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Results of Oocyte Cryopreservation using Choline-Based Freezing Medium
charyya, A. C. Varghese, S. Das, A. Biswas, M. Mandal, A. Agarwal. Department of Biochemistry, University of Calcutta, Calcutta, India; Kalpataru Arogyoniketan, Barasat, India; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: Despite many refinements in cryopreservation techniques, the recovery of post thaw sperm remains sub-optimal. The most commonly reported detrimental effect of freeze-thaw of human sperm is substantial reduction in motility and velocity, which may be associated with extensive damage that occurs during sperm processing methods employed to remove cryoprotectants and seminal plasma. The aim of our study was to evaluate the motility parameters of cryopreserved human sperm following separation by the different sperm preparation methods such as 1) swim-up with and 2) without reduced glutathione (GSH) as an antioxidant, 3) hyaluronate swim-up and 4) density gradient. DESIGN: Prospective in vitro study MATERIALS AND METHODS: Twenty-five normozoospermic semen samples were cryopreserved with glycerol-based cryoprotectant. Thawed semen of each volunteer (n⫽12) was divided into two equal aliquots. Both underwent wash and swim-up procedure using Ham’s F-10 (HF-10) alone or HF-10 with GSH (5mMol/L) respectively. In the other group (n⫽13), one aliquot was processed by neat swim-up using hyaluronic acid (2mg/ml of HF-10) and the other by density gradient centrifugation (DGC, PurespermTM 80/40) followed by a swim up. Post-processing samples were analyzed using a computer assisted semen analyzer (CASA; Hamilton-Thorn, CEROS) for sperm motion parameters. RESULTS: In comparison to HF-10 wash and pellet swim-up, processing with GSH in HF-10 showed significantly higher percentage (%) motility (25.0 ⫾ 6.2 vs. 12.0 ⫾ 5.3; p ⫽ 0.003) and % rapid forms (24.5 ⫾ 5.9 vs. 11.5 ⫾ 5.4; p ⫽ 0.002). However, there were no significant differences in other CASA parameters. Both hyaluronate swim-up and DGC methods were equally good in the recovery of % motile (34.3 ⫾ 3.4 vs. 47.3 ⫾ 24.2: p ⫽ 0.380) and rapid forms (27.0 ⫾ 6.4 vs. 40.5 ⫾ 24.4; p ⫽ 0.391). Average path velocity and VCL were higher in DGC group (p ⫽ 0.037 and 0.041). In contrast ALH showed significantly higher values for hyaluronate group (2.2⫾1.3 vs. 4.8⫾0.5; p ⫽ 0.038). CONCLUSION: The selection procedure for post thaw semen samples should be carried out using optimized techniques so as to avoid further damage to fragile sperm population resulting from freeze-thaw process. Inclusion of GSH in processing media can confer protection against cell damage by oxidants, electrophiles and free radicals. Density gradient centrifugation (DGC) or hyaluronate swim-up offer better alternatives for processing cryopreserved semen. Supported by: None
P-74 Results of Oocyte Cryopreservation using Choline-Based Freezing Medium. R. M. Azambuja, M. Badalotti, L. Okada, J. Michelon, F. Badalotti, A. Petracco. Fertilitat - Centro de Medicina Reprodutiva, Porto Alegre, Brazil. OBJECTIVE: The purpose of this study was to observe the clinical pregnancy rate in patients that used frozen - thawed oocytes. Choline-based freezing medium was used. DESIGN: A total of 32 cycles using frozen - thawed oocytes and ICSI underwent ART at Fertilitat - Centro de Medicina Reprodutiva- Brazil during the period of 2002-2005. The couples did not wish to freeze embryos, therefore, oocyte freezing was selected. After extensive orientation, the appropriate consent forms were signed. MATERIALS AND METHODS: Ovarian stimulation was induced with gonadotropins after pituitary down regulation with leuprolide acetate. Follicular aspiration was performed by the transvaginal route 34⫾ 2 hours following hCG injection, and semen was prepared using a Percoll gradient. The oocytes were aliquoted into two groups, with one group being inseminated and the second group being frozen following the Stachecki et al., (Biology of Reproduction. 59: 396-400, 1998) protocol. Approximately 2 months after the negative bHCG, the endometrium was prepared using Estradiol Benzoate (Estrofen®, Medley, Brazil) with an initial dose of 2mg/day, starting on the 3rd day of the cycle. The maximum dosage used was 6mg/day when the endometrium reached a thickness of 8mm by ultrasound. Three days before the embryo transfer, the patient started using 90mg progesterone gel daily (Crinone 8%®, Serono, Brazil). Two days
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before the replacement of the embryos, the oocytes were thawed following the protocol of Stachecki et al., (Biology of Reproduction. 59: 396-400, 1998), and were inseminated by ICSI routinely. RESULTS: The table shows the results using oocytes frozen with choline-based freezing medium
CONCLUSION: While the number of children born from oocyte cryopreservation is low, the use of low-sodium choline-based media is becoming more popular. These results demonstrate the possibility of obtaining clinical pregnancies and babies born from oocytes cryopreserved in a choline-based medium. It is important to consider patient age and embryo quality, as well as the number of embryos to be replaced when performing this procedure. Supported by: None P-75 Cryopreserved Blastocysts Using Vitrification Protocol Give Excellent Pregnancy and Implantation Rates After Thawing. S. Hong, T. Kim, S. Lee, D. Choi, K. Cha. CHA Fertility Center, Los Angeles, CA; College of Medicine, Pochon CHA University, Infertility Medical Center of CHA General Hospital, Seoul, Republic of Korea. OBJECTIVE: With increasing use of blastocyst culture as a routine protocol in human clinical IVF, cryopreservation of excessive human blastocysts has become an important part of infertility treatment. Vitrification technique for cryopreservation of human blastocysts has been developed and applied in clinical settings. In our previous study, the vitrification protocol showed promising results for both oocytes and blastocysts. In this study we have modified our original method to improve efficiency of vitrification for blastocysts. We report the survival, pregnancy and implantation rats of vitrified blastocysts. DESIGN: Clinical trial of vitrification for human blastocysts MATERIALS AND METHODS: After embryo transfers in IVF cycles, surplus embryos that developed to blastocyst stage were first equilibrated in cryoprotectant solution containing 1.5 mol/L ethylene glycol (EG) for 5min at 37°C followed by introducing vitrification solution containing 5.5 mol/L EG ⫹ 1.0 mol/L sucrose for 20sec at room temperature. Blastocysts were then placed on the electron microscope (EM) grids and immediately plunged to the liquid nitrogen (LN2) directly. A total of 407 blastocysts from 127 egg retrieval cycles were cryopreserved using vitrification technique. For thawing, the EM grid holding the embryos was taken out of the liquid nitrogen container, then immediately transferred to organ culture dish sequentially containing 1.0ml of thawing solutions which is 1.0, 0.5, 0.25, 0.125, 0 mol/L of sucrose at intervals of 2.5 min at 37°C. Vitrified blastocysts were thawed around 4PM the day before embryo transfer, and cultured in G2.2 or G2.3 medium overnight. Re-expanded blastocysts were judged for survival following morning, then transferred to the patients in the afternoon. RESULTS: A total of 89 blastocysts obtained from 19 cycles of oocyte collection were vitrified. Of which, 77 blastocysts were thawed, and 53 embryos (69%) survived. A total of 47 blastocysts were transferred to 19 patients in 20 embryo transfer cycles. The mean number of blastocysts transferred per cycle was 2.4 ⫾ 0.9. Of the 47 embryos transferred, 24(51%) were determined to be implanted about 4 weeks after embryo transfer by ultrasound exam. Of 20 transfers, 16 resulted in clinical pregnancy; the pregnancy rate was 80% per warming cycle and 84% per vitrification cycle. There were no cancelled embryo transfers after thawing. Ten patients delivered 13 babies (3 sets of twins and 7 of singleton deliveries) between November 2003 and March 2005. Two patients are on going. Two patients ended miscarriage and two patients performed therapeutic abortion after amniocentesis. CONCLUSION: Significantly high pregnancy and implantation rates were observed with vitrified blastocysts. This indicates that the vitrification
Vol. 84, Suppl 1, September 2005
P-74 Results of Oocyte Cryopreservation using Choline-Based Freezing Medium. R. M. Azambuja, M. Badalotti, L. Okada, J. Michelon, F. Badalotti, A. Petracco. Fertilitat - Centro de Medicina Reprodutiva, Porto Alegre, Brazil. OBJECTIVE: The purpose of this study was to observe the clinical pregnancy rate in patients that used frozen - thawed oocytes. Choline-based freezing medium was used. DESIGN: A total of 32 cycles using frozen - thawed oocytes and ICSI underwent ART at Fertilitat - Centro de Medicina Reprodutiva- Brazil during the period of 2002-2005. The couples did not wish to freeze embryos, therefore, oocyte freezing was selected. After extensive orientation, the appropriate consent forms were signed. MATERIALS AND METHODS: Ovarian stimulation was induced with gonadotropins after pituitary down regulation with leuprolide acetate. Follicular aspiration was performed by the transvaginal route 34⫾ 2 hours following hCG injection, and semen was prepared using a Percoll gradient. The oocytes were aliquoted into two groups, with one group being inseminated and the second group being frozen following the Stachecki et al., (Biology of Reproduction. 59: 396-400, 1998) protocol. Approximately 2 months after the negative bHCG, the endometrium was prepared using Estradiol Benzoate (Estrofen®, Medley, Brazil) with an initial dose of 2mg/day, starting on the 3rd day of the cycle. The maximum dosage used was 6mg/day when the endometrium reached a thickness of 8mm by ultrasound. Three days before the embryo transfer, the patient started using 90mg progesterone gel daily (Crinone 8%®, Serono, Brazil). Two days
S178
Abstracts
before the replacement of the embryos, the oocytes were thawed following the protocol of Stachecki et al., (Biology of Reproduction. 59: 396-400, 1998), and were inseminated by ICSI routinely. RESULTS: The table shows the results using oocytes frozen with choline-based freezing medium
CONCLUSION: While the number of children born from oocyte cryopreservation is low, the use of low-sodium choline-based media is becoming more popular. These results demonstrate the possibility of obtaining clinical pregnancies and babies born from oocytes cryopreserved in a choline-based medium. It is important to consider patient age and embryo quality, as well as the number of embryos to be replaced when performing this procedure. Supported by: None P-75 Cryopreserved Blastocysts Using Vitrification Protocol Give Excellent Pregnancy and Implantation Rates After Thawing. S. Hong, T. Kim, S. Lee, D. Choi, K. Cha. CHA Fertility Center, Los Angeles, CA; College of Medicine, Pochon CHA University, Infertility Medical Center of CHA General Hospital, Seoul, Republic of Korea. OBJECTIVE: With increasing use of blastocyst culture as a routine protocol in human clinical IVF, cryopreservation of excessive human blastocysts has become an important part of infertility treatment. Vitrification technique for cryopreservation of human blastocysts has been developed and applied in clinical settings. In our previous study, the vitrification protocol showed promising results for both oocytes and blastocysts. In this study we have modified our original method to improve efficiency of vitrification for blastocysts. We report the survival, pregnancy and implantation rats of vitrified blastocysts. DESIGN: Clinical trial of vitrification for human blastocysts MATERIALS AND METHODS: After embryo transfers in IVF cycles, surplus embryos that developed to blastocyst stage were first equilibrated in cryoprotectant solution containing 1.5 mol/L ethylene glycol (EG) for 5min at 37°C followed by introducing vitrification solution containing 5.5 mol/L EG ⫹ 1.0 mol/L sucrose for 20sec at room temperature. Blastocysts were then placed on the electron microscope (EM) grids and immediately plunged to the liquid nitrogen (LN2) directly. A total of 407 blastocysts from 127 egg retrieval cycles were cryopreserved using vitrification technique. For thawing, the EM grid holding the embryos was taken out of the liquid nitrogen container, then immediately transferred to organ culture dish sequentially containing 1.0ml of thawing solutions which is 1.0, 0.5, 0.25, 0.125, 0 mol/L of sucrose at intervals of 2.5 min at 37°C. Vitrified blastocysts were thawed around 4PM the day before embryo transfer, and cultured in G2.2 or G2.3 medium overnight. Re-expanded blastocysts were judged for survival following morning, then transferred to the patients in the afternoon. RESULTS: A total of 89 blastocysts obtained from 19 cycles of oocyte collection were vitrified. Of which, 77 blastocysts were thawed, and 53 embryos (69%) survived. A total of 47 blastocysts were transferred to 19 patients in 20 embryo transfer cycles. The mean number of blastocysts transferred per cycle was 2.4 ⫾ 0.9. Of the 47 embryos transferred, 24(51%) were determined to be implanted about 4 weeks after embryo transfer by ultrasound exam. Of 20 transfers, 16 resulted in clinical pregnancy; the pregnancy rate was 80% per warming cycle and 84% per vitrification cycle. There were no cancelled embryo transfers after thawing. Ten patients delivered 13 babies (3 sets of twins and 7 of singleton deliveries) between November 2003 and March 2005. Two patients are on going. Two patients ended miscarriage and two patients performed therapeutic abortion after amniocentesis. CONCLUSION: Significantly high pregnancy and implantation rates were observed with vitrified blastocysts. This indicates that the vitrification
Vol. 84, Suppl 1, September 2005