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Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application
LESSONS FROM THE LABORATORY Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application Juergen Liebermann, Ph.D.,a and Michael J. Tucker, Ph.D.a,b a
Fertility Centers of Illinois, Chicago, Illinois; and b Georgia Reproductive Specialists, Atlanta, Georgia
Objective: To evaluate implantation of day 5 and day 6 vitrified and slow-frozen blastocysts. Design: Retrospective analysis comparing two cryopreservation techniques. Setting: Private IVF clinic. Patient(s): Five hundred eight cryopreserved embryo transfer candidates. Intervention(s): Supernumerary day 5 and day 6 blastocysts were vitrified or slow-frozen and transfered after warming or thawing. Main Outcome Measure(s): Comparison of two cryopreservation techniques with respect to survival rate, implantation, and pregnancy rates of day 5 and day 6 blastocysts. Result(s): In 254 vitrified transfer cycles, survival, embryonic implantation, and clinical pregnancy rates for day 5 blastocysts were 95.9%, 33.4%, 48.7%, respectively, and for day 6 blastocysts 97.5%, 25.9%, 42.8%. In 254 slow-frozen transfer cycles, survival, embryonic implantation, and clinical pregnancy rates for day 5 blastocysts were 91.4%, 29.6%, 42.8%, respectively, and for day 6 blastocysts 94.8%, 28.2%, 43.1%. Overall there was a slightly, but not significantly, higher outcome regarding implantation and clinical pregnancy with the use of day 5 blastocysts (31.3% and 45.4%, respectively) versus day 6 blastocysts (26.7, and 42.9%, respectively). Conclusion(s): Vitrification technique yields the same implantation and pregnancy rate as slow-frozen blastocyst transfers. Slow growing embryos can be cryopreserved on day 6, because they yield a satisfactory survival, implantation, and pregnancy rate. (Fertil Steril威 2006;86:20 – 6. ©2006 by American Society for Reproductive Medicine.) Key Words: Extended culture, blastocysts, cryopreservation, conventional, vitrification, frozen embryo transfer, Cryotop
Cryopreservation has become an increasingly important therapeutic strategy in reproductive medicine, with the birth of many infants after use of this procedure. It is important for cryopreservation in general to establish consistent outcomes, especially in terms of embryo cryosurvival to allow high chances of success in performing a frozen embryo transfer (FET). This in turn will increase the positive awareness of cryopreservation in assisted reproduction technology (ART) as a means to reduce multiple implantation, thereby expanding its use in all areas of reproductive medicine. However, standard cryopreservation technologies appear to illustrate their ultimate limitations in their lack of consistency in cryosurvival. Consequently, radically different protocols may provide the answer to increased consistent sucReceived September 23, 2005; revised and accepted January 27, 2006. Presented in part at the 61st Annual Meeting of the American Society for Reproductive Medicine, Montreal, Quebec, October 15–19, 2005. Reprint requests: Juergen Liebermann, Ph.D., Fertility Centers of Illinois, 900 N. Kingsbury, River Walk 6, Chicago 60610, Illinois (FAX: 312-4941687; E-mail: [email protected]).
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cess, and interest has recently shifted to vitrification as an attractive alternative to slow-freezing methodology (1). The chief difference between vitrification and conventional cryopreservation procedures is that it is an “open system” defined by [1] direct contact between the solution containing the embryos and liquid nitrogen (LN2), and [2] storage of embryos in only partially sealed containers. To promote extremely rapid cooling, this system lacks any significant thermoinsulating layer around the specimen. If outcomes between both techniques are at least comparable, certain benefits of the vitrification protocol may appeal to clinical embryologists. Vitrification as an ultrarapid cooling technique is simple, potentially faster, and inexpensive; further, it is starting to become clinically established and seems to have the potential to be more reliable and consistent than conventional cryopreservation when carried out properly (2, 3). Further, the need for controlled-rate freezing equipment, which requires routine calibration and maintenance, is eliminated. The cells are placed into the cryoprotectant, then the cells are placed in a very small volume of
Fertility and Sterility姞 Vol. 86, No. 1, July 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2006.01.029
cryoprotectant on a special carrier, and then they are cooled at extreme rates by plunging them directly into LN2. With this method no ice crystals form, avoiding damage to the cells or the tissues. Concerns with the high concentration of cryoprotectant required to achieve vitrification are allayed to some extent by recent publications such as that by Takahashi et al. (4) showing that the use of relatively high concentration of cryoprotectants, such as 15% ethylene glycol (EG) in an equimolar mixture with dimethyl sulfoxide (DMSO), had no negative effect on the perinatal outcome of blastocyst transfer using vitrification when compared with fresh blastocyst transfer. This is an important finding to help confirm the safety of vitrification for clinical use and serves to counter recent published concerns about the use of EG for vitrification and its purported negative effect on organogenesis (5– 6). Our personal preference is blastocyst-stage freezing. There are three major rationales for our stance: [1] successful cryopreservation of human blastocysts is increasingly relevant as extended in vitro culture of human embryos becomes more common, permitting routine use of blastocyst transfer in IVF programs as an optimal course to reduce embryo numbers for uterine transfer, thereby reducing multiple pregnancies; [2] consistent blastocyst cryopreservation is a necessary adjunct to maximize cumulative pregnancy rates from each oocyte retrieval (7); and [3] the superiority of blastocyst stage freezing over earlier stage freezing in terms of implantation per thawed embryo transferred improves overall expectations for the cryopreservation program (8). With an increasing proportion of IVF offspring being born after cryopreservation, any improvement in survival rates and viability of such embryos will only add to the readiness with which cryopreservation is adopted as a frontline option in ART. Comparing the survival rates of blastocysts from slowfreezing and vitrification protocols, Yeoman et al. (9) showed in rhesus monkey blastocysts a postthaw survival rate of 36% with 5% hatching after slow-freezing and 85% with 71% hatching after vitrification. In addition, after vitrification of human blastocysts with an intact zona pellucida using the cryoloop as carrier, survival rates of 72%–90%, clinical pregnancy rates of 37%– 48%, and an implantation rate of 22%–29% have been reported (4, 10 –13). Also, the clinical outcomes using carriers other than the cryoloop, such as French ministraws, hemistraws, and electron microscope grids, have shown that vitrification is universally effective and practical for the cryopreservation of human blastocysts (14 –19). These reports help confirm the effectiveness of vitrification as a feasible cryopreservation option. To date no clinical study has attempted to compare cryopreservation of human day 5 and day 6 blastocysts using either a traditional slow-freezing protocol or a vitrification protocol. We therefore analyzed retrospectively the reproductive outcome of both cryopreservation techniques, and the embryonic implantation and relative pregnancy rates for cryopreseved day 5 and day 6 blastocysts. Fertility and Sterility姞
MATERIALS AND METHODS All patients used standard long or short protocols using GnRH analog and gonadotropins for controlled ovarian hyperstimulation. Embryo cultures were carried out in Sage sequential media (Sage, Trumbull, CT) under mineral oil (Sage), using Nuclon four-well dishes (Nunc, Roskilde, Denmark). Slow-frozen–thawed blastocysts were derived from IVF as well as intracytoplacmic sperm injection (ICSI) procedures, whereas all vitrified-warmed blastocysts were derived from ICSI procedures only. The reason for this is that our IVF program initiated a 100% ICSI insemination protocol from January 2004 onward, and from January 2004 we changed our entire cryopreservation program of day 5 and day 6 blastocysts from conventional freezing to vitrification only. When the patient had fresh embryo transfer on day 3 or 5, supernumerary blastocysts were scored depending on the developmental stage and were graded according to the system of Gardner and Lane (20). This system incorporates assessment of the rate of development and independent measure of the inner cell mass (ICM) and trophectoderm (TE). We used a modification of this system to accommodate a tradition of embryo quality scoring that has graded the best embryos with a low number (21). Each blastocyst has an overall numeric score based on rate of development and expansion: 1 (fully expanded or hatching day 5), 2 (fully expanded or hatching day 6), 3 (moderate expansion day 6 or early cavitation day 5), or 4 (early cavitation day 6 or morula day 5 or 6). We add to this numeric score two alphabetic scores to grade first the ICM and second the TE. Blastocysts were graded A (high cell number with good cell-cell adhesion), B (lower cell number with poorer cell-cell attachment), or C (no ICM apparent, sparse granular cells in the TE). As an example, a good-quality well-expanded blastocyst on day 6 with good ICM and good integrity of TE would be scored as 2AA. Only grade 1 or 2 blastocysts on day 5 or 6 with an AA to BB score for ICM and TE were cryopreserved. Patients not achieving a clinical pregnancy returned for a frozen blastocyst transfer cycle. In both groups the blastocysts were derived from the patient’s own oocytes. All women received GnRH agonist and supplementary estroderm (Ortho-McNeil, Raritan, NJ) for preparation of the endometrium. Intramuscular administration of progesterone in oil (Schein, Florham Park, NJ) was initiated when the endometrial thickness reached 10 mm or more (usually 4 days before the frozen blastocyst transfer was scheduled and applied until the first hCG 10 days after transfer). One to three blastocysts were transfered into the patient’s uterus 5 days after the initiation of progesterone treatment regardless of the blastocyst developmental stage. Between January 2004 and December 2005, we performed 254 cycles of vitrified-warmed blastocyst transfers with patients’ age 34.2 ⫾ 5.0 years. During the same period, we performed 254 cycles of slow-frozen–thawed blastocyst transfers with patients’ age 35.1 ⫾ 4.7 years. 21
Vitrification of blastocysts was undertaken using the Cryotop carrier system (Kitazato Bio Pharma, Fuji-shi, Japan) after a two-step loading with cryoprotectant agents at 24°C. Briefly, blastocysts were placed in equilibration solution, which is the base medium (Hepes-buffered HTF with 20% HSA; Sage) containing 7.5% (v/v) EG and 7.5% (v/v) DMSO. After 5–7 minutes, the blastocysts were washed quickly in vitrification solution, which is the base medium containing 15% (v/v) DMSO, 15% (v/v) EG, and 0.5 mol/L sucrose, for 30 seconds and transferred onto the Cryotop using a micropipette. Immediately after the loading of ⱕ2 blastocysts in a 1-L drop on the Cryotop carrier, it was plunged into fresh clean LN2. The time the blastocysts were exposed to the vitrification solution before cooling was ⱕ30 seconds. After loading the embryos, the Cryotop was capped under the LN2 to seal and protect the vitrified material before cryostorage. To remove the cryoprotectants, blastocysts were warmed and diluted in a two-step process. With the Cryotop submerged in LN2, the protective cap was removed, and then the carrier with the blastocysts was removed from the LN2 and placed directly into a prewarmed (approximately 30°C) organ culture dish containing 1 mL of 1.0 mol/L sucrose. Blastocysts were picked up directly from the Cryotop and placed in a fresh drop of 1.0 mol/L sucrose at 24°C. After 5 minutes, blastocysts were transfered to 0.5 mol/L sucrose solution. After an additional 5 minutes, blastocysts were washed in the base medium and returned to the culture medium (Sage Blastocyst Medium) until transfer. Slow-frozen blastocysts were originally frozen in modified protocols containing glycerol and sucrose based on Menezo’s two-step method previously described (22, 23). Briefly, blastocysts were exposed for 10 minutes in 5% glycerol at 25°C, then moved in 9% glycerol ⫹ 0.2 mol/L sucrose. Blastocysts were loaded into straws (IMV Technologies, L’Aigle, France), sealed, and loaded into a rate-controlled Kryo 10 LN2 freezer (Planar Kryo 10, Perkasie, PA). The initial blastocyst cooling was achieved at a rate of ⫺2°C/min to ⫺7°C. Ice crystallization was initiated manually by touching the straw with cold forceps. The temperature was held for 10 minutes. Further cooling was attained at a rate of ⫺0.3°C/min to ⫺36°C, held for 15 minutes, after which the blastocysts were plunged directly into LN2 (⫺196°C) for cryostorage. Blastocysts were thawed by removing the straw from the LN2, and disappearance of all intracellular ice crystals occurred during initial blastocyst thawing, carried out for 30 seconds at room temperature. The straw was cut open onto a small Petri dish, and the contents expelled into the dish. Once the blastocysts were located, they were placed in serial dilutions of cryoprotectant at 25°C through two concentrations of glycerol (9% and 5%; 3 minutes per dilution) in 0.2 mol/L sucrose. Additional thawing was achieved at 25°C through 0.2 mol/L sucrose for 2 minutes, then the blastocysts were washed in the base medium (Hepes-buffered HTF with 10% HSA; Sage) and re22
Liebermann and Tucker
turned to the culture medium (Sage Blastocyst Medium) until transfer. Laser-assisted hatching, using a 1,480-nm diode laser system (Zilos; Hamilton Thorne Bioscience, Beverly, MA), was carried out on all blastocysts before reexpansion during the dilution steps after warming or thawing. In this way all potential thermal damage to the embryo was easily avoided while allowing the ability to perform extensive zona ablation. In addition, assisted hatching was undertaken routinely on all thawed or warmed blastocysts to compensate both for potential zona hardening following cryopreservation (24) and for the slower development in the case of day 6 blastocysts (25). Laser-assisted hatching was not necessary for those blastocysts that either experienced zona fracture during cryopreservation or were already hatching at the time of cryopreservation. After warming or thawing, blastocysts were cultured for at least 3– 4 hours after warming and assessed for survival based on the morphologic integrity of the ICM, TE, and reexpansion of the blastocele. Then the surviving blastocysts were scored as to their developmental stage and graded for quality as described earlier and transfered to the uteri of the patients. A positive pregnancy is defined as a positive -hCG ⱖ20 mIU/mL 10 days after blastocyst transfer. Implantation rate is defined by the number of gestational sacs per embryo number transferred. Clinical pregnancy refers to the identification of a pregnancy sac in the uterus, whereas ongoing or delivered pregnancy describes pregnancies that continue beyond 20 weeks. Statistical analysis was carried out by means of a 2test using Microsoft Excel 2001 for Macintosh (Redmond, WA). Statistical significance was defined as P⬍.05. RESULTS Table 1 shows the mean age and clinical outcome of patients who completed the vitrified or slow-frozen blastocyst transfer program. The mean age of the women was 34.2 ⫾ 5.0 years in the vitrified group and 35.1 ⫾ 4.7 years in the slow-frozen group. No significant differences could be observed regarding age in the two groups. The blastocyst postwarming survival rates after using either the vitrification technique or the conventional technique are shown in Table 1. A total of 547 vitrified blastocysts (day 5 and day 6) were warmed, of which 528 survived warming (96.5%), whereas in the slow-freezing group (day 5 and day 6), 92.1% of the blastocysts (525/570) survived. Generally, the blastocyst survival rate was slightly higher in the vitrified group than in the slow-frozen group, but no significant difference was noted. Combined, the percentage of surviving blastocysts was 94.3% (1,053/1,117). In the vitrified group, 523 vitrified-warmed blastocysts in 254 cycles of 257 attempted cycles were transferred (mean 2.0 blastocysts per FET). Overall, the implantation and clinical pregnancy rates per transfer were 30.6% and 46.1%, respectively. To date, 41 deliveries have occurred with no
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TABLE 1 Retrospective data from the blastocyst cryopreservation program (Fertility Centers of Illinois, Chicago) where both vitrification (VIT) and conventional (CONV) technologies were applied from January 2004 to December 2005. Technique Patients’ age (y) No. of thawed cycles No. of transfers No. of blastocysts warmed/thawed No. of blastocysts survived (%) No. of blastocysts transferred Mean no. of blastocysts transferred No. of implantations (%) No. of positive pregnancy/thaw (%) No. of positive pregnancy/FET (%) No. of clinical pregnancy/thaw (%) No. of clinical pregnancy/FET (%) Ongoing pregnancies (%) No. of livebirths
VIT
CONV
34.2 ⫾ 5.0 257 254 547 528 (96.5) 523 2.0 160 (30.6) 132 (51.4) 132 (51.9) 117 (45.5) 117 (46.1) 117 (88.6) 54
35.1 ⫾ 4.7 259 254 570 525 (92.1) 518 2.0 152 (28.9) 135 (52.1) 135 (53.1) 109 (42.1) 109 (42.9) 109 (79.6) 79
Note: P ⬎ .05 for every comparison. FET ⫽ frozen embryo transfer. Liebermann. Comparison of vitrification and conventional cryopreservation of blastocysts. Fertil Steril 2006.
reported abnormalities (54 infants: 23 boys and 31 girls). From the 259 slow-frozen–thawed cycles, transfer could be carried out in 254 cycles with 518 blastocysts (mean 2.0 blastocysts per FET). Overall, the implantation and clinical pregnancy rates per transfer were 28.9% and 42.9%, respectively. At this time, 63 deliveries have occurred with no reported abnormalities (79 infants: 34 boys and 45 girls). When the vitrified-warmed blastocysts were divided into day 5 and day 6 groups, 95.9% (331/345) of day 5 blastocysts and 97.5% (197/202) of day 6 blastocysts survived after warming (Table 2), but this difference was not significant. As shown in Table 2, implantation and clinical pregnancy rates per transfer occurring in the day 5 blastocyst group were 33.4% and 48.7% respectively, which were significantly higher than in the day 6 blastocyst group (25.9% and 42.8%; 2: P⬍.01 and P⬍.01, respectively). In slow-frozen–thawed blastocyst transfers; the survival rate between day 5 and day 6 was slightly but not significantly different (91.4% vs. 94.8%). As shown in Table 2, the implantation and clinical pregnancy rates per transfer did not differ in the day 5 group (29.6% and 42.8%, respectively) compared with the day 6 group (28.2% and 43.1%). Table 3 shows that in total the survival rate of cryopreserved day 5 blastocysts was 93.4% (746/799) and did not significantly differ from that of cryopreserved day 6 blastocysts (96.5%; 307/318). The implantation and clinical pregnancy rates per transfer of day 5 blastocysts were 31.3% (230/734) and 45.4% (160/352), respectively. As shown in Table 3, transfer of day 6 blastocysts showed an excellent implantation rate of 26.7% (82/307) and a clinical pregnancy Fertility and Sterility姞
rate per transfer of 42.9% (67/156). There were no statistical differences between groups. DISCUSSION With the introduction of sequential culture media in ART, and driven by the large increase in the rate of multiple pregnancies arising from earlier-stage ET, extended culture to the blastocyst stage has become more common. Consequently, the need to cryopreserve human blastocysts is also increasing. Although the results achieved by conventional slow freezing seem successful (23, 26 –30), clinical results with blastocyst cryopreservation have not necessarily been consistent, owing to the higher potential for damaging ice crystal formation in traditional slow-freezing protocols. Recently there has been an increasing number of reports of successful human blastocyst vitrification (10 –19, 31, 32). We compared 24 months of experience with vitrification of blastocysts, using 15% EG, 15% DMSO, and 0.5 mol/L sucrose, with contemporaneous data using conventional slow cryopreservation with 9% glycerol plus 0.2 mol/L sucrose. From published reports, it seems that there are no apparent differences between the two cryopreservation techniques with respect to survival implantation and pregnancy. The data from the present retrospective clinical study have clarified that vitrification can at least achieve comparable results to those achieved with conventional slow-freeze technology. Furthermore, the results from this study help to prove that vitrification of blastocysts using the Cryotop procedure is effective by achieving high implantation and preg23
TABLE 2 Comparison of retrospective data from the blastocyst cryopreservation program (Fertility Centers of Illinois, Chicago) of day 5 and day 6 blastocysts where both vitrification (VIT) and conventional (CONV) technologies were applied from January 2004 to December 2005. VIT day 5
VIT day 6
Patients’ age (y) 33.9 ⫾ 4.9 34.5 ⫾ 5.3 No. of thawed cycles 158 99 No. of transfers 156 98 No. of blastocysts warmed/thawed 345 202 No. of blastocysts survived (%) 331 (95.9) 197 (97.5) No. of blastocysts transferred 326 197 Mean no. of blastocysts transferred 2.1 2.0 51 (25.9)a No. of implantations (%) 109 (33.4)a No. of positive pregnancy/thaw (%) 86 (54.4) 46 (46.0) No. of positive pregnancy/FET (%) 86 (55.1) 46 (46.5) No. of clinical pregnancy/thaw (%) 76 (48.1) 42 (42.4) No. of clinical pregnancy/FET (%) 76 (48.7)b 42 (42.8)b Ongoing/delivered pregnancies (%) 76 (88.4) 42 (91.3)
CONV day 5
CONV day 6
35.0 ⫾ 4.7 201 196 454 415 (91.4) 408 2.0 121 (29.6) 106 (52.7) 106 (54.0) 84 (41.8) 84 (42.8) 84 (79.2)
35.4 ⫾ 4.8 58 58 116 110 (94.8) 110 1.9 31 (28.2) 29 (50.0) 29 (50.0) 25 (43.1) 25 (43.1) 25 (86.2)
Note: FET ⫽ frozen embryo transfer. a,b P ⬍ .01. Liebermann. Comparison of vitrification and conventional cryopreservation of blastocysts. Fertil Steril 2006.
nancy rates and safe for clinical use by giving rise to healthy infants without abnormalities. The results of the present study help to confirm recently published results with respect to clinical outcomes following blastocyst vitrification with use of a stepwise vitrification protocol using a mixture of 7.5% EG-DMSO (lower-strength
step) followed by 15% EG-DMSO with 0.5 mol/L sucrose (full-strength step) (4). Concerns about introduction of high concentrations of cryoprotectant, which are necessary to prevent mechanical damage from ice, exist with vitrification. The problem of cryoprotectant toxicity is an immediate and practical one, just as it is to a lesser extent in classic slow-
TABLE 3 Overall comparison of retrospective data from the blastocyst cryopreservation program (Fertility Centers of Illinois, Chicago) of day 5 and day 6 blastocysts where both vitrification (VIT) and conventional (CONV) technologies were applied from January 2004 to December 2005.
Patients’ age (y) No. of thawed cycles No. of transfers No. of blastocysts warmed/thawed No. of blastocysts survived (%) No. of blastocysts transferred Mean no. of blastocysts transferred No. of implantations (%) No. of positive pregnancy/thaw (%) No. of positive pregnancy/FET (%) No. of clinical pregnancy/thaw (%) No. of clinical pregnancy/FET (%) Ongoing/delivered pregnancies (%)
VIT ⴙ CONV day 5
VIT ⴙ CONV day 6
34.6 ⫾ 4.8 359 352 799 746 (93.4) 734 2.0 230 (31.3) 192 (53.4) 192 (54.5) 160 (44.5) 160 (45.4) 160 (83.3)
34.9 ⫾ 5.1 157 156 318 307 (96.5) 307 2.0 82 (26.7) 75 (47.7) 75 (48.0) 67 (42.6) 67 (42.9) 67 (89.3)
Note: P ⬎ .05 for every comparison. FET ⫽ frozen embryo transfer. Liebermann. Comparison of vitrification and conventional cryopreservation of blastocysts. Fertil Steril 2006.
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Vitrification during clinical application
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cooling procedures. Extremely rapid cooling allows a decrease to be made in the concentration of the cryoprotectant and thereby a reduction in potential toxicity (33). The greatest advantages of vitrification have been seen in chill-sensitive cells such as oocytes and blastocysts (34). The main characteristic of the blastocyst is its fluid-filled cavity, the blastocele. It seems that with increasing volume of the blastocelic cavity, the survival rate drops with vitrification. This is thought to be due to insufficient permeation of cryoprotectant into the blastocelic cavity, such that residual water may promote ice crystallization during the vitrification process. Several articles report that survival rates in cryopreserved expanded blastocysts could be improved by artificial reduction of the blastocelic cavity (14, 15, 19, 35, 36). In addition, recent publications have shown that survival rates of cryopreserved expanded blastocysts as well as zonafree hatched blastocysts (36, 37) can be improved by artificial reduction of the blastocelic cavity. In our protocol we proceeded without any opening in the zona pellucida before vitrification independent of the size of the blastocelic cavity. Previous investigators have found superior implantation rates with fresh transfers occurring at day 5 as compared with day 6 (38). They reported an almost doubled clinical pregnancy and implantation for fresh day 5 blastocyst compared with fresh day 6 blastocysts (38). Our data comparing vitrified or frozen day 5 and day 6 blastocysts did not confirm this observation to such an extent. However, the reported high embryonic implantation and pregnancy rates following vitrified-warmed as well as slow frozen–thawed transfer of day 6 blastocysts in the present study should encourage cryopreservation of day 6 blastocysts, and even those that do not fully expand until day 7 (23). Although more slowly developing blastocysts may be innately compromised to some extent, the data from this report show that there is profound clinical value in knowing they can be vitrified/frozen as late as day 6, successfully thawed, and result in a live birth. It is plausible that a more synchronous transfer of these warmed/thawed blastocysts contributed to the excellent outcome observed in the present study. There is one issue with vitrification that needs further discussion. A concern has been made that fungi, bacteria, and viruses are able to survive in LN2 (39 – 44). Given that with vitrification the cells are directly plunged into LN2, they therefore have direct contact with LN2 and so the question arises as to whether the LN2 has to be sterilized because it may be a possible source of contamination. Use of clean LN2 for the initial vitrification step, followed by sealing of the carrier, seems to address the concern of potential contamination during cryostorage. To further reduce fears of contamination, it is possible to store material from potentially infectious patients separately from seemingly noninfectious samples. Therefore, it is important to perform routine screening tests for viral infections, including HIV and hepatitis B and C, on all couples undergoing infertility treatment. In the event that a couple screens positive, we offer vitrification of Fertility and Sterility姞
blastocysts. Even though we consider the risk of crosscontamination during storage to be almost infinitesimal, in such cases we nevertheless recommend placing embryos in specially designated tanks, or shipping them off-site. It is worth noting that to date no viral, fungal, or bacterial contamination event has been described from approximately 400 publications related to vitrification since 1985. In conclusion, although some problems remain to be fully addressed with vitrification as a routine cryopreservation technique, we believe that it shows much promise as a successful alternative to conventional freezing technology. Even without significant clinical improvement, the evident advantages of vitrification are that cryosurvival seems more consistent, allowing greater ease of patient management, with transfers being almost certain to occur. Concerns about vitrification are well defined, limited in number, and to our thinking easily surmountable. In general, with much shorter protocols, vitrification [1] is able to be undertaken on a more flexible basis by laboratory staff, [2] allows for the potential reduction in personnel time needed during the entire vitrification process, [3] simplifies laboratory techniques for cryopreservation in human ART, and [4] may enable more optimal timing of embryo cryopreservation, e.g., individual blastocysts may be cryopreserved at their optimal stage of development and expansion. Interest levels will inevitably rise, given the potential benefits of vitrification. This in turn will drive its development to higher levels of clinical efficiency and utilization (1, 45, 46). Acknowledgments: The authors thank the Fertility Centers of Illinois (FCI) and Elissa Knopoff, B.S., Jill Matthews, B.S., Amanda Erman, B.S., Rebecca Brohammer, B.S., Sara Sanchez, B.S., Yuri Wagner, B.S., and Andrew Barker, B.S., the embryologists at the FCI IVF Laboratory River North, for their invaluable contributions and support in pushing vitrification to become the standard protocol for cryopreservation of human blastocysts within our program.
REFERENCES 1. Liebermann J, Nawroth F, Isachenko V, Isachenko E, Rahimi G, Tucker MJ. The potential importance of vitrification in reproductive medicine. Biol Reprod 2002;67:1671– 80. 2. Tucker MJ ,Liebermann J. Oocyte and embryo cryopreservation. In: Patrizio P, Tucker MJ, Guelman V, editors. A color atlas of human assisted reproduction: laboratory and clinical insight. Philadelphia: Lippincott, Williams and Wilkins; 2003. p. 137–59. 3. Liebermann J, Tucker MJ. Vitrifying and warming of human oocytes, embryos, and blastocysts: vitrification procedures as an alternative to conventional cryopreservation methods. In: Schatten H, Germ cell protocols, vol. 2: Molecular embryo analysis, live imaging, transgenesis, and cloning. Totowa, NJ: Humana Press; 2004. p. 345– 64. 4. Takahashi K, Mukaida T, Goto T, Oka C. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertil Steril 2005;84:88 –92. 5. Menezo Y. Cryopreservation of IVF embryos: which stage? Eur J Obstet Gynecol Reprod Biol 2004;113(Suppl 1):S28 –32 (review). 6. Menezo Y. Blastocyst freezing. Eur J Obstet Gynecol Reprod Biol 2004;115(Suppl 1):S12–5 (review). 7. Langley MT, Marek DM, Gardner DK, Doody KM, Doody KJ. Extended embryo culture in human assisted reproduction treatments. Hum Reprod 2001;16:902– 8.
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8. Mandelbaum J, Belaisch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998;13(Suppl 3)161–77. 9. Yeoman RP, Gerami-Naini B, Mitalipov S, Nusser KD, WidmannBrowning AA, Wolf DP. Cryoloop vitrification yields superior survival of rhesus monkey blastocysts. Hum Reprod 2001;16:1965–9. 10. Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K. Successful birth after transfer of vitrified human blastocysts with use of a Cryoloop containerless technique. Fertil Steril 2001;76:618 –23. 11. Reed ML, Lane M, Gardner DK, Jensen NL, Thompson J. Vitrification of human blastocysts using the Cryoloop method: successful clinical application and birth of offspring. J Assist Reprod Genet 2002;19: 304 – 6. 12. Mukaida T, Takahashi K, Kasai M. Blastocyst cryopreservation: ultrarapid vitrification using Cryoloop technique. Reprod Biomed Online 2003;6:221–5. 13. Mukaida T, Nakamura S, Tomiyama T, Wada S, Oka C, Kasai M, et al. Vitrification of human blastocysts using Cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003;18:384 –91. 14. Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, van Roosendaal E, Vandervorst M, et al. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 2002;17:744 –51. 15. Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, Bollen N, van Roosendaal E, et al. Vitrification of human blastocysts with the hemistraw carrier: application of assisted hatching after thawing. Hum Reprod 2003;18:1501–11. 16. Choi DH, Chung HM, Lim JM, Ko JJ, Yoon TK, Cha KY. Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVF-ET program. Fertil Steril 2000;74:838 –9. 17. Yokota Y, Sato S, Yokota M, Ishikawa Y, Makita M, Asada T, et al. Successful pregnancy following blastocyst vitrification. Hum Reprod 2000;15:1802–3. 18. Yokota Y, Sato S, Yokota M, Yokota H, Araki Y. Birth of a healthy baby following vitrification of human blastocysts. Fertil Steril 2001;75: 1027–9. 19. Son WY, Yoon SH, Yoon HJ, Lee SM, Lim JH. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod 2003;18:137–9. 20. Gardner DK, Lane M. Embryo culture systems. In: Trouson AO, Gardner DK, Handbook of in vitro fertilization. 2nd ed. Boca Raton (FL): CRC Press; 2000. p. 205– 64. 21. Tucker MJ, Liebermann J. Morphological Scoring of Human Embryos and Its Relevance to Blastocyst Transfer. In: Patrizio P, Tucker MJ, Guelman V, editors. Color atlas of human assisted reproduction: laboratory and clinical insight. Philadelphia: Lippincott, Williams and Wilkins; 2003. p. 99 –108. 22. Menezo Y, Veiga A. Cryopreservation of blastocysts. Proceedings of the 10th World Congress on IVF and Assisted Reproduction; 1997 May 24 –28;Vancouver, British Columbia. Bologna, Italy: Monduzzi Editore; 1997. p. 41–5. 23. Sills ES, Sweitzer CL, Morton PC, Perloe M, Kaplan CR, Tucker MJ. Dizygotic twin delivery following in vitro fertilization and transfer of thawed blastocysts cryopreserved at day 6 and 7. Fertil Steril 2003;79: 424 –7. 24. Tucker MJ, Cohen J, Massey JB, Mayer MP, Wiker SR, Wright G. Partial dissection of the zona pellucida of frozen/thawed human embryos may enhance blastocyst hatching, implantation and pregnancy outcome. Am J Obstet Gynecol 1991; 165:341–345. 25. Tucker M. Relevance of assisted hatching with blastocyst stage transfer. Proceedings of the 1st World Congress on Controversies in Obstetrics, Gynecology and Infertility; 1999 Oct 28 –31; Prague. Bologna, Italy: Monduzzi Editore; 1999. p. 49 –52. 26. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability. Fertil Steril 2003;79:407–10.
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27. Ding J, Pry M, Rana N, Dmowski WP. Improved outcome of frozenthawed blastocyst transfer with Menezo’s two-step thawing compared to the stepwise thawing protocol. J Assist Reprod Genet 2004;21:203– 10. 28. Veeck LL, Bodine R, Clarke RN, Berrios R, Libraro J, Moschini RM, et al. High pregnancy rates can be achieved after freezing and thawing human blastocysts. Fertil Steril 2004;82:1418 –27. 29. Kosasa TS, McNamee PI, Morton C, Huang TT. Pregnancy rates after transfer of cryopreserved blastocysts cultured in a sequential media. Am J Obstet Gynecol 2005;192:2035–39. 30. Van den Abbeel E, Camus M, Verheyen G, Van Waesberghe L, Devroey P, Van Steirteghem A. Slow controlled-rate freezing of sequentially cultured human blastocysts: an evaluation of two freezing strategies. Hum Reprod 2005;20:2939 – 45. 31. Son WY, Lee SY, Chang MJ, Yoon SH, Chian RC, Lim JH. Pregnancy resulting from transfer of repeat vitrified blastocysts produced by invitro matured oocytes in patient with polycystic ovary syndrome. Reprod Biomed Online 2005;10:398 – 401. 32. Huang CC, Lee TH, Chen SU, Chen HH, Cheng TC, Liu CH, et al. Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Hum Reprod 2005;20:122– 8. 33. Liebermann J, Tucker MJ. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification. Reproduction 2002;124:483–9. 34. Liebermann J, Tucker MJ, Sills ES. Cryoloop vitrification in assisted reproduction: analysis of survival rates in ⬎1000 human oocytes after ultra-rapid cooling with polymer augmented cryoprotectants. Clin Exp Obstet Gynecol 2003;30:125–9. 35. Zech NH, Lejeune B, Zech H, Vanderzwalmen P. Vitrification of hatching and hatched human blastocysts: effect of an opening in the zona pellucida before vitrification. Reprod Biomed Online 2005;355– 61. 36. Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. Case report: successful pregnancy after vitrification of a human blastocyst that had completely escaped from the zona pellucida on day 6. Hum Reprod 2004;19:988 –90. 37. Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod 2004;19:2884 – 8. 38. Shapiro B, Richter K, Harris D, Daneshmand S. A comparison of day 5 and 6 blastocysts transfers. Fertil Steril 2001;75:1126 –30. 39. Tedder RS, Zuckerman MA, Goldstone AH, Hawkins AE, Fielding A, Briggs EM, et al. Hepatitis-B transmission from contaminated cryopreservation tank. Lancet 1995;346:137– 40. 40. Fountain DM, Ralston M, Higgins N, Gorlin JB, Uhl L, Wheeler C, et al. Liquid nitrogen freezer: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 1997;37:585–91. 41. Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiol 2000;40: 110 – 6. 42. Bielanski A, Bergeron H, Lau PC, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiol 2003;46:146 –52. 43. Kyuwa S, Nishikawa T, Kaneko T, Nakashima T, Kawano K, Nakamura N, et al.. Experimental evaluation of cross-contamination between cryotubes containing mouse 2-cell embryos and murine pathogens in liquid nitrogen tanks. Exp Anim 2003;52:67–70. 44. Letur-Konirsch H, Collin G, Sifer C, Devaux A, Kuttenn F, Madelenat P, et al. Safety of cryopreservation straws for human gametes or embryos: a study with human immunodeficiency virus–1 under cryopreservation conditions. Hum Reprod 2003;18:140 – 4 45. Liebermann J, Dietl J, Vanderzwalmen P, Tucker MJ. Recent developments in human oocyte, embryo and blastocyst vitrification: where are we now? Reprod Biomed Online 2003;7:623–33. 46. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online 2005; 11:608 – 614.
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Fertility Centers of Illinois, Chicago, Illinois; and b Georgia Reproductive Specialists, Atlanta, Georgia
Objective: To evaluate implantation of day 5 and day 6 vitrified and slow-frozen blastocysts. Design: Retrospective analysis comparing two cryopreservation techniques. Setting: Private IVF clinic. Patient(s): Five hundred eight cryopreserved embryo transfer candidates. Intervention(s): Supernumerary day 5 and day 6 blastocysts were vitrified or slow-frozen and transfered after warming or thawing. Main Outcome Measure(s): Comparison of two cryopreservation techniques with respect to survival rate, implantation, and pregnancy rates of day 5 and day 6 blastocysts. Result(s): In 254 vitrified transfer cycles, survival, embryonic implantation, and clinical pregnancy rates for day 5 blastocysts were 95.9%, 33.4%, 48.7%, respectively, and for day 6 blastocysts 97.5%, 25.9%, 42.8%. In 254 slow-frozen transfer cycles, survival, embryonic implantation, and clinical pregnancy rates for day 5 blastocysts were 91.4%, 29.6%, 42.8%, respectively, and for day 6 blastocysts 94.8%, 28.2%, 43.1%. Overall there was a slightly, but not significantly, higher outcome regarding implantation and clinical pregnancy with the use of day 5 blastocysts (31.3% and 45.4%, respectively) versus day 6 blastocysts (26.7, and 42.9%, respectively). Conclusion(s): Vitrification technique yields the same implantation and pregnancy rate as slow-frozen blastocyst transfers. Slow growing embryos can be cryopreserved on day 6, because they yield a satisfactory survival, implantation, and pregnancy rate. (Fertil Steril威 2006;86:20 – 6. ©2006 by American Society for Reproductive Medicine.) Key Words: Extended culture, blastocysts, cryopreservation, conventional, vitrification, frozen embryo transfer, Cryotop
Cryopreservation has become an increasingly important therapeutic strategy in reproductive medicine, with the birth of many infants after use of this procedure. It is important for cryopreservation in general to establish consistent outcomes, especially in terms of embryo cryosurvival to allow high chances of success in performing a frozen embryo transfer (FET). This in turn will increase the positive awareness of cryopreservation in assisted reproduction technology (ART) as a means to reduce multiple implantation, thereby expanding its use in all areas of reproductive medicine. However, standard cryopreservation technologies appear to illustrate their ultimate limitations in their lack of consistency in cryosurvival. Consequently, radically different protocols may provide the answer to increased consistent sucReceived September 23, 2005; revised and accepted January 27, 2006. Presented in part at the 61st Annual Meeting of the American Society for Reproductive Medicine, Montreal, Quebec, October 15–19, 2005. Reprint requests: Juergen Liebermann, Ph.D., Fertility Centers of Illinois, 900 N. Kingsbury, River Walk 6, Chicago 60610, Illinois (FAX: 312-4941687; E-mail: [email protected]).
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cess, and interest has recently shifted to vitrification as an attractive alternative to slow-freezing methodology (1). The chief difference between vitrification and conventional cryopreservation procedures is that it is an “open system” defined by [1] direct contact between the solution containing the embryos and liquid nitrogen (LN2), and [2] storage of embryos in only partially sealed containers. To promote extremely rapid cooling, this system lacks any significant thermoinsulating layer around the specimen. If outcomes between both techniques are at least comparable, certain benefits of the vitrification protocol may appeal to clinical embryologists. Vitrification as an ultrarapid cooling technique is simple, potentially faster, and inexpensive; further, it is starting to become clinically established and seems to have the potential to be more reliable and consistent than conventional cryopreservation when carried out properly (2, 3). Further, the need for controlled-rate freezing equipment, which requires routine calibration and maintenance, is eliminated. The cells are placed into the cryoprotectant, then the cells are placed in a very small volume of
Fertility and Sterility姞 Vol. 86, No. 1, July 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
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cryoprotectant on a special carrier, and then they are cooled at extreme rates by plunging them directly into LN2. With this method no ice crystals form, avoiding damage to the cells or the tissues. Concerns with the high concentration of cryoprotectant required to achieve vitrification are allayed to some extent by recent publications such as that by Takahashi et al. (4) showing that the use of relatively high concentration of cryoprotectants, such as 15% ethylene glycol (EG) in an equimolar mixture with dimethyl sulfoxide (DMSO), had no negative effect on the perinatal outcome of blastocyst transfer using vitrification when compared with fresh blastocyst transfer. This is an important finding to help confirm the safety of vitrification for clinical use and serves to counter recent published concerns about the use of EG for vitrification and its purported negative effect on organogenesis (5– 6). Our personal preference is blastocyst-stage freezing. There are three major rationales for our stance: [1] successful cryopreservation of human blastocysts is increasingly relevant as extended in vitro culture of human embryos becomes more common, permitting routine use of blastocyst transfer in IVF programs as an optimal course to reduce embryo numbers for uterine transfer, thereby reducing multiple pregnancies; [2] consistent blastocyst cryopreservation is a necessary adjunct to maximize cumulative pregnancy rates from each oocyte retrieval (7); and [3] the superiority of blastocyst stage freezing over earlier stage freezing in terms of implantation per thawed embryo transferred improves overall expectations for the cryopreservation program (8). With an increasing proportion of IVF offspring being born after cryopreservation, any improvement in survival rates and viability of such embryos will only add to the readiness with which cryopreservation is adopted as a frontline option in ART. Comparing the survival rates of blastocysts from slowfreezing and vitrification protocols, Yeoman et al. (9) showed in rhesus monkey blastocysts a postthaw survival rate of 36% with 5% hatching after slow-freezing and 85% with 71% hatching after vitrification. In addition, after vitrification of human blastocysts with an intact zona pellucida using the cryoloop as carrier, survival rates of 72%–90%, clinical pregnancy rates of 37%– 48%, and an implantation rate of 22%–29% have been reported (4, 10 –13). Also, the clinical outcomes using carriers other than the cryoloop, such as French ministraws, hemistraws, and electron microscope grids, have shown that vitrification is universally effective and practical for the cryopreservation of human blastocysts (14 –19). These reports help confirm the effectiveness of vitrification as a feasible cryopreservation option. To date no clinical study has attempted to compare cryopreservation of human day 5 and day 6 blastocysts using either a traditional slow-freezing protocol or a vitrification protocol. We therefore analyzed retrospectively the reproductive outcome of both cryopreservation techniques, and the embryonic implantation and relative pregnancy rates for cryopreseved day 5 and day 6 blastocysts. Fertility and Sterility姞
MATERIALS AND METHODS All patients used standard long or short protocols using GnRH analog and gonadotropins for controlled ovarian hyperstimulation. Embryo cultures were carried out in Sage sequential media (Sage, Trumbull, CT) under mineral oil (Sage), using Nuclon four-well dishes (Nunc, Roskilde, Denmark). Slow-frozen–thawed blastocysts were derived from IVF as well as intracytoplacmic sperm injection (ICSI) procedures, whereas all vitrified-warmed blastocysts were derived from ICSI procedures only. The reason for this is that our IVF program initiated a 100% ICSI insemination protocol from January 2004 onward, and from January 2004 we changed our entire cryopreservation program of day 5 and day 6 blastocysts from conventional freezing to vitrification only. When the patient had fresh embryo transfer on day 3 or 5, supernumerary blastocysts were scored depending on the developmental stage and were graded according to the system of Gardner and Lane (20). This system incorporates assessment of the rate of development and independent measure of the inner cell mass (ICM) and trophectoderm (TE). We used a modification of this system to accommodate a tradition of embryo quality scoring that has graded the best embryos with a low number (21). Each blastocyst has an overall numeric score based on rate of development and expansion: 1 (fully expanded or hatching day 5), 2 (fully expanded or hatching day 6), 3 (moderate expansion day 6 or early cavitation day 5), or 4 (early cavitation day 6 or morula day 5 or 6). We add to this numeric score two alphabetic scores to grade first the ICM and second the TE. Blastocysts were graded A (high cell number with good cell-cell adhesion), B (lower cell number with poorer cell-cell attachment), or C (no ICM apparent, sparse granular cells in the TE). As an example, a good-quality well-expanded blastocyst on day 6 with good ICM and good integrity of TE would be scored as 2AA. Only grade 1 or 2 blastocysts on day 5 or 6 with an AA to BB score for ICM and TE were cryopreserved. Patients not achieving a clinical pregnancy returned for a frozen blastocyst transfer cycle. In both groups the blastocysts were derived from the patient’s own oocytes. All women received GnRH agonist and supplementary estroderm (Ortho-McNeil, Raritan, NJ) for preparation of the endometrium. Intramuscular administration of progesterone in oil (Schein, Florham Park, NJ) was initiated when the endometrial thickness reached 10 mm or more (usually 4 days before the frozen blastocyst transfer was scheduled and applied until the first hCG 10 days after transfer). One to three blastocysts were transfered into the patient’s uterus 5 days after the initiation of progesterone treatment regardless of the blastocyst developmental stage. Between January 2004 and December 2005, we performed 254 cycles of vitrified-warmed blastocyst transfers with patients’ age 34.2 ⫾ 5.0 years. During the same period, we performed 254 cycles of slow-frozen–thawed blastocyst transfers with patients’ age 35.1 ⫾ 4.7 years. 21
Vitrification of blastocysts was undertaken using the Cryotop carrier system (Kitazato Bio Pharma, Fuji-shi, Japan) after a two-step loading with cryoprotectant agents at 24°C. Briefly, blastocysts were placed in equilibration solution, which is the base medium (Hepes-buffered HTF with 20% HSA; Sage) containing 7.5% (v/v) EG and 7.5% (v/v) DMSO. After 5–7 minutes, the blastocysts were washed quickly in vitrification solution, which is the base medium containing 15% (v/v) DMSO, 15% (v/v) EG, and 0.5 mol/L sucrose, for 30 seconds and transferred onto the Cryotop using a micropipette. Immediately after the loading of ⱕ2 blastocysts in a 1-L drop on the Cryotop carrier, it was plunged into fresh clean LN2. The time the blastocysts were exposed to the vitrification solution before cooling was ⱕ30 seconds. After loading the embryos, the Cryotop was capped under the LN2 to seal and protect the vitrified material before cryostorage. To remove the cryoprotectants, blastocysts were warmed and diluted in a two-step process. With the Cryotop submerged in LN2, the protective cap was removed, and then the carrier with the blastocysts was removed from the LN2 and placed directly into a prewarmed (approximately 30°C) organ culture dish containing 1 mL of 1.0 mol/L sucrose. Blastocysts were picked up directly from the Cryotop and placed in a fresh drop of 1.0 mol/L sucrose at 24°C. After 5 minutes, blastocysts were transfered to 0.5 mol/L sucrose solution. After an additional 5 minutes, blastocysts were washed in the base medium and returned to the culture medium (Sage Blastocyst Medium) until transfer. Slow-frozen blastocysts were originally frozen in modified protocols containing glycerol and sucrose based on Menezo’s two-step method previously described (22, 23). Briefly, blastocysts were exposed for 10 minutes in 5% glycerol at 25°C, then moved in 9% glycerol ⫹ 0.2 mol/L sucrose. Blastocysts were loaded into straws (IMV Technologies, L’Aigle, France), sealed, and loaded into a rate-controlled Kryo 10 LN2 freezer (Planar Kryo 10, Perkasie, PA). The initial blastocyst cooling was achieved at a rate of ⫺2°C/min to ⫺7°C. Ice crystallization was initiated manually by touching the straw with cold forceps. The temperature was held for 10 minutes. Further cooling was attained at a rate of ⫺0.3°C/min to ⫺36°C, held for 15 minutes, after which the blastocysts were plunged directly into LN2 (⫺196°C) for cryostorage. Blastocysts were thawed by removing the straw from the LN2, and disappearance of all intracellular ice crystals occurred during initial blastocyst thawing, carried out for 30 seconds at room temperature. The straw was cut open onto a small Petri dish, and the contents expelled into the dish. Once the blastocysts were located, they were placed in serial dilutions of cryoprotectant at 25°C through two concentrations of glycerol (9% and 5%; 3 minutes per dilution) in 0.2 mol/L sucrose. Additional thawing was achieved at 25°C through 0.2 mol/L sucrose for 2 minutes, then the blastocysts were washed in the base medium (Hepes-buffered HTF with 10% HSA; Sage) and re22
Liebermann and Tucker
turned to the culture medium (Sage Blastocyst Medium) until transfer. Laser-assisted hatching, using a 1,480-nm diode laser system (Zilos; Hamilton Thorne Bioscience, Beverly, MA), was carried out on all blastocysts before reexpansion during the dilution steps after warming or thawing. In this way all potential thermal damage to the embryo was easily avoided while allowing the ability to perform extensive zona ablation. In addition, assisted hatching was undertaken routinely on all thawed or warmed blastocysts to compensate both for potential zona hardening following cryopreservation (24) and for the slower development in the case of day 6 blastocysts (25). Laser-assisted hatching was not necessary for those blastocysts that either experienced zona fracture during cryopreservation or were already hatching at the time of cryopreservation. After warming or thawing, blastocysts were cultured for at least 3– 4 hours after warming and assessed for survival based on the morphologic integrity of the ICM, TE, and reexpansion of the blastocele. Then the surviving blastocysts were scored as to their developmental stage and graded for quality as described earlier and transfered to the uteri of the patients. A positive pregnancy is defined as a positive -hCG ⱖ20 mIU/mL 10 days after blastocyst transfer. Implantation rate is defined by the number of gestational sacs per embryo number transferred. Clinical pregnancy refers to the identification of a pregnancy sac in the uterus, whereas ongoing or delivered pregnancy describes pregnancies that continue beyond 20 weeks. Statistical analysis was carried out by means of a 2test using Microsoft Excel 2001 for Macintosh (Redmond, WA). Statistical significance was defined as P⬍.05. RESULTS Table 1 shows the mean age and clinical outcome of patients who completed the vitrified or slow-frozen blastocyst transfer program. The mean age of the women was 34.2 ⫾ 5.0 years in the vitrified group and 35.1 ⫾ 4.7 years in the slow-frozen group. No significant differences could be observed regarding age in the two groups. The blastocyst postwarming survival rates after using either the vitrification technique or the conventional technique are shown in Table 1. A total of 547 vitrified blastocysts (day 5 and day 6) were warmed, of which 528 survived warming (96.5%), whereas in the slow-freezing group (day 5 and day 6), 92.1% of the blastocysts (525/570) survived. Generally, the blastocyst survival rate was slightly higher in the vitrified group than in the slow-frozen group, but no significant difference was noted. Combined, the percentage of surviving blastocysts was 94.3% (1,053/1,117). In the vitrified group, 523 vitrified-warmed blastocysts in 254 cycles of 257 attempted cycles were transferred (mean 2.0 blastocysts per FET). Overall, the implantation and clinical pregnancy rates per transfer were 30.6% and 46.1%, respectively. To date, 41 deliveries have occurred with no
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TABLE 1 Retrospective data from the blastocyst cryopreservation program (Fertility Centers of Illinois, Chicago) where both vitrification (VIT) and conventional (CONV) technologies were applied from January 2004 to December 2005. Technique Patients’ age (y) No. of thawed cycles No. of transfers No. of blastocysts warmed/thawed No. of blastocysts survived (%) No. of blastocysts transferred Mean no. of blastocysts transferred No. of implantations (%) No. of positive pregnancy/thaw (%) No. of positive pregnancy/FET (%) No. of clinical pregnancy/thaw (%) No. of clinical pregnancy/FET (%) Ongoing pregnancies (%) No. of livebirths
VIT
CONV
34.2 ⫾ 5.0 257 254 547 528 (96.5) 523 2.0 160 (30.6) 132 (51.4) 132 (51.9) 117 (45.5) 117 (46.1) 117 (88.6) 54
35.1 ⫾ 4.7 259 254 570 525 (92.1) 518 2.0 152 (28.9) 135 (52.1) 135 (53.1) 109 (42.1) 109 (42.9) 109 (79.6) 79
Note: P ⬎ .05 for every comparison. FET ⫽ frozen embryo transfer. Liebermann. Comparison of vitrification and conventional cryopreservation of blastocysts. Fertil Steril 2006.
reported abnormalities (54 infants: 23 boys and 31 girls). From the 259 slow-frozen–thawed cycles, transfer could be carried out in 254 cycles with 518 blastocysts (mean 2.0 blastocysts per FET). Overall, the implantation and clinical pregnancy rates per transfer were 28.9% and 42.9%, respectively. At this time, 63 deliveries have occurred with no reported abnormalities (79 infants: 34 boys and 45 girls). When the vitrified-warmed blastocysts were divided into day 5 and day 6 groups, 95.9% (331/345) of day 5 blastocysts and 97.5% (197/202) of day 6 blastocysts survived after warming (Table 2), but this difference was not significant. As shown in Table 2, implantation and clinical pregnancy rates per transfer occurring in the day 5 blastocyst group were 33.4% and 48.7% respectively, which were significantly higher than in the day 6 blastocyst group (25.9% and 42.8%; 2: P⬍.01 and P⬍.01, respectively). In slow-frozen–thawed blastocyst transfers; the survival rate between day 5 and day 6 was slightly but not significantly different (91.4% vs. 94.8%). As shown in Table 2, the implantation and clinical pregnancy rates per transfer did not differ in the day 5 group (29.6% and 42.8%, respectively) compared with the day 6 group (28.2% and 43.1%). Table 3 shows that in total the survival rate of cryopreserved day 5 blastocysts was 93.4% (746/799) and did not significantly differ from that of cryopreserved day 6 blastocysts (96.5%; 307/318). The implantation and clinical pregnancy rates per transfer of day 5 blastocysts were 31.3% (230/734) and 45.4% (160/352), respectively. As shown in Table 3, transfer of day 6 blastocysts showed an excellent implantation rate of 26.7% (82/307) and a clinical pregnancy Fertility and Sterility姞
rate per transfer of 42.9% (67/156). There were no statistical differences between groups. DISCUSSION With the introduction of sequential culture media in ART, and driven by the large increase in the rate of multiple pregnancies arising from earlier-stage ET, extended culture to the blastocyst stage has become more common. Consequently, the need to cryopreserve human blastocysts is also increasing. Although the results achieved by conventional slow freezing seem successful (23, 26 –30), clinical results with blastocyst cryopreservation have not necessarily been consistent, owing to the higher potential for damaging ice crystal formation in traditional slow-freezing protocols. Recently there has been an increasing number of reports of successful human blastocyst vitrification (10 –19, 31, 32). We compared 24 months of experience with vitrification of blastocysts, using 15% EG, 15% DMSO, and 0.5 mol/L sucrose, with contemporaneous data using conventional slow cryopreservation with 9% glycerol plus 0.2 mol/L sucrose. From published reports, it seems that there are no apparent differences between the two cryopreservation techniques with respect to survival implantation and pregnancy. The data from the present retrospective clinical study have clarified that vitrification can at least achieve comparable results to those achieved with conventional slow-freeze technology. Furthermore, the results from this study help to prove that vitrification of blastocysts using the Cryotop procedure is effective by achieving high implantation and preg23
TABLE 2 Comparison of retrospective data from the blastocyst cryopreservation program (Fertility Centers of Illinois, Chicago) of day 5 and day 6 blastocysts where both vitrification (VIT) and conventional (CONV) technologies were applied from January 2004 to December 2005. VIT day 5
VIT day 6
Patients’ age (y) 33.9 ⫾ 4.9 34.5 ⫾ 5.3 No. of thawed cycles 158 99 No. of transfers 156 98 No. of blastocysts warmed/thawed 345 202 No. of blastocysts survived (%) 331 (95.9) 197 (97.5) No. of blastocysts transferred 326 197 Mean no. of blastocysts transferred 2.1 2.0 51 (25.9)a No. of implantations (%) 109 (33.4)a No. of positive pregnancy/thaw (%) 86 (54.4) 46 (46.0) No. of positive pregnancy/FET (%) 86 (55.1) 46 (46.5) No. of clinical pregnancy/thaw (%) 76 (48.1) 42 (42.4) No. of clinical pregnancy/FET (%) 76 (48.7)b 42 (42.8)b Ongoing/delivered pregnancies (%) 76 (88.4) 42 (91.3)
CONV day 5
CONV day 6
35.0 ⫾ 4.7 201 196 454 415 (91.4) 408 2.0 121 (29.6) 106 (52.7) 106 (54.0) 84 (41.8) 84 (42.8) 84 (79.2)
35.4 ⫾ 4.8 58 58 116 110 (94.8) 110 1.9 31 (28.2) 29 (50.0) 29 (50.0) 25 (43.1) 25 (43.1) 25 (86.2)
Note: FET ⫽ frozen embryo transfer. a,b P ⬍ .01. Liebermann. Comparison of vitrification and conventional cryopreservation of blastocysts. Fertil Steril 2006.
nancy rates and safe for clinical use by giving rise to healthy infants without abnormalities. The results of the present study help to confirm recently published results with respect to clinical outcomes following blastocyst vitrification with use of a stepwise vitrification protocol using a mixture of 7.5% EG-DMSO (lower-strength
step) followed by 15% EG-DMSO with 0.5 mol/L sucrose (full-strength step) (4). Concerns about introduction of high concentrations of cryoprotectant, which are necessary to prevent mechanical damage from ice, exist with vitrification. The problem of cryoprotectant toxicity is an immediate and practical one, just as it is to a lesser extent in classic slow-
TABLE 3 Overall comparison of retrospective data from the blastocyst cryopreservation program (Fertility Centers of Illinois, Chicago) of day 5 and day 6 blastocysts where both vitrification (VIT) and conventional (CONV) technologies were applied from January 2004 to December 2005.
Patients’ age (y) No. of thawed cycles No. of transfers No. of blastocysts warmed/thawed No. of blastocysts survived (%) No. of blastocysts transferred Mean no. of blastocysts transferred No. of implantations (%) No. of positive pregnancy/thaw (%) No. of positive pregnancy/FET (%) No. of clinical pregnancy/thaw (%) No. of clinical pregnancy/FET (%) Ongoing/delivered pregnancies (%)
VIT ⴙ CONV day 5
VIT ⴙ CONV day 6
34.6 ⫾ 4.8 359 352 799 746 (93.4) 734 2.0 230 (31.3) 192 (53.4) 192 (54.5) 160 (44.5) 160 (45.4) 160 (83.3)
34.9 ⫾ 5.1 157 156 318 307 (96.5) 307 2.0 82 (26.7) 75 (47.7) 75 (48.0) 67 (42.6) 67 (42.9) 67 (89.3)
Note: P ⬎ .05 for every comparison. FET ⫽ frozen embryo transfer. Liebermann. Comparison of vitrification and conventional cryopreservation of blastocysts. Fertil Steril 2006.
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cooling procedures. Extremely rapid cooling allows a decrease to be made in the concentration of the cryoprotectant and thereby a reduction in potential toxicity (33). The greatest advantages of vitrification have been seen in chill-sensitive cells such as oocytes and blastocysts (34). The main characteristic of the blastocyst is its fluid-filled cavity, the blastocele. It seems that with increasing volume of the blastocelic cavity, the survival rate drops with vitrification. This is thought to be due to insufficient permeation of cryoprotectant into the blastocelic cavity, such that residual water may promote ice crystallization during the vitrification process. Several articles report that survival rates in cryopreserved expanded blastocysts could be improved by artificial reduction of the blastocelic cavity (14, 15, 19, 35, 36). In addition, recent publications have shown that survival rates of cryopreserved expanded blastocysts as well as zonafree hatched blastocysts (36, 37) can be improved by artificial reduction of the blastocelic cavity. In our protocol we proceeded without any opening in the zona pellucida before vitrification independent of the size of the blastocelic cavity. Previous investigators have found superior implantation rates with fresh transfers occurring at day 5 as compared with day 6 (38). They reported an almost doubled clinical pregnancy and implantation for fresh day 5 blastocyst compared with fresh day 6 blastocysts (38). Our data comparing vitrified or frozen day 5 and day 6 blastocysts did not confirm this observation to such an extent. However, the reported high embryonic implantation and pregnancy rates following vitrified-warmed as well as slow frozen–thawed transfer of day 6 blastocysts in the present study should encourage cryopreservation of day 6 blastocysts, and even those that do not fully expand until day 7 (23). Although more slowly developing blastocysts may be innately compromised to some extent, the data from this report show that there is profound clinical value in knowing they can be vitrified/frozen as late as day 6, successfully thawed, and result in a live birth. It is plausible that a more synchronous transfer of these warmed/thawed blastocysts contributed to the excellent outcome observed in the present study. There is one issue with vitrification that needs further discussion. A concern has been made that fungi, bacteria, and viruses are able to survive in LN2 (39 – 44). Given that with vitrification the cells are directly plunged into LN2, they therefore have direct contact with LN2 and so the question arises as to whether the LN2 has to be sterilized because it may be a possible source of contamination. Use of clean LN2 for the initial vitrification step, followed by sealing of the carrier, seems to address the concern of potential contamination during cryostorage. To further reduce fears of contamination, it is possible to store material from potentially infectious patients separately from seemingly noninfectious samples. Therefore, it is important to perform routine screening tests for viral infections, including HIV and hepatitis B and C, on all couples undergoing infertility treatment. In the event that a couple screens positive, we offer vitrification of Fertility and Sterility姞
blastocysts. Even though we consider the risk of crosscontamination during storage to be almost infinitesimal, in such cases we nevertheless recommend placing embryos in specially designated tanks, or shipping them off-site. It is worth noting that to date no viral, fungal, or bacterial contamination event has been described from approximately 400 publications related to vitrification since 1985. In conclusion, although some problems remain to be fully addressed with vitrification as a routine cryopreservation technique, we believe that it shows much promise as a successful alternative to conventional freezing technology. Even without significant clinical improvement, the evident advantages of vitrification are that cryosurvival seems more consistent, allowing greater ease of patient management, with transfers being almost certain to occur. Concerns about vitrification are well defined, limited in number, and to our thinking easily surmountable. In general, with much shorter protocols, vitrification [1] is able to be undertaken on a more flexible basis by laboratory staff, [2] allows for the potential reduction in personnel time needed during the entire vitrification process, [3] simplifies laboratory techniques for cryopreservation in human ART, and [4] may enable more optimal timing of embryo cryopreservation, e.g., individual blastocysts may be cryopreserved at their optimal stage of development and expansion. Interest levels will inevitably rise, given the potential benefits of vitrification. This in turn will drive its development to higher levels of clinical efficiency and utilization (1, 45, 46). Acknowledgments: The authors thank the Fertility Centers of Illinois (FCI) and Elissa Knopoff, B.S., Jill Matthews, B.S., Amanda Erman, B.S., Rebecca Brohammer, B.S., Sara Sanchez, B.S., Yuri Wagner, B.S., and Andrew Barker, B.S., the embryologists at the FCI IVF Laboratory River North, for their invaluable contributions and support in pushing vitrification to become the standard protocol for cryopreservation of human blastocysts within our program.
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