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Birth of a healthy baby after transfer of embryos that were cryopreserved for 8.9 years
FERTILITY AND STERILITY威 VOL. 77, NO. 5, MAY 2002 Copyright ©2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Birth of a healthy baby after transfer of embryos that were cryopreserved for 8.9 years Carlos J. Quintans, Ph.D., Mo´nica J. Donaldson, D.V.M., Marı´a Victoria Bertolino, M.D., Hugo Godoy, M.D., and R. Sergio Pasqualini, M.D. Halitus Instituto Me´dico Afiliado a la Facultad de Medicina de la Universidad de Buenos Aires, Buenos Aires, Argentina
Objective: To report successful pregnancy from embryos that had been cryopreserved for 8.9 years. Design: Case report. Setting: Fertility clinic of the University of Buenos Aires School of Medicine. Patient(s): A 43-year-old woman with ovarian failure and a 45-year-old man with nonobstructive azoospermia underwent embryo donation. Intervention(s): Five embryos that had been cryopreserved and stored since 1989 were thawed in 1998 and donated to an infertile couple. Endometrial preparation was performed with 17-estradiol and progesterone. The four embryos with better morphologic characteristics were transferred into the uterus. Main Outcome Measure(s): Post-thaw embryo survival, pregnancy, and birth. Result(s): Embryo survival was satisfactory as assessed by morphology. Pregnancy was confirmed by ultrasonography. A healthy baby weighing 2120 g was delivered by cesarean section at 36 weeks. Conclusion(s): Human 8-cell embryos may be viable after extended storage and can result in successful pregnancy. (Fertil Steril威 2002;77:1074 – 6. ©2002 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, embryo, embryo donation, extended cryopreservation, duration of cryostorage, human, pregnancy
Received July 19, 2001; revised and accepted November 9, 2001. Reprint requests: Carlos J. Quintans, Ph.D., Halitus Instituto Me´dico Afiliado a la Facultad de Medicina de la Universidad de Buenos Aires, Marcelo T. de Alvear 2084 (C1122AAF), Buenos Aires, Argentina (FAX: 5411-4963-4000; E-mail: [email protected]). 0015-0282/02/$22.00 PII S0015-0282(02)03066-2
1074
Cryopreservation is necessary in assisted reproductive programs because there is no accurate way to predict fertilization and implantation rates in a given IVF cycle. Production of a surplus of embryos and storage of those that are not to be immediately transferred is somewhat controversial from ethical and practical standpoints. Oocyte cryopreservation appears to be a good alternative to embryo freezing, despite having shown only limited success. Some countries have regulations on the duration of embryo storage (1–3), but there are some cases in which longer storage periods should be considered. It would be useful to know for how long embryos can be cryopreserved without being harmed. Some cryobiologists believe that cells may survive for up to 1000 years after cryopreservation (4). Research in mice showed that a cumulative dose of background radiation applied to frozen mouse embryos during a simulated 2000-year storage
period had no detectable effect on their viability (5). Experiments in sheep indicate that embryos may safely survive for up to 13 years (6). Few reports are available on survival of human embryos after long-term storage. Only two studies have reported healthy pregnancies after transfer of embryos that had been cryopreserved for more than 7 years (7, 8). To our knowledge, there are no reports on healthy offspring from 8-cell embryos stored in liquid nitrogen for more than 8 years. We report a successful live birth after transfer of 8-cell embryos that remained cryopreserved for 8.9 years.
CASE REPORT An infertile couple who wished to conceive attended our clinic for embryo donation. The 43-year-old woman had complete ovarian failure, and her 45-year-old husband had secretory
azoospermia. Their request was revised and approved by the institutional review board.
donor sperm fertilized and were cultured to the 8-cell stage in human tubal fluid medium.
On September 28, 1998, five donated 8-cell embryos were thawed. Four of the embryos showed more than 75% blastomere survival and were selected for transfer; the remaining embryo had only one blastomere intact.
On November 7th, 1989, the embryos were cryopreserved in heat-sealed French straws with 1.5 M of 1.2-propanediol and 0.1 M of sucrose (10), by using a manually controlled alcohol bath that was slowly cooled to ⫺30°C in liquid nitrogen vapor (11). Cooling from room temperature to ⫺7°C was achieved at a rate of 1.5°C/min. At ⫺7°C, seeding was performed by using tweezers that had been kept in liquid nitrogen. After a 10-minute holding period, cooling was restarted at a rate of 0.3°C/min until ⫺30°C was reached. At this point, the straws were plunged into liquid nitrogen for storage.
Endometrial preparation was performed by stepwise administration of oral 17-estradiol (Ronfase; Rontag, Buenos Aires, Argentina), 4 mg/d from day 2 to 4 of the menstrual cycle and 6 mg/d from day 5 onward. On day 15, progesterone (Proluton, Schering, Buenos Aires, Argentina), 100 mg/d intramuscularly, was added. Intrauterine transfer was performed on the day of thawing by using a Frydman catheter. Pregnancy was confirmed 15 days after transfer by a -hCG level of 360 IU/L. Four weeks later, ultrasonography showed two gestational sacs with cardiac activity; however, only one sac remained alive after 2 weeks. Hormonal support with estrogen and progesterone was maintained until the 15th week of gestation. Pregnancy developed without complications, and labor started spontaneously at 36 weeks of gestation. A healthy female baby weighing 2120 g was delivered by cesarean section owing to poor cervical progression. Although the infant’s Apgar score was 9/10, she stayed in the neonatal care unit for 10 days for temperature regulation. At 2 years and 4 months of age, the girl showed normal development and was healthy. The five donated embryos were left over from an IVF cycle performed on November 4th, 1989. The oocytes belonged to a healthy 21-year-old woman with no apparent impairment of fertility. She underwent an IVF cycle with epididymal spermatozoa because her husband had infertility due to obstructive azoospermia secondary to vas deferens agenesis. The couple requested that some of the oocytes be fertilized with donor sperm as a safeguard against complete fertilization failure. Ovarian stimulation consisted of administration of human FSH (Metrodin; Serono, Buenos Aires, Argentina), and hMG (Pergonal, Serono) after a long down-regulation protocol with leuprolide acetate (Lupron; Abbott, Buenos Aires, Argentina). Eighteen metaphase II oocytes were retrieved by ultrasonography 36 hours after 10,000 IU of intramuscular hMG (Profasi; Serono) was administered. Oocytes were inseminated in human tubal fluid medium supplemented with 10% maternal serum in four-well Nunc dishes (Roskilde, Denmark). Thirteen oocytes were inseminated with the husband’s epididymal spermatozoa, and five were inseminated with 200,000/mL motile spermatozoa from a fertile sperm bank donor. Only one oocyte inseminated with the husband’s sperm fertilized normally and produced a term pregnancy after embryo transfer (9). All five oocytes inseminated with FERTILITY & STERILITY威
Strict procedures are used to identify and record the donors of embryos. The straws, the goblet holding the straws, and the cane to which the goblet is attached are all marked with the patient’s name and identification number. Records are kept in both print (two copies) and electronic formats and include the patient’s name and identification number, the date of freezing, and embryo characteristics and their location. These records are archived in several different places to prevent accidental losses. The storage tank is kept under continuous surveillance to maintain safe levels of liquid nitrogen, and the tank is refilled manually when needed. Almost 9 years later, embryos were thawed for transfer after both the donating and recipient couple gave informed consent. Rapid thawing was accomplished by keeping the straw at room temperature for 40 seconds and then rotating it between the palms until the contents melted completely. Embryos were then washed in a stepwise manner at room temperature in phosphate-buffered saline with 20% serum substitute supplement (Irvine Scientific, Irvine, CA) containing progressively lower concentrations of cryoprotectant, as follows: 1.0 M of 1.2-propanediol and 0.2 M of sucrose for 5 minutes, 0.5 M of 1.2-propanediol and 0.2 M of sucrose for 5 minutes, and 0.2 of sucrose for 5 minutes. At this point, embryos were cultured for 2 hours in equilibrated human tubal fluid medium with 10% serum substitute supplement. Before transfer, assisted hatching was performed by using acid Tyrode solution (12).
DISCUSSION Few articles have been published on pregnancy and delivery after transfer of human embryos that had been cryopreserved for long periods (7, 8), probably because human embryo cryopreservation was not as widely available 12 years ago as it is today. We believe that our case is the first in which pregnancy was accomplished after transfer of cleavage embryos cryopreserved for almost 9 years. In the cases described by Go et al. (7) and Ben-Ozer and Vermesh (8), stored zygotes were used. Zygote cryopreser1075
vation and storage of cleaved embryos each have advantages and disadvantages. In brief, freezing of zygotes has shown better outcome in terms of implantation and pregnancy rates compared with later-stage embryos. However, cryopreservation of cleaved embryos may allow selection of embryos with better developmental potential. Experimental work in sheep indicates that cryopreservation for up to 13 years may be considered safe for embryo survival (6). Likewise, Glenister and Thornton (5) reported normal live births from frozen mouse embryos that were stored in liquid nitrogen for more than 20 years. The little published data in humans (7, 8) seem to indicate that duration of cryopreservation and storage similar to the one that we report may be safe, although further studies with larger samples are needed. Embryo quality and storage safety affect embryo survival. For storage to be secure, the straws or vials must remain leak-proof and perfectly sealed, and they must be stored at a constant temperature of ⫺196°C. The level of liquid nitrogen in the tank must be checked often to prevent suboptimal storage conditions, which may occur if cryopreserved samples remain exposed only to nitrogen vapor owing to leaking containers or carelessness in refilling them. As pointed out by Go et al. (7), the efficacy and safety of long-term embryo storage is as yet only an assumption based on few previous experiences. Long-term storage may represent an option for couples who wish to postpone childbearing for personal reasons. Moreover, if couples decide to
1076 Quintans et al.
Birth from embryos stored for 8.9 years
donate their embryos, cryopreservation may offer them time to find an appropriate adoptive couple. We believe that our report, together with those of Go et al. (7) and Ben-Ozer and Vermesh (8), will encourage use of embryos that were cryopreserved and stored for extended periods. This report also provides data to help establish the safety of the procedure. We also hope that our report will prevent decisions to dispose of frozen embryos too soon. References 1. Oghoetuoma JO, McKeating C, Horne G, Brison DR, Lieberman BA. Use of in-vitro fertilisation embryos cryopreserved for 5 years or more. Lancet 2000;355:1336. 2. Ruiz A, Pe´ rez I, Pellicer A. Cryostorage of human embryos: time to decide. Hum Reprod 1996;11:703–5. 3. Yding Andersen C, Westergaard LG, Grinsted J, Petersen K, Nyboe Andersen A. Frozen embryos: too cold to touch? Frozen pre-embryos in Denmark. Hum Reprod 1996;11:703. 4. Mazur P. Stopping biological time. The freezing of living cells. Ann N Y Acad Sci 1988;541:514 –31. 5. Glenister PH, Thornton CE. Cryoconservation—archiving for the future. Mamm Genome 2000:11:565–71. 6. Fogarty NM, Maxwell WM, Eppleston J, Evans G. The viability of transferred sheep embryos after long-term cryopreservation. Reprod Fertil Dev 2000;12:31–7. 7. Go KJ, Corson SL, Batzer FR, Walters JL. Live birth from a zygote cryopreserved for 8 years. Hum Reprod 1998;13:2970 –1. 8. Ben-Ozer S, Vermesh M. Full-term delivery following cryopreservation of human embryos for 7.5 years. Hum Reprod 1999;14:1650 –2. 9. Pasqualini RS, Rey Valzacchi G, Quintans CJ, Donaldson M, Matarasso N. Pregnancy with sperm aspiration from the head of the epididymis in vas deferens agenesis Medicina (B Aires) 1993;53:137– 40. 10. Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD, et al. High pregnancy rate after early human embryo freezing. Fertil Steril 1986;46:268 –72. 11. Leibo SP, Mazur P. Methods for the preservation of mammalian embryos by freezing. In: Daniel JC Jr (ed). Methods in mammalian reproduction. New York: Academic Press; 1978:179 –201. 12. Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992;7:685–91.
Vol. 77, No. 5, May 2002
Birth of a healthy baby after transfer of embryos that were cryopreserved for 8.9 years Carlos J. Quintans, Ph.D., Mo´nica J. Donaldson, D.V.M., Marı´a Victoria Bertolino, M.D., Hugo Godoy, M.D., and R. Sergio Pasqualini, M.D. Halitus Instituto Me´dico Afiliado a la Facultad de Medicina de la Universidad de Buenos Aires, Buenos Aires, Argentina
Objective: To report successful pregnancy from embryos that had been cryopreserved for 8.9 years. Design: Case report. Setting: Fertility clinic of the University of Buenos Aires School of Medicine. Patient(s): A 43-year-old woman with ovarian failure and a 45-year-old man with nonobstructive azoospermia underwent embryo donation. Intervention(s): Five embryos that had been cryopreserved and stored since 1989 were thawed in 1998 and donated to an infertile couple. Endometrial preparation was performed with 17-estradiol and progesterone. The four embryos with better morphologic characteristics were transferred into the uterus. Main Outcome Measure(s): Post-thaw embryo survival, pregnancy, and birth. Result(s): Embryo survival was satisfactory as assessed by morphology. Pregnancy was confirmed by ultrasonography. A healthy baby weighing 2120 g was delivered by cesarean section at 36 weeks. Conclusion(s): Human 8-cell embryos may be viable after extended storage and can result in successful pregnancy. (Fertil Steril威 2002;77:1074 – 6. ©2002 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, embryo, embryo donation, extended cryopreservation, duration of cryostorage, human, pregnancy
Received July 19, 2001; revised and accepted November 9, 2001. Reprint requests: Carlos J. Quintans, Ph.D., Halitus Instituto Me´dico Afiliado a la Facultad de Medicina de la Universidad de Buenos Aires, Marcelo T. de Alvear 2084 (C1122AAF), Buenos Aires, Argentina (FAX: 5411-4963-4000; E-mail: [email protected]). 0015-0282/02/$22.00 PII S0015-0282(02)03066-2
1074
Cryopreservation is necessary in assisted reproductive programs because there is no accurate way to predict fertilization and implantation rates in a given IVF cycle. Production of a surplus of embryos and storage of those that are not to be immediately transferred is somewhat controversial from ethical and practical standpoints. Oocyte cryopreservation appears to be a good alternative to embryo freezing, despite having shown only limited success. Some countries have regulations on the duration of embryo storage (1–3), but there are some cases in which longer storage periods should be considered. It would be useful to know for how long embryos can be cryopreserved without being harmed. Some cryobiologists believe that cells may survive for up to 1000 years after cryopreservation (4). Research in mice showed that a cumulative dose of background radiation applied to frozen mouse embryos during a simulated 2000-year storage
period had no detectable effect on their viability (5). Experiments in sheep indicate that embryos may safely survive for up to 13 years (6). Few reports are available on survival of human embryos after long-term storage. Only two studies have reported healthy pregnancies after transfer of embryos that had been cryopreserved for more than 7 years (7, 8). To our knowledge, there are no reports on healthy offspring from 8-cell embryos stored in liquid nitrogen for more than 8 years. We report a successful live birth after transfer of 8-cell embryos that remained cryopreserved for 8.9 years.
CASE REPORT An infertile couple who wished to conceive attended our clinic for embryo donation. The 43-year-old woman had complete ovarian failure, and her 45-year-old husband had secretory
azoospermia. Their request was revised and approved by the institutional review board.
donor sperm fertilized and were cultured to the 8-cell stage in human tubal fluid medium.
On September 28, 1998, five donated 8-cell embryos were thawed. Four of the embryos showed more than 75% blastomere survival and were selected for transfer; the remaining embryo had only one blastomere intact.
On November 7th, 1989, the embryos were cryopreserved in heat-sealed French straws with 1.5 M of 1.2-propanediol and 0.1 M of sucrose (10), by using a manually controlled alcohol bath that was slowly cooled to ⫺30°C in liquid nitrogen vapor (11). Cooling from room temperature to ⫺7°C was achieved at a rate of 1.5°C/min. At ⫺7°C, seeding was performed by using tweezers that had been kept in liquid nitrogen. After a 10-minute holding period, cooling was restarted at a rate of 0.3°C/min until ⫺30°C was reached. At this point, the straws were plunged into liquid nitrogen for storage.
Endometrial preparation was performed by stepwise administration of oral 17-estradiol (Ronfase; Rontag, Buenos Aires, Argentina), 4 mg/d from day 2 to 4 of the menstrual cycle and 6 mg/d from day 5 onward. On day 15, progesterone (Proluton, Schering, Buenos Aires, Argentina), 100 mg/d intramuscularly, was added. Intrauterine transfer was performed on the day of thawing by using a Frydman catheter. Pregnancy was confirmed 15 days after transfer by a -hCG level of 360 IU/L. Four weeks later, ultrasonography showed two gestational sacs with cardiac activity; however, only one sac remained alive after 2 weeks. Hormonal support with estrogen and progesterone was maintained until the 15th week of gestation. Pregnancy developed without complications, and labor started spontaneously at 36 weeks of gestation. A healthy female baby weighing 2120 g was delivered by cesarean section owing to poor cervical progression. Although the infant’s Apgar score was 9/10, she stayed in the neonatal care unit for 10 days for temperature regulation. At 2 years and 4 months of age, the girl showed normal development and was healthy. The five donated embryos were left over from an IVF cycle performed on November 4th, 1989. The oocytes belonged to a healthy 21-year-old woman with no apparent impairment of fertility. She underwent an IVF cycle with epididymal spermatozoa because her husband had infertility due to obstructive azoospermia secondary to vas deferens agenesis. The couple requested that some of the oocytes be fertilized with donor sperm as a safeguard against complete fertilization failure. Ovarian stimulation consisted of administration of human FSH (Metrodin; Serono, Buenos Aires, Argentina), and hMG (Pergonal, Serono) after a long down-regulation protocol with leuprolide acetate (Lupron; Abbott, Buenos Aires, Argentina). Eighteen metaphase II oocytes were retrieved by ultrasonography 36 hours after 10,000 IU of intramuscular hMG (Profasi; Serono) was administered. Oocytes were inseminated in human tubal fluid medium supplemented with 10% maternal serum in four-well Nunc dishes (Roskilde, Denmark). Thirteen oocytes were inseminated with the husband’s epididymal spermatozoa, and five were inseminated with 200,000/mL motile spermatozoa from a fertile sperm bank donor. Only one oocyte inseminated with the husband’s sperm fertilized normally and produced a term pregnancy after embryo transfer (9). All five oocytes inseminated with FERTILITY & STERILITY威
Strict procedures are used to identify and record the donors of embryos. The straws, the goblet holding the straws, and the cane to which the goblet is attached are all marked with the patient’s name and identification number. Records are kept in both print (two copies) and electronic formats and include the patient’s name and identification number, the date of freezing, and embryo characteristics and their location. These records are archived in several different places to prevent accidental losses. The storage tank is kept under continuous surveillance to maintain safe levels of liquid nitrogen, and the tank is refilled manually when needed. Almost 9 years later, embryos were thawed for transfer after both the donating and recipient couple gave informed consent. Rapid thawing was accomplished by keeping the straw at room temperature for 40 seconds and then rotating it between the palms until the contents melted completely. Embryos were then washed in a stepwise manner at room temperature in phosphate-buffered saline with 20% serum substitute supplement (Irvine Scientific, Irvine, CA) containing progressively lower concentrations of cryoprotectant, as follows: 1.0 M of 1.2-propanediol and 0.2 M of sucrose for 5 minutes, 0.5 M of 1.2-propanediol and 0.2 M of sucrose for 5 minutes, and 0.2 of sucrose for 5 minutes. At this point, embryos were cultured for 2 hours in equilibrated human tubal fluid medium with 10% serum substitute supplement. Before transfer, assisted hatching was performed by using acid Tyrode solution (12).
DISCUSSION Few articles have been published on pregnancy and delivery after transfer of human embryos that had been cryopreserved for long periods (7, 8), probably because human embryo cryopreservation was not as widely available 12 years ago as it is today. We believe that our case is the first in which pregnancy was accomplished after transfer of cleavage embryos cryopreserved for almost 9 years. In the cases described by Go et al. (7) and Ben-Ozer and Vermesh (8), stored zygotes were used. Zygote cryopreser1075
vation and storage of cleaved embryos each have advantages and disadvantages. In brief, freezing of zygotes has shown better outcome in terms of implantation and pregnancy rates compared with later-stage embryos. However, cryopreservation of cleaved embryos may allow selection of embryos with better developmental potential. Experimental work in sheep indicates that cryopreservation for up to 13 years may be considered safe for embryo survival (6). Likewise, Glenister and Thornton (5) reported normal live births from frozen mouse embryos that were stored in liquid nitrogen for more than 20 years. The little published data in humans (7, 8) seem to indicate that duration of cryopreservation and storage similar to the one that we report may be safe, although further studies with larger samples are needed. Embryo quality and storage safety affect embryo survival. For storage to be secure, the straws or vials must remain leak-proof and perfectly sealed, and they must be stored at a constant temperature of ⫺196°C. The level of liquid nitrogen in the tank must be checked often to prevent suboptimal storage conditions, which may occur if cryopreserved samples remain exposed only to nitrogen vapor owing to leaking containers or carelessness in refilling them. As pointed out by Go et al. (7), the efficacy and safety of long-term embryo storage is as yet only an assumption based on few previous experiences. Long-term storage may represent an option for couples who wish to postpone childbearing for personal reasons. Moreover, if couples decide to
1076 Quintans et al.
Birth from embryos stored for 8.9 years
donate their embryos, cryopreservation may offer them time to find an appropriate adoptive couple. We believe that our report, together with those of Go et al. (7) and Ben-Ozer and Vermesh (8), will encourage use of embryos that were cryopreserved and stored for extended periods. This report also provides data to help establish the safety of the procedure. We also hope that our report will prevent decisions to dispose of frozen embryos too soon. References 1. Oghoetuoma JO, McKeating C, Horne G, Brison DR, Lieberman BA. Use of in-vitro fertilisation embryos cryopreserved for 5 years or more. Lancet 2000;355:1336. 2. Ruiz A, Pe´ rez I, Pellicer A. Cryostorage of human embryos: time to decide. Hum Reprod 1996;11:703–5. 3. Yding Andersen C, Westergaard LG, Grinsted J, Petersen K, Nyboe Andersen A. Frozen embryos: too cold to touch? Frozen pre-embryos in Denmark. Hum Reprod 1996;11:703. 4. Mazur P. Stopping biological time. The freezing of living cells. Ann N Y Acad Sci 1988;541:514 –31. 5. Glenister PH, Thornton CE. Cryoconservation—archiving for the future. Mamm Genome 2000:11:565–71. 6. Fogarty NM, Maxwell WM, Eppleston J, Evans G. The viability of transferred sheep embryos after long-term cryopreservation. Reprod Fertil Dev 2000;12:31–7. 7. Go KJ, Corson SL, Batzer FR, Walters JL. Live birth from a zygote cryopreserved for 8 years. Hum Reprod 1998;13:2970 –1. 8. Ben-Ozer S, Vermesh M. Full-term delivery following cryopreservation of human embryos for 7.5 years. Hum Reprod 1999;14:1650 –2. 9. Pasqualini RS, Rey Valzacchi G, Quintans CJ, Donaldson M, Matarasso N. Pregnancy with sperm aspiration from the head of the epididymis in vas deferens agenesis Medicina (B Aires) 1993;53:137– 40. 10. Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD, et al. High pregnancy rate after early human embryo freezing. Fertil Steril 1986;46:268 –72. 11. Leibo SP, Mazur P. Methods for the preservation of mammalian embryos by freezing. In: Daniel JC Jr (ed). Methods in mammalian reproduction. New York: Academic Press; 1978:179 –201. 12. Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992;7:685–91.
Vol. 77, No. 5, May 2002