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Comparison of aneuploidy rates of blastocyst stage embryos derived from fresh and vitrified oocytes
P-358 Wednesday, October 21, 2009
P-360 Wednesday, October 21, 2009
COMPLEX PROTEIN SUPPLEMENTATION IMPROVES POSTTHAW RE-EXPANSION AND IMPLANTATION OF BLASTOCYSTS. J. M. Kramer, L. Ali, A. Rhoton-Vlasak, O. Bukulmez, R. S. Williams, K. C. Drury. Animal Molecular and Cellular Biology Program Department of Animal Sciences, University of Florida, Gainesville, FL; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Florida, Gainesville, FL.
HUMAN OOCYTE PRESERVATION EXPERIENCE (HOPE): PRELIMINARY RESULTS. Z. P. Nagy, D. G. Diaz, R. Bennett, A. AL-Sabbagh, R. V. Maamari. Reproductive Biology Associates, Atlanta, GA; West Coast Fertility Centers, Fountain Valley, CA; EMD Serono, Inc., Rockland, MA.
OBJECTIVE: The affects of globulin-rich protein supplementation of cryopreservation solutions on blastocyst post-thaw survival and potential is unknown. The purpose of this study was to test whether the supplementation of freeze and thaw solutions with a commercial protein source containing globulins would improve post-thaw blastocyst developmental potential. DESIGN: Animal experimental research and human retrospective study. MATERIALS AND METHODS: Animal studies compared thaw survival of control-rate cryopreserved murine blastocysts using the globulin-rich protein source, Serum Substitute Supplement (SSS, 10mg/ml HSA þ 2mg/ml globulins; Irvine Scientific), or human serum albumin (HSA, 10mg/ml) alone in solutions for either freeze and/or thaw, as well as the additional supplementation of post-thaw culture medium. The retrospective human study analyzed 28 frozen embryo transfer (FET) cycles in which frozen blastocysts were thawed in solutions supplemented with HSA or SSS. Statistical analysis included fisher’s exact test, chi-square test, and student’s t-test. RESULTS: More murine blastocysts re-expanded if both frozen and thawed in solutions containing SSS compared to HSA alone or frozen with SSS and thawed in HSA (P<0.05). Surprisingly, these observed differences dissipated when post-thaw culture medium was supplemented with SSS, with all treatments demonstrating high rates of re-expansion. Retrospective analysis of human clinical FET cycles revealed higher rates of blastocyst survival (83.3% vs 43.9%), re-expansion (66.7% vs 15.8%), clinical pregnancy per thaw (55.6% vs 10.5%) and implantation (53.8% vs 13.3%) for blastocysts thawed in solutions supplemented with SSS compared to HSA (P<0.05). CONCLUSIONS: The inclusion of a globulin-rich protein source, such as SSS, in thaw solutions improves cryopreservation survival and implantation. Additionally, post-thaw culture medium supplemented with SSS can restore potential for blastocyst re-expansion even if frozen or thawed in solutions devoid of globulins.
P-359 Wednesday, October 21, 2009 COMPARISON OF ANEUPLOIDY RATES OF BLASTOCYST STAGE EMBRYOS DERIVED FROM FRESH AND VITRIFIED OOCYTES. L. F. A. Colturato, C. C. Chang, G. Wright, S. M. Slayden, D. Mitchell-Leef, Z. P. Nagy. Reproductive Biology Associates, Atlanta, GA. OBJECTIVE: Evaluate if the cryopreservation process is associated with a possible increase in the aneuploidy rates in vitrified/warmed oocytes. DESIGN: Prospective, observational study. MATERIALS AND METHODS: The study group included blastocyst stage embryos (donated to research) originated from vitrified oocytes. The control group included blastocysts from a fresh cycle. All cells from each embryo were fixed. Fluorescent in situ hybridization was performed using a 5 chromosome probe mixture (13, 16, 18, 21, and 22). Fisher’s exact test was performed, at the level of P<0.05. RESULTS: Ten embryos were obtained from the vitrified oocytes and 16 embryos were obtained from fresh oocytes. A total of 1068 cells were analyzed, 380 in the vitrification group and 688 in the control group. The control group presented a mean of 43.0þ/-13.4 cells and the vitrification group 38.0 þ/-32.8 cells (NS). The average number of cells with euploid chromosome content was 23.0þ/-14.7 (53.5%) in the control group and 20.0þ/-28.5 (52.6%) in the vitrification group (NS). In the control group, all embryos were found to be mosaic diploid/aneuploid. Thirteen out of 16 embryos had chaotic distribution. Out of the remaining embryos, 2 had some polyploidy cells; 1 had chromosome 21 monosomy and 1 had chromosome 22 nullisomy/trisomy. The vitrification group, also, all of the embryos were mosaic diploid/aneuploid, however 7 out of the 10 embryos had a chaotic complement and the remaining 3 embryos had haploid content. CONCLUSIONS: Blastocysts obtained from fresh oocytes presented similar cell number as embryos obtained from vitrified oocytes. Also, there was no difference in the distribution of the type and frequency of chromosomal abnormalities, regardless whether it was obtained from fresh or from vitrified oocytes. The results of the current study do not indicate an increase of chromosome abnormality in embryos obtained after oocyte vitrification, further studies including a larger number of embryos is warranted to confirm present findings.
FERTILITY & STERILITY
OBJECTIVE: The ASRM considers oocyte cryopreservation as experimental because of limited efficacy and safety data. To address this need, EMD Serono has launched the HOPE Registry. The present aim is to report preliminary data from the initial cohort of enrolled patients from participating US IVF Centers. DESIGN: Phase IV, prospective, 5-year observational registry with a 3-year enrollment period and a baby follow-up at birth and at one year of age. MATERIALS AND METHODS: 75 women have already been enrolled. Descriptive statistics from 48 cycles are reported below. RESULTS: Table 1.
Outcomes by Technique (cycles)
Slow-Freezing (16)
Vitrification (32)
41.6 4.0 22.1 5.3 21.9 5.5 6.4 1.2 89.8% 12.0 79.4% 3.8 0.7 0.3 0.7 25.4% 30.2 57.1% 12.5%
40.6 4.4 31.1 16.2 30.1 15.5 7.2 2.9 89.0% 14.3 73.8% 2.0 0.7 2.0 1.8 50% 43.9 63.6% 0%
Age (yr), (Mean SD) Oocytes Retrieved Oocytes Frozen, Oocytes Thawed Oocyte Survival% 2PN Oocytes (% of Thawed) Embryos Transferred Embryos Cryopreserved Implantation Rate % Clinical Pregnancy (%) Miscarriage Rate (%) Mean SD per cycle
CONCLUSIONS: Oocyte survival, fertilization and embryo development were similarly high in both groups. Both cryopreservation techniques had similarly high clinical pregnancy rates, though after slow-freeze there were more embryos transferred than after vitrification, a difference also reflected by the implantation rates. These initial excellent results may be obtained as a combination of type of patients (majority in both groups were recipients of donated frozen eggs) and advanced cryopreservation techniques. With the continuing incorporation of new patients, future inclusion of the data on offspring and a statistical comparison of the two techniques, the HOPE Registry will provide standardized data regarding efficiency and safety of oocyte cryopreservation to satisfy unmet needs from the medical community. Supported by: Supported by EMD Serono, Inc.
P-361 Wednesday, October 21, 2009 HIGH SURVIVAL AND HATCHING RATES FOLLOWING VITRIFICATION OF HUMAN BLASTOCYSTS WITH CBS— HIGH-SECURITY STRAWS. Y. Shu, J. Watt, J. Gebhardt, J. Dasig, Q. Zhao, B. Behr. IVF Program, Stanford University Medical Center, Palo Alto, CA. OBJECTIVE: Sealing a straw at the end or keeping a carrier containing the specimen inside an outer protecting container provides a strategy to eliminate the potential contamination associated with direct contact with liquid nitrogen; however, it does not allow for rapid cooling/warming of the vitrified samples. Inconstistent results have been reported by using closed carrier system for vitrification of human gamates and embryos. In this study, CBS Highsecurity Straw was used to vitrify human blastocysts. DESIGN: Academic IVF Program. MATERIALS AND METHODS: Day 6 or day 7 human blastocysts unsuitable for transfer and cryopreservation were included. A total of 53 blastocysts were vitrified by either CBS High-security Straw or home-made Open Hemi-straw. Artificial shrinkage was performed by using a single laser pulse before vitrification. Sage Vitrification and Warming Media was used. To increase the cooling/warming rate of the closed system, we have minimized the microdrops containing embryos as small as 0.1 ml in volume. The total time period required for loading, sealing, and plunging was less than 60 s. Immediate blastocyst survival and blastocoele re-expansion were evaluated after warming of vitrified blastocysts.
S189
P-360 Wednesday, October 21, 2009
COMPLEX PROTEIN SUPPLEMENTATION IMPROVES POSTTHAW RE-EXPANSION AND IMPLANTATION OF BLASTOCYSTS. J. M. Kramer, L. Ali, A. Rhoton-Vlasak, O. Bukulmez, R. S. Williams, K. C. Drury. Animal Molecular and Cellular Biology Program Department of Animal Sciences, University of Florida, Gainesville, FL; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Florida, Gainesville, FL.
HUMAN OOCYTE PRESERVATION EXPERIENCE (HOPE): PRELIMINARY RESULTS. Z. P. Nagy, D. G. Diaz, R. Bennett, A. AL-Sabbagh, R. V. Maamari. Reproductive Biology Associates, Atlanta, GA; West Coast Fertility Centers, Fountain Valley, CA; EMD Serono, Inc., Rockland, MA.
OBJECTIVE: The affects of globulin-rich protein supplementation of cryopreservation solutions on blastocyst post-thaw survival and potential is unknown. The purpose of this study was to test whether the supplementation of freeze and thaw solutions with a commercial protein source containing globulins would improve post-thaw blastocyst developmental potential. DESIGN: Animal experimental research and human retrospective study. MATERIALS AND METHODS: Animal studies compared thaw survival of control-rate cryopreserved murine blastocysts using the globulin-rich protein source, Serum Substitute Supplement (SSS, 10mg/ml HSA þ 2mg/ml globulins; Irvine Scientific), or human serum albumin (HSA, 10mg/ml) alone in solutions for either freeze and/or thaw, as well as the additional supplementation of post-thaw culture medium. The retrospective human study analyzed 28 frozen embryo transfer (FET) cycles in which frozen blastocysts were thawed in solutions supplemented with HSA or SSS. Statistical analysis included fisher’s exact test, chi-square test, and student’s t-test. RESULTS: More murine blastocysts re-expanded if both frozen and thawed in solutions containing SSS compared to HSA alone or frozen with SSS and thawed in HSA (P<0.05). Surprisingly, these observed differences dissipated when post-thaw culture medium was supplemented with SSS, with all treatments demonstrating high rates of re-expansion. Retrospective analysis of human clinical FET cycles revealed higher rates of blastocyst survival (83.3% vs 43.9%), re-expansion (66.7% vs 15.8%), clinical pregnancy per thaw (55.6% vs 10.5%) and implantation (53.8% vs 13.3%) for blastocysts thawed in solutions supplemented with SSS compared to HSA (P<0.05). CONCLUSIONS: The inclusion of a globulin-rich protein source, such as SSS, in thaw solutions improves cryopreservation survival and implantation. Additionally, post-thaw culture medium supplemented with SSS can restore potential for blastocyst re-expansion even if frozen or thawed in solutions devoid of globulins.
P-359 Wednesday, October 21, 2009 COMPARISON OF ANEUPLOIDY RATES OF BLASTOCYST STAGE EMBRYOS DERIVED FROM FRESH AND VITRIFIED OOCYTES. L. F. A. Colturato, C. C. Chang, G. Wright, S. M. Slayden, D. Mitchell-Leef, Z. P. Nagy. Reproductive Biology Associates, Atlanta, GA. OBJECTIVE: Evaluate if the cryopreservation process is associated with a possible increase in the aneuploidy rates in vitrified/warmed oocytes. DESIGN: Prospective, observational study. MATERIALS AND METHODS: The study group included blastocyst stage embryos (donated to research) originated from vitrified oocytes. The control group included blastocysts from a fresh cycle. All cells from each embryo were fixed. Fluorescent in situ hybridization was performed using a 5 chromosome probe mixture (13, 16, 18, 21, and 22). Fisher’s exact test was performed, at the level of P<0.05. RESULTS: Ten embryos were obtained from the vitrified oocytes and 16 embryos were obtained from fresh oocytes. A total of 1068 cells were analyzed, 380 in the vitrification group and 688 in the control group. The control group presented a mean of 43.0þ/-13.4 cells and the vitrification group 38.0 þ/-32.8 cells (NS). The average number of cells with euploid chromosome content was 23.0þ/-14.7 (53.5%) in the control group and 20.0þ/-28.5 (52.6%) in the vitrification group (NS). In the control group, all embryos were found to be mosaic diploid/aneuploid. Thirteen out of 16 embryos had chaotic distribution. Out of the remaining embryos, 2 had some polyploidy cells; 1 had chromosome 21 monosomy and 1 had chromosome 22 nullisomy/trisomy. The vitrification group, also, all of the embryos were mosaic diploid/aneuploid, however 7 out of the 10 embryos had a chaotic complement and the remaining 3 embryos had haploid content. CONCLUSIONS: Blastocysts obtained from fresh oocytes presented similar cell number as embryos obtained from vitrified oocytes. Also, there was no difference in the distribution of the type and frequency of chromosomal abnormalities, regardless whether it was obtained from fresh or from vitrified oocytes. The results of the current study do not indicate an increase of chromosome abnormality in embryos obtained after oocyte vitrification, further studies including a larger number of embryos is warranted to confirm present findings.
FERTILITY & STERILITY
OBJECTIVE: The ASRM considers oocyte cryopreservation as experimental because of limited efficacy and safety data. To address this need, EMD Serono has launched the HOPE Registry. The present aim is to report preliminary data from the initial cohort of enrolled patients from participating US IVF Centers. DESIGN: Phase IV, prospective, 5-year observational registry with a 3-year enrollment period and a baby follow-up at birth and at one year of age. MATERIALS AND METHODS: 75 women have already been enrolled. Descriptive statistics from 48 cycles are reported below. RESULTS: Table 1.
Outcomes by Technique (cycles)
Slow-Freezing (16)
Vitrification (32)
41.6 4.0 22.1 5.3 21.9 5.5 6.4 1.2 89.8% 12.0 79.4% 3.8 0.7 0.3 0.7 25.4% 30.2 57.1% 12.5%
40.6 4.4 31.1 16.2 30.1 15.5 7.2 2.9 89.0% 14.3 73.8% 2.0 0.7 2.0 1.8 50% 43.9 63.6% 0%
Age (yr), (Mean SD) Oocytes Retrieved Oocytes Frozen, Oocytes Thawed Oocyte Survival% 2PN Oocytes (% of Thawed) Embryos Transferred Embryos Cryopreserved Implantation Rate % Clinical Pregnancy (%) Miscarriage Rate (%) Mean SD per cycle
CONCLUSIONS: Oocyte survival, fertilization and embryo development were similarly high in both groups. Both cryopreservation techniques had similarly high clinical pregnancy rates, though after slow-freeze there were more embryos transferred than after vitrification, a difference also reflected by the implantation rates. These initial excellent results may be obtained as a combination of type of patients (majority in both groups were recipients of donated frozen eggs) and advanced cryopreservation techniques. With the continuing incorporation of new patients, future inclusion of the data on offspring and a statistical comparison of the two techniques, the HOPE Registry will provide standardized data regarding efficiency and safety of oocyte cryopreservation to satisfy unmet needs from the medical community. Supported by: Supported by EMD Serono, Inc.
P-361 Wednesday, October 21, 2009 HIGH SURVIVAL AND HATCHING RATES FOLLOWING VITRIFICATION OF HUMAN BLASTOCYSTS WITH CBS— HIGH-SECURITY STRAWS. Y. Shu, J. Watt, J. Gebhardt, J. Dasig, Q. Zhao, B. Behr. IVF Program, Stanford University Medical Center, Palo Alto, CA. OBJECTIVE: Sealing a straw at the end or keeping a carrier containing the specimen inside an outer protecting container provides a strategy to eliminate the potential contamination associated with direct contact with liquid nitrogen; however, it does not allow for rapid cooling/warming of the vitrified samples. Inconstistent results have been reported by using closed carrier system for vitrification of human gamates and embryos. In this study, CBS Highsecurity Straw was used to vitrify human blastocysts. DESIGN: Academic IVF Program. MATERIALS AND METHODS: Day 6 or day 7 human blastocysts unsuitable for transfer and cryopreservation were included. A total of 53 blastocysts were vitrified by either CBS High-security Straw or home-made Open Hemi-straw. Artificial shrinkage was performed by using a single laser pulse before vitrification. Sage Vitrification and Warming Media was used. To increase the cooling/warming rate of the closed system, we have minimized the microdrops containing embryos as small as 0.1 ml in volume. The total time period required for loading, sealing, and plunging was less than 60 s. Immediate blastocyst survival and blastocoele re-expansion were evaluated after warming of vitrified blastocysts.
S189