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Birth of a healthy baby following vitrification of human blastocysts
FERTILITY AND STERILITY威 VOL. 75, NO. 5, MAY 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Birth of a healthy baby following vitrification of human blastocysts Yoshimasa Yokota, M.D.,a Setsuko Sato, M.T.,a Mikako Yokota, M.D.,a Hidemi Yokota, M.D.,a and Yasuhisa Araki, Ph.D.b Yokota Ob/Gyn Clinic, Maebashi, Gunma, Japan
Objective: To assess vitrification of human blastocysts. Design: Retrospective study of blastocyst vitrification. Setting: A private clinic. Patient(s): Twenty couples with different types of infertility. Intervention(s): Blastocysts were frozen with rapid vitrification and then transferred after thawing. We vitrified blastocysts using a modification of Ishimori’s vitrification solution of ethylene glycol and dimethyl sulfoxide (VSED). Main Outcome Measure(s): After thawing, survival was defined by the embryo’s development morphology after 6 hours or overnight culture. Result(s): Eighteen of 20 patients underwent treatment. Of 45 vitrified blastocysts, 36 survived, for a survival rate of 80% (36 of 45). The implantation rate was 21.9% (7 of 32), and the pregnancy rate (per embryo transfer cycle) was 33.3% (6 of 18). One of the pregnancies resulted in the delivery of a healthy baby. Conclusion(s): Supernumerary embryos were grown in culture to blastocysts, and the survival rate of vitrified-thawed blastocysts was the same as that for slow freezing of early stage embryos. Blastocyst vitrification should prove effective for clinical treatment. The present results strongly suggest that this rapid and successful vitrification procedure will replace conventional cryopreservation in the future. (Fertil Steril威 2001;75:1027–9. ©2001 by American Society for Reproductive Medicine.) Key Words: ART, human blastocyst, vitrification
Received August 11, 2000; revised and accepted November 9, 2000. Reprint requests: Yoshimasa Yokota, M.D., Yokota Ob/Gyn Clinic, 116-5, Shimokoide, Maebashi, Gunma 3710031, Japan (FAX: ⫹81-27234-4136; E-mail: [email protected]). a Yokota Ob/Gyn Clinic, Maebashi. b The Institute for ARMT, Maebashi. 0015-0282/01/$20.00 PII S0015-0282(01)01685-5
Cryopreservation has become a necessary part of in vitro fertilization (IVF) programs because it helps to avoid the risk of multiple pregnancies after the transfer of large numbers of embryos as well as to prevent wastage of supernumerary embryos from the large number of oocytes primed by ovarian stimulation. Current technology allows us to grow blastocysts in culture with comparative ease and enables a high success rate for the implantation of transferred blastocysts. The resultant small number of blastocysts transferred in turn minimizes the risk of multiple pregnancies, but necessitates the development of a simple method for cryopreservation of supernumerary blastocysts for infertility treatment. In the present study, we attempted simple rapid freezing vitrification of human blastocysts, which resulted in successful pregnancy and delivery.
MATERIALS AND METHODS The 20 couples enrolled in this study had different types of infertility and were treated with conventional IVF (10 couples) or intracytoplasmic sperm injection (10 couples). Each couple had each experienced one or two unsuccessful IVF cycles before enrolling in the present study. Ovarian stimulation, oocyte recovery, IVF, and intracytoplasmic sperm injection (ICSI) were performed following a standard protocol (1). We used human tubal fluid (HTF) containing 10% serum medium on day 2 (D2) or day 3 (D3), and the embryos were transferred. Supernumerary embryos were cultured until day 5 (D5) in Sydney blastocyst medium. The embryos were then vitrified. Before vitrification, embryos of various stages of morphological development from morulae-compaction to expanded blastocysts were used. We vitrified the 1027
weighed 3026 grams and was 51 cm. No obvious anomalies were detected. His karyotype was a normal 46,XY. Of the other five pregnancies, four are proceeding normally and one resulted in a miscarriage.
TABLE 1 Results of human blastocyst vitrification. Variables
Value
No. of patients (no. of treatment cycles) No. of vitrified blastocysts No. of surviving blastocysts Survival rate Average no. of transferred blastocysts Implantation rate Pregnancy rate (per embryo transfer cycle)
20 (18) 45 36 36/45 (80.0%) 1.8 7/32 (21.9%) 6/18 (33.3%)
Yokota. Human blastocyst vitrification. Fertil Steril 2001.
blastocysts using a modification of Ishimori’s vitrification solution containing ethylene glycol and dimethyl sulfoxide (VSED) (2). We used VSED containing modified human tubal fluid (m-HTF) with 20% serum, ethylene glycol (EG), and dimethyl sulfoxide (DMSO) at a 2:1:1 ratio. The blastocysts were exposed to 10% EG for 5 minutes. They were then placed into 50% VSED for 1 minute and finally loaded within 30 seconds into straws containing VSED at room temperature. The straws were placed in liquid nitrogen (LN2) vapor for 2 minutes and then plunged immediately into LN2. The average duration of freezing was 3 months (range: 2 to 5 months). After warming in a 25°C water bath, and after a one-step dilution of the cryoprotectant using 0.5 M sucrose solution, the blastocysts were cultured in vitro for either 6 hours or overnight in Sydney blastocyst medium. Almost all blastocysts used for embryo transfer had normal morphology. The blastocysts used included 30% that were graded as excellent and also included expanded blastocysts. The blastocysts were transferred to the uterus. During the transfer, patients were treated with clomiphene citrate or cyclophenyl plus low-dose hMG, and the blastocysts were transferred on day 5 or day 6 after ovulation. For luteal support, three successive injections of 5000 units of hCG were administered, one every other day. Informed consent was obtained from all 20 couples before the use of VSED.
RESULTS Table 1 shows the clinical results of the human blastocyst vitrification. The survival rate of blastocysts was 80.0% (36 of 45). The implantation and pregnancy rates were 21.9% (7 of 32) and 33.3% (6 of 18), respectively. These results compare well with those of a previously used slow-cooling method. Moreover, we successfully used VSED solution at various stages, from the four-cell to eight-cell cleavage of embryo development (data not shown). One of the mothers delivered a healthy baby at 39 weeks of gestation on June 23, 2000, by cesarean section. The baby 1028 Yokota et al.
Human blastocyst vitrification
DISCUSSION The 1990s saw the first reports of successful vitrification of human cleavage-stage embryos and deliveries (3). The most recent vitrification of human blastocysts resulted in successful delivery of healthy infants (4). However, VSED was not used as the as the cryoprotectant in those studies. To the best of our knowledge, no reports of successful delivery after human blastocyst vitrification with VSED have been published until now. We previously reported the first successful blastocyst vitrification pregnancy in Japan (1), and in the present study, we document the second reported case of a successful delivery of a baby following human blastocyst vitrification. Our survival rate with blastocyst vitrification was high at 80% (36 of 45). The factor responsible for our high survival rate might have been the use of 10% EG and the 5-minute procedure. Embryos were first suspended in a solution containing a lower concentration of cryoprotectant at room temperature for permeation without causing toxic injury, and then exposed to a vitrification solution for a short period of time at room temperature. Therefore, blastocysts had a large blastocele; the protection from injury from toxicity before sufficient permeation was attained could account for the high survival rate. Another factor underlying the success might have been that the cryoprotectant mixture used [EG and dimethyl sulfoxide (DMSO)]) promoted permeation for blastocyst vitrification. Before placing them into LN2, the straws were placed in LN2 vapor for 2 minutes. The temperature was approximately ⫺180°C. We believe this procedure is superior than immediately placing the straws containing VSED into the LN2. Thawing was carried out for 6 hours or overnight for several different stages of blastocyst formation. It might be better to transfer the blastocysts before hatching. When we transferred the majority of blastocysts, they were expanded but had not hatched. The one blastocyst that hatched during the 6 hours of culture was implanted, resulting in a pregnancy. For luteal support, three successive injections of 5000 units of hCG were administered, one every other day. For the patient who had a successful delivery, the blastocysts were transferred on day 6. These results suggest that various stages of early to expanded blastocysts are needed to ensure a successful pregnancy after culture. Although these preliminary findings need to be confirmed by future studies, we believe that these Vol. 75, No. 5, May 2001
clinical results strongly suggest that this rapid and successful vitrification procedure will replace conventional cryopreservation in the near future. References 1. 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.
FERTILITY & STERILITY威
2. Ishimori H, Saeki K, Inai M, Nagao Y, Itasaka J, Miki N, et al. Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide. Theriogenology 1993;40:427–33. 3. Gordts S, Roziers P, Campo R, Noto V. Survival and pregnancy outcome after ultrarapid freezing of human embryos. Fertil Steril 1990;53: 469 –72. 4. 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.
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Birth of a healthy baby following vitrification of human blastocysts Yoshimasa Yokota, M.D.,a Setsuko Sato, M.T.,a Mikako Yokota, M.D.,a Hidemi Yokota, M.D.,a and Yasuhisa Araki, Ph.D.b Yokota Ob/Gyn Clinic, Maebashi, Gunma, Japan
Objective: To assess vitrification of human blastocysts. Design: Retrospective study of blastocyst vitrification. Setting: A private clinic. Patient(s): Twenty couples with different types of infertility. Intervention(s): Blastocysts were frozen with rapid vitrification and then transferred after thawing. We vitrified blastocysts using a modification of Ishimori’s vitrification solution of ethylene glycol and dimethyl sulfoxide (VSED). Main Outcome Measure(s): After thawing, survival was defined by the embryo’s development morphology after 6 hours or overnight culture. Result(s): Eighteen of 20 patients underwent treatment. Of 45 vitrified blastocysts, 36 survived, for a survival rate of 80% (36 of 45). The implantation rate was 21.9% (7 of 32), and the pregnancy rate (per embryo transfer cycle) was 33.3% (6 of 18). One of the pregnancies resulted in the delivery of a healthy baby. Conclusion(s): Supernumerary embryos were grown in culture to blastocysts, and the survival rate of vitrified-thawed blastocysts was the same as that for slow freezing of early stage embryos. Blastocyst vitrification should prove effective for clinical treatment. The present results strongly suggest that this rapid and successful vitrification procedure will replace conventional cryopreservation in the future. (Fertil Steril威 2001;75:1027–9. ©2001 by American Society for Reproductive Medicine.) Key Words: ART, human blastocyst, vitrification
Received August 11, 2000; revised and accepted November 9, 2000. Reprint requests: Yoshimasa Yokota, M.D., Yokota Ob/Gyn Clinic, 116-5, Shimokoide, Maebashi, Gunma 3710031, Japan (FAX: ⫹81-27234-4136; E-mail: [email protected]). a Yokota Ob/Gyn Clinic, Maebashi. b The Institute for ARMT, Maebashi. 0015-0282/01/$20.00 PII S0015-0282(01)01685-5
Cryopreservation has become a necessary part of in vitro fertilization (IVF) programs because it helps to avoid the risk of multiple pregnancies after the transfer of large numbers of embryos as well as to prevent wastage of supernumerary embryos from the large number of oocytes primed by ovarian stimulation. Current technology allows us to grow blastocysts in culture with comparative ease and enables a high success rate for the implantation of transferred blastocysts. The resultant small number of blastocysts transferred in turn minimizes the risk of multiple pregnancies, but necessitates the development of a simple method for cryopreservation of supernumerary blastocysts for infertility treatment. In the present study, we attempted simple rapid freezing vitrification of human blastocysts, which resulted in successful pregnancy and delivery.
MATERIALS AND METHODS The 20 couples enrolled in this study had different types of infertility and were treated with conventional IVF (10 couples) or intracytoplasmic sperm injection (10 couples). Each couple had each experienced one or two unsuccessful IVF cycles before enrolling in the present study. Ovarian stimulation, oocyte recovery, IVF, and intracytoplasmic sperm injection (ICSI) were performed following a standard protocol (1). We used human tubal fluid (HTF) containing 10% serum medium on day 2 (D2) or day 3 (D3), and the embryos were transferred. Supernumerary embryos were cultured until day 5 (D5) in Sydney blastocyst medium. The embryos were then vitrified. Before vitrification, embryos of various stages of morphological development from morulae-compaction to expanded blastocysts were used. We vitrified the 1027
weighed 3026 grams and was 51 cm. No obvious anomalies were detected. His karyotype was a normal 46,XY. Of the other five pregnancies, four are proceeding normally and one resulted in a miscarriage.
TABLE 1 Results of human blastocyst vitrification. Variables
Value
No. of patients (no. of treatment cycles) No. of vitrified blastocysts No. of surviving blastocysts Survival rate Average no. of transferred blastocysts Implantation rate Pregnancy rate (per embryo transfer cycle)
20 (18) 45 36 36/45 (80.0%) 1.8 7/32 (21.9%) 6/18 (33.3%)
Yokota. Human blastocyst vitrification. Fertil Steril 2001.
blastocysts using a modification of Ishimori’s vitrification solution containing ethylene glycol and dimethyl sulfoxide (VSED) (2). We used VSED containing modified human tubal fluid (m-HTF) with 20% serum, ethylene glycol (EG), and dimethyl sulfoxide (DMSO) at a 2:1:1 ratio. The blastocysts were exposed to 10% EG for 5 minutes. They were then placed into 50% VSED for 1 minute and finally loaded within 30 seconds into straws containing VSED at room temperature. The straws were placed in liquid nitrogen (LN2) vapor for 2 minutes and then plunged immediately into LN2. The average duration of freezing was 3 months (range: 2 to 5 months). After warming in a 25°C water bath, and after a one-step dilution of the cryoprotectant using 0.5 M sucrose solution, the blastocysts were cultured in vitro for either 6 hours or overnight in Sydney blastocyst medium. Almost all blastocysts used for embryo transfer had normal morphology. The blastocysts used included 30% that were graded as excellent and also included expanded blastocysts. The blastocysts were transferred to the uterus. During the transfer, patients were treated with clomiphene citrate or cyclophenyl plus low-dose hMG, and the blastocysts were transferred on day 5 or day 6 after ovulation. For luteal support, three successive injections of 5000 units of hCG were administered, one every other day. Informed consent was obtained from all 20 couples before the use of VSED.
RESULTS Table 1 shows the clinical results of the human blastocyst vitrification. The survival rate of blastocysts was 80.0% (36 of 45). The implantation and pregnancy rates were 21.9% (7 of 32) and 33.3% (6 of 18), respectively. These results compare well with those of a previously used slow-cooling method. Moreover, we successfully used VSED solution at various stages, from the four-cell to eight-cell cleavage of embryo development (data not shown). One of the mothers delivered a healthy baby at 39 weeks of gestation on June 23, 2000, by cesarean section. The baby 1028 Yokota et al.
Human blastocyst vitrification
DISCUSSION The 1990s saw the first reports of successful vitrification of human cleavage-stage embryos and deliveries (3). The most recent vitrification of human blastocysts resulted in successful delivery of healthy infants (4). However, VSED was not used as the as the cryoprotectant in those studies. To the best of our knowledge, no reports of successful delivery after human blastocyst vitrification with VSED have been published until now. We previously reported the first successful blastocyst vitrification pregnancy in Japan (1), and in the present study, we document the second reported case of a successful delivery of a baby following human blastocyst vitrification. Our survival rate with blastocyst vitrification was high at 80% (36 of 45). The factor responsible for our high survival rate might have been the use of 10% EG and the 5-minute procedure. Embryos were first suspended in a solution containing a lower concentration of cryoprotectant at room temperature for permeation without causing toxic injury, and then exposed to a vitrification solution for a short period of time at room temperature. Therefore, blastocysts had a large blastocele; the protection from injury from toxicity before sufficient permeation was attained could account for the high survival rate. Another factor underlying the success might have been that the cryoprotectant mixture used [EG and dimethyl sulfoxide (DMSO)]) promoted permeation for blastocyst vitrification. Before placing them into LN2, the straws were placed in LN2 vapor for 2 minutes. The temperature was approximately ⫺180°C. We believe this procedure is superior than immediately placing the straws containing VSED into the LN2. Thawing was carried out for 6 hours or overnight for several different stages of blastocyst formation. It might be better to transfer the blastocysts before hatching. When we transferred the majority of blastocysts, they were expanded but had not hatched. The one blastocyst that hatched during the 6 hours of culture was implanted, resulting in a pregnancy. For luteal support, three successive injections of 5000 units of hCG were administered, one every other day. For the patient who had a successful delivery, the blastocysts were transferred on day 6. These results suggest that various stages of early to expanded blastocysts are needed to ensure a successful pregnancy after culture. Although these preliminary findings need to be confirmed by future studies, we believe that these Vol. 75, No. 5, May 2001
clinical results strongly suggest that this rapid and successful vitrification procedure will replace conventional cryopreservation in the near future. References 1. 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.
FERTILITY & STERILITY威
2. Ishimori H, Saeki K, Inai M, Nagao Y, Itasaka J, Miki N, et al. Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide. Theriogenology 1993;40:427–33. 3. Gordts S, Roziers P, Campo R, Noto V. Survival and pregnancy outcome after ultrarapid freezing of human embryos. Fertil Steril 1990;53: 469 –72. 4. 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.
1029