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Update on Clinical Efficiency of the Vitrification Method for Human Oocytes in an In Vitro Fertilization Program
three days in a sequential media. This study included a total 134 patients who underwent cryotransfer of vitrified blastocysts after failure of the previous elective fresh embryo transfer of PGD (n⫽40), coculture day-5/6 (n⫽29), or a day-3 (n⫽65). A total of 289 human blastocysts: 82, 87 and 120 were warmed for each respective group. Blastocyst vitrification/warming procedure was performed following the Yokota and co-workers procedure with some modifications. Warmed blastocysts were considered to survive when after a period of 12 hours of culture had no sign of degeneration and were fully expanded. Statistical evaluation was performed using one way ANOVA test and -square test analysis. RESULTS: The results are shown in the following table. CONCLUSION: Although, as expected, less implantation rates were obtained in the group of embryos coming from suboptimal day-3 embryos, that were able to get to blastocyst stage, their survival rate after warming is comparable to the survival rate achieved by the good prognosis human blastocyts, namely PGD blastocysts and blastocysts come from our extended coculture system. Supported by: None
P-63 Update on Clinical Efficiency of the Vitrification Method for Human Oocytes in an In Vitro Fertilization Program. T. Okimura, K. Kato, Q. Zhan, M. Kuwayama, J. Zhang, O. Kato. New Hope Fertility Center, New York, NY; R&D Division, Kato Ladies Clinic, Tokyo, Japan. OBJECTIVE: Oocyte cryopreservation can be a useful tool in IVF/ ICSI-ET cycles of human ART. Time allowance between oocyte retrieval and insemination by cryopreservation gives chances for certain female cancer patients to avoid iatrogenic sterility from chemo/radiotherapy. For patients who desire to delay their child bearing, long term preservation provides an opportunity to have the babies of their owns. Present experiments were conducted to evaluate the clinical efficiency of the minimum volume cooling (MVC) vitrification method for human oocytes in IVF cycles. DESIGN: Retrospective clinical study MATERIALS AND METHODS: Metaphase II Oocytes were denuded of the cumulus cells on the day of retrieval before vitrification. The Cryotop method was used to vitrify human oocytes. Oocytes were first equilibrated in 7.5% ethylene glycol (EG) and 7.5% DMSO in modified medium 199 (M-199) for 10-15min before being transferred into the vitrification solution (VS) for 30 sec. After replacing the extracellular solution by pipetting, oocytes were then transferred into the vitrification container with minimum volume of VS, and immediately submerged into liquid nitrogen. Thawing was performed by plunging the Cryotop containing the oocytes into 1M sucrose in M-199 at 37°C for 1 min, and the embryos were then placed in 0.5M Sucrose in M-199 for 3 min. The cryoprotective agents were diluted out by incubating in an isotonic culture medium for 5 min twice. After the recovery of oocytes they were cultured for 3 h in 5% CO2 in air at 37.0 C. Those oocytes considered to have survived were inseminated by intracytoplasmic sperm injection. Those embryos were cultured for 2 or 5days before transfer. RESULTS: Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection 52 of them became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescent in situ hybridization of five blastocysts showed that all were normal diploid embryos. 29 embryo transfers with mean number of 2.2 embryos per transfer on D2 or D5 resulted in 12 pregnancies; 7 full term deliveries with 3 ongoing pregnancies. CONCLUSION: Our results suggest that vitrification by the Cryotop method is the most efficient method ever published for human oocyte cryopreservation. Furthermore, oocytes cryopreservation will make oocyte/ ooplasm donation more flexible. Supported by: None
P-64 The Effect of Laser Assisted Hatching on Pregnancy Rates With Cryopreserved-Thawed Embryos. A. Y. Baratz, T. Di Berardino, E. M. Greenblatt. Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada; Division of Reproductive Sciences, Mount Sinai Hospital and University of Toronto, Toronto, ON, Canada.
S174
Abstracts
OBJECTIVE: To compare clinical pregnancy and implantation rates after transfer of 72 hour cryopreserved-thawed embryos that were or were not exposed to laser assisted hatching (LAH). DESIGN: Retrospective case-control study. MATERIALS AND METHODS: Cryopreserved-thawed embryos have a lower implantation rate and clinical pregnancy rate than fresh embryos. This difference may be the consequence of a change in the thickness or the quality of the zona pellucida. Different types of assisted hatching have been proposed to improve cryopreserved-thawed embryo cycle outcomes. LAH was initiated at our clinic in August 2003, and began to be performed routinely for 72 hour cryopreserved embryos at that time. We identified 106 cryopreserved-thawed embryo transfer cycles from August 2003 to March 2005 in whom LAH was performed (Group 1; cases). Prior to the introduction of LAH, we identified 19 cycles of cryopreserved-thawed 72 hour embryo transfer (March 2003-August 2003) in which identical culture media and freeze/thaw media and protocols had been applied, but in which LAH was not performed (Group 2; controls). All of the embryos were cultured in media for twenty four hours prior to embryo transfer. Immediately prior to transfer, LAH was performed using the OCTAX laser. Exposure time was adjusted to create an area of zona thinning that was 1.5 X the thickness of the zona pellucida. Only cryopreserved embryo transfers where all the intended embryos were hatched were included in the study. Routine statistical analysis was performed using the chi-square test to compare the two groups. RESULTS: In Group 1, of the 106 cycles (251 embryos) in which LAH was performed for cryopreserved-thawed embryo transfers, 37 (14.7%) implanted with a clinical pregnancy rate of 34.9%. In Group 2 (controls not hatched), 4 (8.5%) of the embryos implanted with a clinical pregnancy rate of 21.1%. The difference between the laser assisted hatching group and the non-assisted hatching group was not found to be statistically significant. CONCLUSION: In this retrospective study of LAH versus non-assisted hatching for 72 hour cryopreserved-thawed embryo transfers an improvement in implantation and clinical pregnancy rates did not reach statistical significance. However, the trend that emerges from this small retrospective study warrants a prospective randomized study with a larger sample size to more accurately evaluate the routine use of LAH to improve implantation and pregnancy rates during cryopreserved-thawed transfer cycles. Supported by: None.
P-65 An Ongoing Pregnancy After Frozen Thawed Embryo Transfer in a Patient with Klinefelter Syndrome. H. Yarali, G. Bozdag. Hacettepe University School of Medicine, Ankara, Turkey; Hacettepe University, School of Medicine, Department of Obstetrics and Gynecology, Ankara, Turkey. OBJECTIVE: To describe a healthy ongoing pregnancy achieved with frozen thawed embryo transfer in a patient with non-mosaic Klinefelter Syndrome. DESIGN: A case report from tertiary center for assisted reproductive technologies. MATERIALS AND METHODS: A 34-year-old man with azospermia and non-mosaic Klinefelter Syndrome and his 26-year-old wife presented with primary infertility of eight-year duration. The physical examination of the man revealed a normal male habitus (height, 167 cm; weight, 72 kg) with usual facial hair. The testes were atrophic at physical examination. The patient had elevated FSH (35.0 IU/L) and LH concentrations (14.0 IU/L) but low testosterone concentration (2.1ng/mL, 0.073nmol/L; conversion factor⫽ 0.03467). Repeated semen analyses revealed azospermia. Intracytoplasmic sperm injection using surgically retrieved spermatozoa was planned. Luteal-long leuprolide acetate with oral contraceptive pre-treatment and recombinant-FSH were used for controlled ovarian hyperstimulation. No spermatozoa was noted at the initial wet prep with the TESE procedure one day preceding the oocyte pick-up. However, at total of 15 motile and 10 immotile spermatozoa were identified after a thorough inspection of the washed specimen drops early in the morning after overnight incubation under oil. Twenty-three oocyte-cumulus complexes were retrieved, 18 of which were metaphase-II. Since pre-implantation genetic diagnosis (PGD) is not available at our center and large percentage of sperm produced by patients with Klinefelter Syndrome are normal (23,X or 23,Y), PGD was not performed. Intracytoplasmic sperm injection using surgically retrieved spermatozoa was planned. The patient did not conceive with the
Vol. 84, Suppl 1, September 2005
P-63 Update on Clinical Efficiency of the Vitrification Method for Human Oocytes in an In Vitro Fertilization Program. T. Okimura, K. Kato, Q. Zhan, M. Kuwayama, J. Zhang, O. Kato. New Hope Fertility Center, New York, NY; R&D Division, Kato Ladies Clinic, Tokyo, Japan. OBJECTIVE: Oocyte cryopreservation can be a useful tool in IVF/ ICSI-ET cycles of human ART. Time allowance between oocyte retrieval and insemination by cryopreservation gives chances for certain female cancer patients to avoid iatrogenic sterility from chemo/radiotherapy. For patients who desire to delay their child bearing, long term preservation provides an opportunity to have the babies of their owns. Present experiments were conducted to evaluate the clinical efficiency of the minimum volume cooling (MVC) vitrification method for human oocytes in IVF cycles. DESIGN: Retrospective clinical study MATERIALS AND METHODS: Metaphase II Oocytes were denuded of the cumulus cells on the day of retrieval before vitrification. The Cryotop method was used to vitrify human oocytes. Oocytes were first equilibrated in 7.5% ethylene glycol (EG) and 7.5% DMSO in modified medium 199 (M-199) for 10-15min before being transferred into the vitrification solution (VS) for 30 sec. After replacing the extracellular solution by pipetting, oocytes were then transferred into the vitrification container with minimum volume of VS, and immediately submerged into liquid nitrogen. Thawing was performed by plunging the Cryotop containing the oocytes into 1M sucrose in M-199 at 37°C for 1 min, and the embryos were then placed in 0.5M Sucrose in M-199 for 3 min. The cryoprotective agents were diluted out by incubating in an isotonic culture medium for 5 min twice. After the recovery of oocytes they were cultured for 3 h in 5% CO2 in air at 37.0 C. Those oocytes considered to have survived were inseminated by intracytoplasmic sperm injection. Those embryos were cultured for 2 or 5days before transfer. RESULTS: Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection 52 of them became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescent in situ hybridization of five blastocysts showed that all were normal diploid embryos. 29 embryo transfers with mean number of 2.2 embryos per transfer on D2 or D5 resulted in 12 pregnancies; 7 full term deliveries with 3 ongoing pregnancies. CONCLUSION: Our results suggest that vitrification by the Cryotop method is the most efficient method ever published for human oocyte cryopreservation. Furthermore, oocytes cryopreservation will make oocyte/ ooplasm donation more flexible. Supported by: None
P-64 The Effect of Laser Assisted Hatching on Pregnancy Rates With Cryopreserved-Thawed Embryos. A. Y. Baratz, T. Di Berardino, E. M. Greenblatt. Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada; Division of Reproductive Sciences, Mount Sinai Hospital and University of Toronto, Toronto, ON, Canada.
S174
Abstracts
OBJECTIVE: To compare clinical pregnancy and implantation rates after transfer of 72 hour cryopreserved-thawed embryos that were or were not exposed to laser assisted hatching (LAH). DESIGN: Retrospective case-control study. MATERIALS AND METHODS: Cryopreserved-thawed embryos have a lower implantation rate and clinical pregnancy rate than fresh embryos. This difference may be the consequence of a change in the thickness or the quality of the zona pellucida. Different types of assisted hatching have been proposed to improve cryopreserved-thawed embryo cycle outcomes. LAH was initiated at our clinic in August 2003, and began to be performed routinely for 72 hour cryopreserved embryos at that time. We identified 106 cryopreserved-thawed embryo transfer cycles from August 2003 to March 2005 in whom LAH was performed (Group 1; cases). Prior to the introduction of LAH, we identified 19 cycles of cryopreserved-thawed 72 hour embryo transfer (March 2003-August 2003) in which identical culture media and freeze/thaw media and protocols had been applied, but in which LAH was not performed (Group 2; controls). All of the embryos were cultured in media for twenty four hours prior to embryo transfer. Immediately prior to transfer, LAH was performed using the OCTAX laser. Exposure time was adjusted to create an area of zona thinning that was 1.5 X the thickness of the zona pellucida. Only cryopreserved embryo transfers where all the intended embryos were hatched were included in the study. Routine statistical analysis was performed using the chi-square test to compare the two groups. RESULTS: In Group 1, of the 106 cycles (251 embryos) in which LAH was performed for cryopreserved-thawed embryo transfers, 37 (14.7%) implanted with a clinical pregnancy rate of 34.9%. In Group 2 (controls not hatched), 4 (8.5%) of the embryos implanted with a clinical pregnancy rate of 21.1%. The difference between the laser assisted hatching group and the non-assisted hatching group was not found to be statistically significant. CONCLUSION: In this retrospective study of LAH versus non-assisted hatching for 72 hour cryopreserved-thawed embryo transfers an improvement in implantation and clinical pregnancy rates did not reach statistical significance. However, the trend that emerges from this small retrospective study warrants a prospective randomized study with a larger sample size to more accurately evaluate the routine use of LAH to improve implantation and pregnancy rates during cryopreserved-thawed transfer cycles. Supported by: None.
P-65 An Ongoing Pregnancy After Frozen Thawed Embryo Transfer in a Patient with Klinefelter Syndrome. H. Yarali, G. Bozdag. Hacettepe University School of Medicine, Ankara, Turkey; Hacettepe University, School of Medicine, Department of Obstetrics and Gynecology, Ankara, Turkey. OBJECTIVE: To describe a healthy ongoing pregnancy achieved with frozen thawed embryo transfer in a patient with non-mosaic Klinefelter Syndrome. DESIGN: A case report from tertiary center for assisted reproductive technologies. MATERIALS AND METHODS: A 34-year-old man with azospermia and non-mosaic Klinefelter Syndrome and his 26-year-old wife presented with primary infertility of eight-year duration. The physical examination of the man revealed a normal male habitus (height, 167 cm; weight, 72 kg) with usual facial hair. The testes were atrophic at physical examination. The patient had elevated FSH (35.0 IU/L) and LH concentrations (14.0 IU/L) but low testosterone concentration (2.1ng/mL, 0.073nmol/L; conversion factor⫽ 0.03467). Repeated semen analyses revealed azospermia. Intracytoplasmic sperm injection using surgically retrieved spermatozoa was planned. Luteal-long leuprolide acetate with oral contraceptive pre-treatment and recombinant-FSH were used for controlled ovarian hyperstimulation. No spermatozoa was noted at the initial wet prep with the TESE procedure one day preceding the oocyte pick-up. However, at total of 15 motile and 10 immotile spermatozoa were identified after a thorough inspection of the washed specimen drops early in the morning after overnight incubation under oil. Twenty-three oocyte-cumulus complexes were retrieved, 18 of which were metaphase-II. Since pre-implantation genetic diagnosis (PGD) is not available at our center and large percentage of sperm produced by patients with Klinefelter Syndrome are normal (23,X or 23,Y), PGD was not performed. Intracytoplasmic sperm injection using surgically retrieved spermatozoa was planned. The patient did not conceive with the
Vol. 84, Suppl 1, September 2005