- Email: [email protected]
Vitrified blastocysts yield equivalent pregnancy rates as compared to fresh blastocysts
DESIGN: Validation trial. MATERIALS AND METHODS: 166 plasma samples from women, drawn at R9 weeks gestation, including 20 aneuploid pregnancies, were analyzed using a novel method, Parental Support (PS). Cell-free DNA from maternal plasma was isolated, amplified using targeted PCR focusing on more than 10,000 SNPs on chromosomes 13, 18, 21, X, and Y, and sequenced, along with genetic samples from the mother and father. A Bayesian-based Maximum Likelihood (ML) informatics method was applied to the data to assess the chromosomal count of the five chromosomes of interest. A confidence was also calculated, which is analogous to a personalized accuracy for each sample and enables determination of whether a redraw is necessary for high reliability on all chromosomes. Confirmation of the chromosome complement was achieved by obtaining a karyotype by prenatal diagnosis or from child samples post-delivery. RESULTS: The algorithm returned a call at all five chromosomes for 148 cases (89.2%); all calls in this group were correct (740/740) with an average confidence of 99.83% per copy number call, including 11 trisomy 21, 3 trisomy 18, 2 trisomy 13, 1 45,X, and 2 47,XXY calls, with an average confidence of 99.97%. CONCLUSION: In contrast to other methods, PS is able to calculate a precise confidence in each sample, allowing for blood draws earlier in pregnancy without increasing the rate of false positives or false negatives. PS is able to detect fetuses having an abnormality from a maternal blood sample with similarly high accuracy across chromosomes 13, 18, 21, X, and Y.This can allow for IVF clinics to offer early and accurate chromosomal screening to their patients. Supported by: Supported, in part, by a grant from the NIH, National Institute of Child Health and Human Development (4R44HD062114-02).
O-94 Monday, October 22, 2012 04:30 PM FACTORS THAT AFFECT MINERAL OIL TOXICITY: ROLE OF OXYGEN AND PROTEIN SUPPLEMENT. D. E. Morbeck, J. R. Fredrickson, D. L. Walker. Obstetrics and Gynecology, Mayo Clinic, Rochester, MN. OBJECTIVE: Though mineral oil is commonly filtered, washed and bioassay tested, three lots from three different companies recently demonstrated toxicity in IVF labs and were recalled by the suppliers. Differences between supplier and clinical laboratory conditions and QC methods may explain why toxicity was not originally detected. The objective of this study was to compare 2 incubator conditions and different protein supplements on sensitivity to toxicity of these 3 lots of oil. DESIGN: Animal research study. MATERIALS AND METHODS: Zygotes were collected from superovulated, inbred mice per IACUC protocol. Experiment 1 tested incubator conditions. Embryos were cultured in HTF plus PVA in a CO2 Forma incubator with room air (20% O2) or a Planer BT-37 with 5% O2 medical grade air. Experiment 2 tested protein x oil interaction. Embryos were cultured in HTF with 5 mg/ml HSA or 10% SSS. Control oil (Fisher) was compared to 3 recalled lots of oil: L1, L2 and L3. Expanded blastocyst rate at 96 and 120 hours were the endpoints. RESULTS: Blastocyst rates (BR) in control media were not different at 96 hours in 5% O2 but were lower for L1, L2 and L3 in the 20% O2 incubator (P<0.05). At 120 hours, BR were lower for affected oils in both incubators. When HSA was added to the culture, BR was similar to control, whereas embryos cultured in SSS arrested and blastomeres lysed by the 8 cell stage in L2, had lower BR in L1 or were not affected in L3. Peroxide
Blastocyst Rates
96h
Control L1 L2 L3
120h
96h/20% O2
5% O2
20% O2
5% O2
20% O2
HSA
SSS
85.0 73.8 71.7 80.0
70.9 47.5* 7.5* 43.5*
73.3 44.4* 40.0* 30.0*
62.2 8.3* 0* 22.9*
76.9 89.4 60.0 65.0
86.7 36.7* 0* 80.0
* P<0.05.
FERTILITY & STERILITYÒ
testing indicated that L1 and L2 had elevated peroxide levels whereas L3 was similar to the control. CONCLUSION: Oil is the most variable product in the IVF laboratory and its toxicity continues to affect clinical outcomes. These results demonstrate interactions between oxygen and protein and illustrate the need for more sophisticated mineral oil screening. Supported by: OB/GYN Research Committee.
O-95 Monday, October 22, 2012 04:45 PM 12-HOUR PREIMPLANTATION GENETIC SCREENING (PGS) BY TARGETED SEQUENCING AND HIGHLY MULTIPLEX PCR WITH 5% ADO ON BENCHTOP SEQUENCERS. M. Hill, J. Baner, B. Zimmermann, A. Ryan, G. Gemelos, M. Rabinowitz. Natera, Inc., San Carlos, CA. OBJECTIVE: Demonstrate proof of concept for a rapid turnaround PGS sequencing technology that could be performed in-clinic in 12 hours using the MiSeq small-footprint push-button sequencer. DESIGN: Thirty single cell samples were isolated from a trisomy 21 (47,XX,+21; 10 replicate cells) and five karyotypically normal cell lines (two 46,XY and three 46,XX; four replicate cells each). Cell lines corresponding to parents of the test lines were also evaluated. MATERIALS AND METHODS: In 12 hours we performed multiplex PCR and sequencing of 1200 SNPs on chromosomes 1, 21, and X on an Illumina MiSeq with an average depth of read of 350 reads per SNP. The ploidy state of each chromosome was determined by a Maximum Likelihood estimator, comparing the observed read count of each allele against a theoretical model of ploidy hypotheses (monosomy, disomy, trisomy). The model combines a Monte Carlo simulation of the PCR noise processes with a binomial model to incorporate variations in read depth. Performance with fewer measured loci per chromosome was measured by randomly subsampling from the 1200-plex measurements. RESULTS: Among 90 distinct disomy, trisomy, or X chromosome calls made on 30 cells, accuracy was 100% [95% CI: 96.0% - 100%]. Furthermore, random resampling of the data to simulate fewer loci indicated that 100 loci per chromosome would be sufficient for >99% accuracy with our method. The allele dropout rate in SNPs known to be heterozygous was approximately 5%. To our knowledge, this is the lowest level of allele dropouts ever reported from a single cell in a highly multiplex reaction. CONCLUSION: This is the first demonstration of multiplex PCR amplification and sequencing on a push-button sequencer capable of the rapid turnaround required for PGS with Day 5 biopsy and Day 6 transfer. The accuracy of the statistical model and extremely low allele drop-out enables highly accurate ploidy calling from single cells.
O-96 Monday, October 22, 2012 05:00 PM VITRIFIED BLASTOCYSTS YIELD EQUIVALENT PREGNANCY RATES AS COMPARED TO FRESH BLASTOCYSTS. D. A. Kelk, E. L. Paganetti, M. P. Leondires, M. E. Moore, C. M. Murdock, S. S. Richlin. Reproductive Medicine Associates of Connecticut, Norwalk, CT. OBJECTIVE: Fresh embryos have historically yielded higher pregnancy rates than frozen embryos. For many programs, vitrification has afforded increased pregnancy and implantation rates over classical slow freeze protocols. This study seeks to evaluate if vitrified blastocyst pregnancy and implantation rates can match those attained with fresh blastocysts. DESIGN: A retrospective analysis of 399 fresh blastocyst and 115 vitrified blastocyst transfers in patients under 38 years of age. MATERIALS AND METHODS: All fresh IVF cycles from Jan 2010 through March 2012 for patients <38 years old with fresh blastocyst transfers were compared to vitrified blastocyst FET’s. Donor egg cycles were not included. Embryos were group cultured in 50ml drops of Global culture media in 5-well dishes in MINC incubators with a 6.5% CO2/5.0% O2 gas mixture. Blastocyst stage embryos were vitrified on Day 5 and/or Day 6 using Irvine Scientific Vit Kit and HSV straws. Statistical analysis of pregnancy and implantation rates were performed using Chi-square. Statistical significance was set at P<0.05. RESULTS: Mean maternal age, pregnancy and implantation rates are displayed in Table 1.
S29
Fresh vs Vitrified Blastocyst Transfers
Fresh Blastocyst ET
Vitrified Blastocyst ET
399 33.1 1.85 72.4% 60.2% 50.1%
115 32.1 1.91 76.5% 67.0% 50.5%
# of Transfers Mean Maternal Age Avg # Embryos Transferred Positive BhCG Rate Clinical Pregnancy Rate Implantation Rate
CONCLUSION: Pregnancy and implantation rates were not statistically different between fresh and vitrified blastocysts (P>0.05). These findings should be validated in a prospective randomized study. These data support that there is little, if any compromise to the viability of the embryo with vitrification. When timed appropriately, frozen embryo transfers allow for synchrony between the embryo and the uterine window of implantation. Programs should be comfortable in vitrifying blastocysts in freeze-all cycles, OHSS patients, PGD/PGS cases, donor egg recipients, and gestational carriers.
O-97 Monday, October 22, 2012 05:15 PM ADVANCES IN QUALITY CONTROL: MOUSE EMBRYO MORPHOKINETICS ARE SENSITIVE MARKERS OF IN VITRO STRESS. H. S. Wolff, J. R. Fredrickson, D. L. Walker, D. E. Morbeck. Reproductive Endocrinology and Infertility, Mayo Clinic, Rochester, MN. OBJECTIVE: Poor conditions for embryo culture have adverse effects on outcomes and can result in lower live birth rates. Morphokinetics (MK) of embryo development may detect variations in culture conditions. The aim of this study was to determine if toxins at levels that do not affect blastocyst formation in mouse embryos could be detected by MK analysis. DESIGN: Academic center animal study. MATERIALS AND METHODS: Cryopreserved 1-cell embryos from F1 hybrid mice were cultured with cumene hydroperoxide (CP; 0, 2, 4, 6, and 8 mM) and Triton X-100 (TX100; 0, 0.0008, 0.0012, 0.0016, 0.002%). Using the Embryoscope, time-lapse images were obtained every 20 minutes for 120 hours. Endpoints were timing of cell division and embryo development. The blastocyst rate (BR) was defined as the percentage of embryos that developed to the expanded blastocyst stage within 96 hours. RESULTS: BR was not affected for embryos cultured in CP at 2, 4, or 6 mm. In contrast, time of compaction (tMor) was significantly earlier in the control compared to treated embryos. For TX100, BR was not affected at 0.0008 or 0.0012%. In contrast, tMor was earlier in the control group compared to treated embryos. CONCLUSION: Our findings indicate that MK of mouse embryos is a sensitive method for quality control testing of in vitro culture media.
TABLE 1.
Control - CP 2 uM 4 uM 6 uM 8 uM Control - TX 0.0008% 0.0012% 0.0016% 0.002%
BR (%)
t4
t8
tMor
93.65.5 87.16.1 80.011.8 75.013.3 3.418.6* 93.87.8 85.57.7 90.06.9 68.112.1* 61.913.3*
29.02.5 29.76.2 33.05.7 32.65.0* 34.44.0* 28.02.7 30.93.2 30.62.5 33.56.0* 36.05.4*
40.23.5 44.07.7 46.66.0* 48.15.2* 48.15.2* 37.93.4 42.14.8* 41.23.2 43.54.6* 47.84.7*
52.84.5 56.86.4* 58.64.8* 59.65.0* 68.26.3* 50.74.6 56.65.1* 55.63.3* 60.45.9* 66.26.8*
* P<0.01.
S30
ASRM Abstracts
Compared to our previous work, MK sensitivity is similar to mouse embryo assay (MEA) using outbred mice. An MK algorithm should enhance assay sensitivity while providing results in half the time of the traditional MEA. Supported by: Mayo Clinic Dept OB/GYN Research Grant.
O-98 Monday, October 22, 2012 05:30 PM TIME LAPSE OBSERVATION OF EMBRYO DEVELOPMENT IDENTIFIES LATER STAGE MORPHOLOGY BASED PARAMETERS ASSOCIATED WITH BLASTOCYST QUALITY BUT NOT M. Rawlins,a CHROMOSOME CONSTITUTION. J. Stevens,a A. Janesch,a N. Treff,b W. B. Schoolcraft,a M. G. Katz-Jaffe.a aColorado Center for Reproductive Medicine, Lone Tree, CO; bReproductive Medicine Associates of New Jersey, Morristown, NJ. OBJECTIVE: Time lapse observation of embryo development allows for continual assessment during this dynamic process. The aim of this study was to examine morphology based parameters during embryo development in association with blastocyst quality and chromosome constitution. DESIGN: Research study. MATERIALS AND METHODS: A total of 53 oocytes were randomly selected from 35 infertile couples for time lapse observation (EmbryoScopeTM). All oocytes completed normal fertilization and underwent trophectoderm biopsy for chromosome analysis by quantitative PCR. Groups were classified by blastocyst quality: Group A¼expanded blastocyst at <110 hours after ICSI (n¼25); Group B¼slower blastocyst development with expansion completed R110 hours after ICSI (n¼28). RESULTS: There were no significant differences between the groups for pronuclear appearance, disappearance or size, exact time of early cleavage divisions, degree of blastomere asynchrony, and presence/absence of fragmentation. Conversely, significant differences were observed at later stages of embryonic development for timing of the fourth cleavage division (A¼53.06.2 vs. B¼58.39.8 hrs; P<0.05), progression to morula (A¼91.43.9 vs. B¼96.37.5 hrs; P<0.01), appearance of a blastocele cavity (A¼95.23.5 vs. B¼104.28.5 hrs; P<0.0001), and the presence of an inner cell mass (A¼99.73.5 vs. B¼112.78.6 hrs; P<0.0001). In relation to blastocyst chromosome constitution (euploid vs. aneuploid) no significant association was observed for any early or later stage time lapse morphology based parameters. CONCLUSION: Dynamic time lapse evaluation of embryonic development revealed significant later stage morphology based parameters that correlated with excellent blastocyst quality, although not with chromosome constitution. Data indicates slower euploid blastocysts exhibit poorer IVF outcomes. Ongoing time lapse studies may assist in the improvement of embryo selection techniques but would appear not able to replace embryo biopsy for chromosome analysis. Supported by: Unisense Fertilitech.
O-99 Monday, October 22, 2012 05:45 PM OVERNIGHT SURVIVAL OF THAWED AND TROPHECTODERM BIOPSIED BLASTOCYSTS. C. F. Boylan, M. P. Portmann, L. S. Morrison, S. M. Carney, G. Kovalevsky, R. F. Feinberg. Reproductive Associates of Delaware, Newark, DE. OBJECTIVE: To assess survival of blasts that were vitrified, thawed, TE biopsied and cultured overnight. DESIGN: Patients having two or more day 5 blasts vitrified between 2005 and 2011, who have opted to donate embryos via consent, were included in the study. Donor egg and prior PGS embryos were excluded from the study. MATERIALS AND METHODS: Oocytes were retrieved, placed in Fert Media (Sage), hyaluronidased after 2 to 3 hours, ICSI’ed 1 to 3 hours following hyaluronidase and placed into Cleav Media (Sage). All embryos were cultured individually. On day 3, embryos were placed into Blast Media (Sage). Untransferred blasts were vitrified for future use. Upon thaw, embryos were placed in 20%SPS Blast Media (Sage). Approx. four hours
N (# Thawed) Initial Survival Overnight Survival
56 53 (94.6%) 49 (92.5%)
Vol. 98, No. 3, Supplement, September 2012
O-94 Monday, October 22, 2012 04:30 PM FACTORS THAT AFFECT MINERAL OIL TOXICITY: ROLE OF OXYGEN AND PROTEIN SUPPLEMENT. D. E. Morbeck, J. R. Fredrickson, D. L. Walker. Obstetrics and Gynecology, Mayo Clinic, Rochester, MN. OBJECTIVE: Though mineral oil is commonly filtered, washed and bioassay tested, three lots from three different companies recently demonstrated toxicity in IVF labs and were recalled by the suppliers. Differences between supplier and clinical laboratory conditions and QC methods may explain why toxicity was not originally detected. The objective of this study was to compare 2 incubator conditions and different protein supplements on sensitivity to toxicity of these 3 lots of oil. DESIGN: Animal research study. MATERIALS AND METHODS: Zygotes were collected from superovulated, inbred mice per IACUC protocol. Experiment 1 tested incubator conditions. Embryos were cultured in HTF plus PVA in a CO2 Forma incubator with room air (20% O2) or a Planer BT-37 with 5% O2 medical grade air. Experiment 2 tested protein x oil interaction. Embryos were cultured in HTF with 5 mg/ml HSA or 10% SSS. Control oil (Fisher) was compared to 3 recalled lots of oil: L1, L2 and L3. Expanded blastocyst rate at 96 and 120 hours were the endpoints. RESULTS: Blastocyst rates (BR) in control media were not different at 96 hours in 5% O2 but were lower for L1, L2 and L3 in the 20% O2 incubator (P<0.05). At 120 hours, BR were lower for affected oils in both incubators. When HSA was added to the culture, BR was similar to control, whereas embryos cultured in SSS arrested and blastomeres lysed by the 8 cell stage in L2, had lower BR in L1 or were not affected in L3. Peroxide
Blastocyst Rates
96h
Control L1 L2 L3
120h
96h/20% O2
5% O2
20% O2
5% O2
20% O2
HSA
SSS
85.0 73.8 71.7 80.0
70.9 47.5* 7.5* 43.5*
73.3 44.4* 40.0* 30.0*
62.2 8.3* 0* 22.9*
76.9 89.4 60.0 65.0
86.7 36.7* 0* 80.0
* P<0.05.
FERTILITY & STERILITYÒ
testing indicated that L1 and L2 had elevated peroxide levels whereas L3 was similar to the control. CONCLUSION: Oil is the most variable product in the IVF laboratory and its toxicity continues to affect clinical outcomes. These results demonstrate interactions between oxygen and protein and illustrate the need for more sophisticated mineral oil screening. Supported by: OB/GYN Research Committee.
O-95 Monday, October 22, 2012 04:45 PM 12-HOUR PREIMPLANTATION GENETIC SCREENING (PGS) BY TARGETED SEQUENCING AND HIGHLY MULTIPLEX PCR WITH 5% ADO ON BENCHTOP SEQUENCERS. M. Hill, J. Baner, B. Zimmermann, A. Ryan, G. Gemelos, M. Rabinowitz. Natera, Inc., San Carlos, CA. OBJECTIVE: Demonstrate proof of concept for a rapid turnaround PGS sequencing technology that could be performed in-clinic in 12 hours using the MiSeq small-footprint push-button sequencer. DESIGN: Thirty single cell samples were isolated from a trisomy 21 (47,XX,+21; 10 replicate cells) and five karyotypically normal cell lines (two 46,XY and three 46,XX; four replicate cells each). Cell lines corresponding to parents of the test lines were also evaluated. MATERIALS AND METHODS: In 12 hours we performed multiplex PCR and sequencing of 1200 SNPs on chromosomes 1, 21, and X on an Illumina MiSeq with an average depth of read of 350 reads per SNP. The ploidy state of each chromosome was determined by a Maximum Likelihood estimator, comparing the observed read count of each allele against a theoretical model of ploidy hypotheses (monosomy, disomy, trisomy). The model combines a Monte Carlo simulation of the PCR noise processes with a binomial model to incorporate variations in read depth. Performance with fewer measured loci per chromosome was measured by randomly subsampling from the 1200-plex measurements. RESULTS: Among 90 distinct disomy, trisomy, or X chromosome calls made on 30 cells, accuracy was 100% [95% CI: 96.0% - 100%]. Furthermore, random resampling of the data to simulate fewer loci indicated that 100 loci per chromosome would be sufficient for >99% accuracy with our method. The allele dropout rate in SNPs known to be heterozygous was approximately 5%. To our knowledge, this is the lowest level of allele dropouts ever reported from a single cell in a highly multiplex reaction. CONCLUSION: This is the first demonstration of multiplex PCR amplification and sequencing on a push-button sequencer capable of the rapid turnaround required for PGS with Day 5 biopsy and Day 6 transfer. The accuracy of the statistical model and extremely low allele drop-out enables highly accurate ploidy calling from single cells.
O-96 Monday, October 22, 2012 05:00 PM VITRIFIED BLASTOCYSTS YIELD EQUIVALENT PREGNANCY RATES AS COMPARED TO FRESH BLASTOCYSTS. D. A. Kelk, E. L. Paganetti, M. P. Leondires, M. E. Moore, C. M. Murdock, S. S. Richlin. Reproductive Medicine Associates of Connecticut, Norwalk, CT. OBJECTIVE: Fresh embryos have historically yielded higher pregnancy rates than frozen embryos. For many programs, vitrification has afforded increased pregnancy and implantation rates over classical slow freeze protocols. This study seeks to evaluate if vitrified blastocyst pregnancy and implantation rates can match those attained with fresh blastocysts. DESIGN: A retrospective analysis of 399 fresh blastocyst and 115 vitrified blastocyst transfers in patients under 38 years of age. MATERIALS AND METHODS: All fresh IVF cycles from Jan 2010 through March 2012 for patients <38 years old with fresh blastocyst transfers were compared to vitrified blastocyst FET’s. Donor egg cycles were not included. Embryos were group cultured in 50ml drops of Global culture media in 5-well dishes in MINC incubators with a 6.5% CO2/5.0% O2 gas mixture. Blastocyst stage embryos were vitrified on Day 5 and/or Day 6 using Irvine Scientific Vit Kit and HSV straws. Statistical analysis of pregnancy and implantation rates were performed using Chi-square. Statistical significance was set at P<0.05. RESULTS: Mean maternal age, pregnancy and implantation rates are displayed in Table 1.
S29
Fresh vs Vitrified Blastocyst Transfers
Fresh Blastocyst ET
Vitrified Blastocyst ET
399 33.1 1.85 72.4% 60.2% 50.1%
115 32.1 1.91 76.5% 67.0% 50.5%
# of Transfers Mean Maternal Age Avg # Embryos Transferred Positive BhCG Rate Clinical Pregnancy Rate Implantation Rate
CONCLUSION: Pregnancy and implantation rates were not statistically different between fresh and vitrified blastocysts (P>0.05). These findings should be validated in a prospective randomized study. These data support that there is little, if any compromise to the viability of the embryo with vitrification. When timed appropriately, frozen embryo transfers allow for synchrony between the embryo and the uterine window of implantation. Programs should be comfortable in vitrifying blastocysts in freeze-all cycles, OHSS patients, PGD/PGS cases, donor egg recipients, and gestational carriers.
O-97 Monday, October 22, 2012 05:15 PM ADVANCES IN QUALITY CONTROL: MOUSE EMBRYO MORPHOKINETICS ARE SENSITIVE MARKERS OF IN VITRO STRESS. H. S. Wolff, J. R. Fredrickson, D. L. Walker, D. E. Morbeck. Reproductive Endocrinology and Infertility, Mayo Clinic, Rochester, MN. OBJECTIVE: Poor conditions for embryo culture have adverse effects on outcomes and can result in lower live birth rates. Morphokinetics (MK) of embryo development may detect variations in culture conditions. The aim of this study was to determine if toxins at levels that do not affect blastocyst formation in mouse embryos could be detected by MK analysis. DESIGN: Academic center animal study. MATERIALS AND METHODS: Cryopreserved 1-cell embryos from F1 hybrid mice were cultured with cumene hydroperoxide (CP; 0, 2, 4, 6, and 8 mM) and Triton X-100 (TX100; 0, 0.0008, 0.0012, 0.0016, 0.002%). Using the Embryoscope, time-lapse images were obtained every 20 minutes for 120 hours. Endpoints were timing of cell division and embryo development. The blastocyst rate (BR) was defined as the percentage of embryos that developed to the expanded blastocyst stage within 96 hours. RESULTS: BR was not affected for embryos cultured in CP at 2, 4, or 6 mm. In contrast, time of compaction (tMor) was significantly earlier in the control compared to treated embryos. For TX100, BR was not affected at 0.0008 or 0.0012%. In contrast, tMor was earlier in the control group compared to treated embryos. CONCLUSION: Our findings indicate that MK of mouse embryos is a sensitive method for quality control testing of in vitro culture media.
TABLE 1.
Control - CP 2 uM 4 uM 6 uM 8 uM Control - TX 0.0008% 0.0012% 0.0016% 0.002%
BR (%)
t4
t8
tMor
93.65.5 87.16.1 80.011.8 75.013.3 3.418.6* 93.87.8 85.57.7 90.06.9 68.112.1* 61.913.3*
29.02.5 29.76.2 33.05.7 32.65.0* 34.44.0* 28.02.7 30.93.2 30.62.5 33.56.0* 36.05.4*
40.23.5 44.07.7 46.66.0* 48.15.2* 48.15.2* 37.93.4 42.14.8* 41.23.2 43.54.6* 47.84.7*
52.84.5 56.86.4* 58.64.8* 59.65.0* 68.26.3* 50.74.6 56.65.1* 55.63.3* 60.45.9* 66.26.8*
* P<0.01.
S30
ASRM Abstracts
Compared to our previous work, MK sensitivity is similar to mouse embryo assay (MEA) using outbred mice. An MK algorithm should enhance assay sensitivity while providing results in half the time of the traditional MEA. Supported by: Mayo Clinic Dept OB/GYN Research Grant.
O-98 Monday, October 22, 2012 05:30 PM TIME LAPSE OBSERVATION OF EMBRYO DEVELOPMENT IDENTIFIES LATER STAGE MORPHOLOGY BASED PARAMETERS ASSOCIATED WITH BLASTOCYST QUALITY BUT NOT M. Rawlins,a CHROMOSOME CONSTITUTION. J. Stevens,a A. Janesch,a N. Treff,b W. B. Schoolcraft,a M. G. Katz-Jaffe.a aColorado Center for Reproductive Medicine, Lone Tree, CO; bReproductive Medicine Associates of New Jersey, Morristown, NJ. OBJECTIVE: Time lapse observation of embryo development allows for continual assessment during this dynamic process. The aim of this study was to examine morphology based parameters during embryo development in association with blastocyst quality and chromosome constitution. DESIGN: Research study. MATERIALS AND METHODS: A total of 53 oocytes were randomly selected from 35 infertile couples for time lapse observation (EmbryoScopeTM). All oocytes completed normal fertilization and underwent trophectoderm biopsy for chromosome analysis by quantitative PCR. Groups were classified by blastocyst quality: Group A¼expanded blastocyst at <110 hours after ICSI (n¼25); Group B¼slower blastocyst development with expansion completed R110 hours after ICSI (n¼28). RESULTS: There were no significant differences between the groups for pronuclear appearance, disappearance or size, exact time of early cleavage divisions, degree of blastomere asynchrony, and presence/absence of fragmentation. Conversely, significant differences were observed at later stages of embryonic development for timing of the fourth cleavage division (A¼53.06.2 vs. B¼58.39.8 hrs; P<0.05), progression to morula (A¼91.43.9 vs. B¼96.37.5 hrs; P<0.01), appearance of a blastocele cavity (A¼95.23.5 vs. B¼104.28.5 hrs; P<0.0001), and the presence of an inner cell mass (A¼99.73.5 vs. B¼112.78.6 hrs; P<0.0001). In relation to blastocyst chromosome constitution (euploid vs. aneuploid) no significant association was observed for any early or later stage time lapse morphology based parameters. CONCLUSION: Dynamic time lapse evaluation of embryonic development revealed significant later stage morphology based parameters that correlated with excellent blastocyst quality, although not with chromosome constitution. Data indicates slower euploid blastocysts exhibit poorer IVF outcomes. Ongoing time lapse studies may assist in the improvement of embryo selection techniques but would appear not able to replace embryo biopsy for chromosome analysis. Supported by: Unisense Fertilitech.
O-99 Monday, October 22, 2012 05:45 PM OVERNIGHT SURVIVAL OF THAWED AND TROPHECTODERM BIOPSIED BLASTOCYSTS. C. F. Boylan, M. P. Portmann, L. S. Morrison, S. M. Carney, G. Kovalevsky, R. F. Feinberg. Reproductive Associates of Delaware, Newark, DE. OBJECTIVE: To assess survival of blasts that were vitrified, thawed, TE biopsied and cultured overnight. DESIGN: Patients having two or more day 5 blasts vitrified between 2005 and 2011, who have opted to donate embryos via consent, were included in the study. Donor egg and prior PGS embryos were excluded from the study. MATERIALS AND METHODS: Oocytes were retrieved, placed in Fert Media (Sage), hyaluronidased after 2 to 3 hours, ICSI’ed 1 to 3 hours following hyaluronidase and placed into Cleav Media (Sage). All embryos were cultured individually. On day 3, embryos were placed into Blast Media (Sage). Untransferred blasts were vitrified for future use. Upon thaw, embryos were placed in 20%SPS Blast Media (Sage). Approx. four hours
N (# Thawed) Initial Survival Overnight Survival
56 53 (94.6%) 49 (92.5%)
Vol. 98, No. 3, Supplement, September 2012