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Birth after cryopreservation of immature oocytes with subsequent in vitro maturation
FERTILITY AND STERILITYt VOL. 70, NO. 3, SEPTEMBER 1998 Copyright ©1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Birth after cryopreservation of immature oocytes with subsequent in vitro maturation Michael J. Tucker, Ph.D., Graham Wright, B.Sc., Paula C. Morton, B.S., and Joe B. Massey, M.D. Reproductive Biology Associates, Atlanta, Georgia
Objective: To establish the clinical feasibility of using cryostored germinal vesicle oocytes for IVF and ET. Design: Case report. Setting: Private infertility clinic. Patient(s): A 28-year-old woman with tubal infertility undergoing IVF therapy. Intervention(s): Oocytes collected after ovarian stimulation were frozen without insemination or were inseminated, fertilized, and frozen as cleavage stage embryos. No fresh oocyte or embryo transfer was undertaken. All oocytes were thawed, and those that survived were used for IVF-ET. Main Outcome Measure(s): Oocyte cryosurvival, in vitro maturation, fertilization, embryo development, and pregnancy outcome. Result(s): None of 16 mature oocytes survived thawing; however, three of 13 germinal vesicle oocytes survived. After 30 hours in vitro maturation two oocytes had matured and underwent intracytoplasmic sperm injection with the partner’s sperm. Both fertilized normally and were transferred to the patient. The woman delivered an apparently healthy female infant at 40 weeks. Conclusion(s): This case report proves the feasibility if not the efficiency of using immature oocytes for cryostorage, coupling both cryopreservation and in vitro maturation. (Fertil Sterilt 1998;70:578 –9. ©1998 by American Society for Reproductive Medicine.) Key Words: Germinal vesicle oocytes, cryopreservation, in vitro maturation, ICSI, pregnancy
Received and accepted May 13, 1998. Reprint requests: Michael J. Tucker, Ph.D., 3245 Bywater Trail, Roswell, Georgia 30075 (FAX: 770992-9946). 0015-0282/98/$19.00 PII S0015-0282(98)00205-2
578
Frozen storage of both human semen and embryos has become routine, although cryopreservation of oocytes has remained an elusive goal. Only a small number of pregnancies have been achieved in the last 12 years from cryopreservation of mature metaphase II oocytes, including a set of twins from our own clinic. More recently, however, we have taken a different approach, by investigating the feasibility of cryostorage of immature prophase I oocytes. Infants have been born after in vitro maturation, fertilization and transfer of embryos from immature oocytes, but without cryopreservation (1). Prophase I, or germinal vesicle oocytes as they are called, have been frozenthawed and fertilized but without transfer (2).
CASE REPORT We report a case in which 61 oocytes were retrieved from a 28-year-old woman after ovar-
ian stimulation with gonadotropins. The woman had tubal infertility after three ectopic pregnancies. Of the 61 oocytes, 9 were atretic and were discarded. Twenty-three oocytes were placed in culture, and 15 of these that were mature were inseminated by intracytoplasmic sperm injection (ICSI). Twenty-nine oocytes were frozen, of which 16 were mature and 13 were immature at the germinal vesicle stage. Nine embryos were generated from the freshly inseminated oocytes, of which eight were deemed suitable for freezing at the cleavage cell stage and remain frozen still. All oocytes that were to be frozen, regardless of stage of maturity, were stripped of cumulus and coronal cells with hyaluronidase and by mechanical dissection. They were exposed to the cryoprotectants 1,2-propanediol and sucrose at room temperature before being cooled in a
controlled rate freezer at a rate of 22°C per minute to 27.0°C per minute, when ice crystallization was induced manually with chilled forceps. Cooling at 0.3°C per minute continued to 238°C, after which oocytes were plunged directly into liquid nitrogen at 2196°C for cryostorage. No fresh transfer of embryos was performed according to an experimental protocol approved by our institutional review board; however, the patient returned 40 days later, at which time all frozen oocytes were thawed at 31°C in a water bath and cryoprotectant was removed at room temperature by passage of oocytes through a series of dilutions of cryoprotectant. No mature oocytes survived this procedure, whereas 3 of 13 germinal vesicle oocytes survived. After 30 hours in regular culture in vitro, two germinal vesicle oocytes reached full maturity having extruded their first polar bodies at the metaphase II stage of meiosis. These two eggs underwent ICSI and both fertilized normally, and after 28 hours additional culture both cleaved adequately, allowing uterine embryo transfer to be undertaken. At the time of ET the woman was receiving hormone replacement therapy with micronized oral estrogen and intramuscular progesterone, and subsequently one embryo implanted. At 18 weeks of pregnancy an amniocentesis indicated an apparently healthy normal female fetus. The pregnancy continued successfully to term at 40 weeks gestation, when a 3,300-g female infant was born.
oocyte quality can be suppressed significantly in women with such polycystic ovarian type responses (3). The competency of germinal vesicle oocytes retrieved after standard ovarian stimulation for IVF may not be optimal given that most of the best quality oocytes would be the mature ones. Consequently, differing approaches may be needed to optimize germinal vesicle oocyte quality to improve chances of in vitro maturation postthaw. Retention of some of the cumulus or coronal cell complex may be necessary also to enhance postthaw maturation rates (4). Concerns have been raised that immature oocytes after cryopreservation may have higher rates of chromosomal abnormalities (5). Although this needs more study, there are techniques that permit chromosomal screening of all embryos before transfer (6). It is of interest that other investigators have found that the immature human oocyte can withstand profound changes in nuclear, nucleolar, and cytoplasmic organization associated with cryopreservation (7). The pregnancy we report herein, however, prompts cautious optimism for the future of consistent human oocyte cryopreservation. Such technology may allow elective oocyte cryostorage for women contemplating cancer therapy or for those who would like to defer reproduction until after 35 years of age. In addition, it will permit more cost-effective oocyte donation and potentially act as a convenient source of donor ooplasm.
DISCUSSION References We believe this to be the first report of cryopreservation of immature oocytes at the germinal vesicle stage to give rise to a pregnancy in the human. It is possible that prophase I oocytes are a more suitable stage at which to freeze human oocytes. Conceivably, germinal vesicle oocytes are more robust before formation of the chromosomal spindle present in metaphase II mature oocytes and also have different membrane permeability characteristics more conducive to passage of cryoprotectant into the egg cell. In this case report, albeit with few oocytes to compare, it is intriguing that no mature oocytes survived but three germinal vesicle oocytes did, while undergoing identical protocols. It is not surprising that oocyte quality was poor in this particular case, given the extreme ovarian response to exogenous stimulation that occurred. It has been reported that
FERTILITY & STERILITYt
1. Cha KY, Koo JJ, Ko JJ, Choi DH, Han SY, Yoon TK. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991;55:109 –13. 2. Toth TL, Jones HW, Baka SG, Jones HW, Muasher S, Veeck LL, et al. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril 1994;61:891– 4. 3. Aboulghar MA, Mansour RT, Serour GI, Ramzy AM, Amin YM. Oocyte quality in patients with severe ovarian hyperstimulation syndrome. Fertil Steril 1997;68:1017–21. 4. Oktay K, Newton H, Aubard Y, Salha O, Gosden RG. Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology? Fertil Steril 1998;69:1–7. 5. Park SE, Son WY, Lee SH, Lee KA, Ko JJ, Cha KY. Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil Steril 1997;68:920 – 6. 6. Munne´ S, Magli C, Adler A, Wright G, de Boer K, Mortimer D, et al. Treatment-related chromosome abnormalities in human embryos. Hum Reprod 1997;12:780 – 4. 7. Van Blerkom J, Davis PW. Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Tech 1994;27:165–93.
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Birth after cryopreservation of immature oocytes with subsequent in vitro maturation Michael J. Tucker, Ph.D., Graham Wright, B.Sc., Paula C. Morton, B.S., and Joe B. Massey, M.D. Reproductive Biology Associates, Atlanta, Georgia
Objective: To establish the clinical feasibility of using cryostored germinal vesicle oocytes for IVF and ET. Design: Case report. Setting: Private infertility clinic. Patient(s): A 28-year-old woman with tubal infertility undergoing IVF therapy. Intervention(s): Oocytes collected after ovarian stimulation were frozen without insemination or were inseminated, fertilized, and frozen as cleavage stage embryos. No fresh oocyte or embryo transfer was undertaken. All oocytes were thawed, and those that survived were used for IVF-ET. Main Outcome Measure(s): Oocyte cryosurvival, in vitro maturation, fertilization, embryo development, and pregnancy outcome. Result(s): None of 16 mature oocytes survived thawing; however, three of 13 germinal vesicle oocytes survived. After 30 hours in vitro maturation two oocytes had matured and underwent intracytoplasmic sperm injection with the partner’s sperm. Both fertilized normally and were transferred to the patient. The woman delivered an apparently healthy female infant at 40 weeks. Conclusion(s): This case report proves the feasibility if not the efficiency of using immature oocytes for cryostorage, coupling both cryopreservation and in vitro maturation. (Fertil Sterilt 1998;70:578 –9. ©1998 by American Society for Reproductive Medicine.) Key Words: Germinal vesicle oocytes, cryopreservation, in vitro maturation, ICSI, pregnancy
Received and accepted May 13, 1998. Reprint requests: Michael J. Tucker, Ph.D., 3245 Bywater Trail, Roswell, Georgia 30075 (FAX: 770992-9946). 0015-0282/98/$19.00 PII S0015-0282(98)00205-2
578
Frozen storage of both human semen and embryos has become routine, although cryopreservation of oocytes has remained an elusive goal. Only a small number of pregnancies have been achieved in the last 12 years from cryopreservation of mature metaphase II oocytes, including a set of twins from our own clinic. More recently, however, we have taken a different approach, by investigating the feasibility of cryostorage of immature prophase I oocytes. Infants have been born after in vitro maturation, fertilization and transfer of embryos from immature oocytes, but without cryopreservation (1). Prophase I, or germinal vesicle oocytes as they are called, have been frozenthawed and fertilized but without transfer (2).
CASE REPORT We report a case in which 61 oocytes were retrieved from a 28-year-old woman after ovar-
ian stimulation with gonadotropins. The woman had tubal infertility after three ectopic pregnancies. Of the 61 oocytes, 9 were atretic and were discarded. Twenty-three oocytes were placed in culture, and 15 of these that were mature were inseminated by intracytoplasmic sperm injection (ICSI). Twenty-nine oocytes were frozen, of which 16 were mature and 13 were immature at the germinal vesicle stage. Nine embryos were generated from the freshly inseminated oocytes, of which eight were deemed suitable for freezing at the cleavage cell stage and remain frozen still. All oocytes that were to be frozen, regardless of stage of maturity, were stripped of cumulus and coronal cells with hyaluronidase and by mechanical dissection. They were exposed to the cryoprotectants 1,2-propanediol and sucrose at room temperature before being cooled in a
controlled rate freezer at a rate of 22°C per minute to 27.0°C per minute, when ice crystallization was induced manually with chilled forceps. Cooling at 0.3°C per minute continued to 238°C, after which oocytes were plunged directly into liquid nitrogen at 2196°C for cryostorage. No fresh transfer of embryos was performed according to an experimental protocol approved by our institutional review board; however, the patient returned 40 days later, at which time all frozen oocytes were thawed at 31°C in a water bath and cryoprotectant was removed at room temperature by passage of oocytes through a series of dilutions of cryoprotectant. No mature oocytes survived this procedure, whereas 3 of 13 germinal vesicle oocytes survived. After 30 hours in regular culture in vitro, two germinal vesicle oocytes reached full maturity having extruded their first polar bodies at the metaphase II stage of meiosis. These two eggs underwent ICSI and both fertilized normally, and after 28 hours additional culture both cleaved adequately, allowing uterine embryo transfer to be undertaken. At the time of ET the woman was receiving hormone replacement therapy with micronized oral estrogen and intramuscular progesterone, and subsequently one embryo implanted. At 18 weeks of pregnancy an amniocentesis indicated an apparently healthy normal female fetus. The pregnancy continued successfully to term at 40 weeks gestation, when a 3,300-g female infant was born.
oocyte quality can be suppressed significantly in women with such polycystic ovarian type responses (3). The competency of germinal vesicle oocytes retrieved after standard ovarian stimulation for IVF may not be optimal given that most of the best quality oocytes would be the mature ones. Consequently, differing approaches may be needed to optimize germinal vesicle oocyte quality to improve chances of in vitro maturation postthaw. Retention of some of the cumulus or coronal cell complex may be necessary also to enhance postthaw maturation rates (4). Concerns have been raised that immature oocytes after cryopreservation may have higher rates of chromosomal abnormalities (5). Although this needs more study, there are techniques that permit chromosomal screening of all embryos before transfer (6). It is of interest that other investigators have found that the immature human oocyte can withstand profound changes in nuclear, nucleolar, and cytoplasmic organization associated with cryopreservation (7). The pregnancy we report herein, however, prompts cautious optimism for the future of consistent human oocyte cryopreservation. Such technology may allow elective oocyte cryostorage for women contemplating cancer therapy or for those who would like to defer reproduction until after 35 years of age. In addition, it will permit more cost-effective oocyte donation and potentially act as a convenient source of donor ooplasm.
DISCUSSION References We believe this to be the first report of cryopreservation of immature oocytes at the germinal vesicle stage to give rise to a pregnancy in the human. It is possible that prophase I oocytes are a more suitable stage at which to freeze human oocytes. Conceivably, germinal vesicle oocytes are more robust before formation of the chromosomal spindle present in metaphase II mature oocytes and also have different membrane permeability characteristics more conducive to passage of cryoprotectant into the egg cell. In this case report, albeit with few oocytes to compare, it is intriguing that no mature oocytes survived but three germinal vesicle oocytes did, while undergoing identical protocols. It is not surprising that oocyte quality was poor in this particular case, given the extreme ovarian response to exogenous stimulation that occurred. It has been reported that
FERTILITY & STERILITYt
1. Cha KY, Koo JJ, Ko JJ, Choi DH, Han SY, Yoon TK. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991;55:109 –13. 2. Toth TL, Jones HW, Baka SG, Jones HW, Muasher S, Veeck LL, et al. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril 1994;61:891– 4. 3. Aboulghar MA, Mansour RT, Serour GI, Ramzy AM, Amin YM. Oocyte quality in patients with severe ovarian hyperstimulation syndrome. Fertil Steril 1997;68:1017–21. 4. Oktay K, Newton H, Aubard Y, Salha O, Gosden RG. Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology? Fertil Steril 1998;69:1–7. 5. Park SE, Son WY, Lee SH, Lee KA, Ko JJ, Cha KY. Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil Steril 1997;68:920 – 6. 6. Munne´ S, Magli C, Adler A, Wright G, de Boer K, Mortimer D, et al. Treatment-related chromosome abnormalities in human embryos. Hum Reprod 1997;12:780 – 4. 7. Van Blerkom J, Davis PW. Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Tech 1994;27:165–93.
579