO, 0.65 mol/l sucrose and 25 µmol/l Ficoll, proved to be ineffective. This report (Yeoman et al., 2001
) indicated that a higher cryoprotectant concentration was desirable for successful vitrification even in the presence of a cryoloop. In our work, we used a cryosolution composed of 20% (3.6 mol/l) EG, 20% (2.4 mol/l) DMSO and 0.5 mol/l sucrose, which could have been construed ineffective given the findings from the previous study. This further suggests that when adopting an ultra-rapid cooling rate for blastocysts, the cryosolution viscosity used could be decreased while still preserving its function, and thus it would be less toxic to embryos or gametes. We observed that there was less dehydration in mouse blastocysts in early or expanding embryos during cryosolution treatment or the warming process. However, the different sized human blastocysts exhibited a different degree of dehydration. According to observation of the shrinkage of blastocoel, those grade A or B blastocysts exhibiting a greater degree of dehydration showed smaller blastocoels (<20% the original size of the blastocoel) or unexpanded blastocoels during treatment in the cryoprotectants or warming solutions. A large or expanding and hatching blastocyst (grade C and D) showed a lower degree of dehydration (retaining >90% of the original size of the blastocoel) during treatments and had a better survival rate than early blastocysts (90 and 78%; data not shown). Those blastocysts with greater dehydration showed a lower expanding ability and degenerated easily after warming. It was suggested that adjusted concentrations of protectants and treatment times for small sized human blastocysts are needed. A previous study reported that survival of blastocysts was associated with larger size in rabbit blastocysts vitrified using a two-step procedure. After warming, the larger blastocysts (200300 µm) showed significantly more attachment (5464%) and trophectoderm outgrowth (4458%) rates than the smaller blastocysts (attachment, 29%; trophectoderm outgrowth, 25%) (Cervera and Garcia-Ximenez, 2003
). This suggests that early blastocysts may need a shorter treatment time or a modified higher cryosolution concentration.
To decrease the vitrified solution volume, Arav et al. (2002) developed a bovine embryo vitrification method that included a minimum drop size (MDS) of only 0.10.5 µl of vitrification solution in super-cooled liquid nitrogen (210°C). This approach maximized the cooling rate to the highest physically possible (24 000130 000°C/min). Cryoloops are a refined strategy that can be adopted to reduce the cryosolution volume used (Lane et al., 1999a
,b
). The cryosolution volume within the thin film layer in a cryoloop is
0.10.2 µl. Therefore, we adopted the cryoloop for super-cooled vitrification. The cryopreservation fluid volume used during cryoloop-supported vitrification is such that the volume is sufficiently small to enhance the success of vitrification and also avoid/reduce the potential for blastocyst damage during the freezing process (Kasai et al., 2002
).
In experiment II, we compared the slow freezing procedure with super-cooled vitrification for human blastocyst cryopreservation. The slow freezing method is associated with chill injury and ice crystal formation during the freezing and warming process. Between 5 and approximately 15°C, the cells' contents remain unfrozen and super-cooled but ice forms in the external medium, and water flows out of the cells osmotically and freezes externally (Goa and Critser, 2000). Intracellular ice formation causing injury was induced by the water flux across the cell membrane during the cooling or warming (Muldrew and McGann, 1994
). The subsequent physical events in the cells depend on the cooling rate. If the cells become increasingly super-cooled, intracellular water is not lost fast enough, eventually attaining equilibrium by freezing intracellularly (Mazur, 1963
, 1990
). When the cells are slow cooled, they experience severe shrinkage by losing water rapidly, concentrating the intercellular solution, resulting in long-term exposure to high concentration of cryoprotectant. According to those cases, cell injury may be caused by the increasing ice-crystal formation (Goa and Critser, 2000
). Human blastocysts with blastocoele present are much less permeable to cryoprotectant and also to water (Mukaida et al., 2001
). The slow freezing process for embryos at the cleavage stage may not be appropriate given that it typically requires more time for cryoprotectant and water exchange in the blastocysts during the freezing procedure than for cleavage embryos. In super-cooled vitrification, the chance of ice crystal formation within the blastocoel cavity would probably be decreased further compared with slow cooling procedures (Vajta et al., 1997
). Our study suggested that human blastocysts could most probably avoid ice crystal injury during the super-cooled vitrification and warming procedures compared with slower cooling and warming techniques.
Here we presented seven successful pregnancies following human blastocysts LN super-cooling vitrification combined with cryoloops. A moderately low expanded blastocyst survival rate subsequent to cryopreservation and warming was noted by previous investigators (Ludwig et al., 1999). The fluid-filled cavity within the expanded blastocyst previously has been reported to be a factor affecting the potential blastocyst survival rate following vitrification by some (Ludwig et al., 1999
) but not all (Vanderzwalmen et al., 2002
) previous investigators. A possible explanation for this is that the likelihood of ice crystal formation during blastocyst freezing increases proportionally with the blastocoel volume increase. In the current thirteen human cases in this study, cryopreservation was performed at the early and expanded blastocyst stages. Seventy-four embryos survived. This desirable outcome was facilitated by the ultra-rapid cooling rate adopted. Ultra-rapid cooling decreases the chance of ice crystal formation. The cryoloopsuper-cooled LN combination greatly decreases ice crystal formation. The clinical application efficiency of this technique for expanded blastocysts, however, appears to warrant further investigation.
Mouse and human blastocysts were shown to be cryopreserved effectively using super-cooled LN combined with a cryoloop in this study. The proposed method is rapid, easy and effective for the cryostorage of supernumerary blastocysts in an IVF programme. The developmental potential for human blastocysts appears unharmed by blastocyst passage through the proposed freezing and warming technique.
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Notes |
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References |
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