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Morphokinetic assessment of the early embryo development
O-206 Tuesday, October 18, 2011 05:00 PM PROSPECTIVE RANDOMIZED COMPARISON OF NON-CRYOPRESERVED AND VITRIFIED SIBLING OOCYTE FUNCTION AND RESULTING EMBRYO DEVELOPMENT. A. Monteiro da Rocha, M. Nichi, A. L. Rossi, P. Serafini, E. Motta, G. D. Smith. Ob/Gyn, Physiology, Urology, University of Michigan, Ann Arbor, MI; Huntington Center for Reproductive Medicine of Brazil, Sao Paulo, Brazil. OBJECTIVE: Vitrification of donor oocytes and establishment of a ‘‘Donor Oocyte Bank’’ has practical, quarantine/disease-testing, and economic advantages. Study aims were to compare oocyte function and embryo development in a pool of sibling oocytes used both as non-cryopreserved oocytes and following vitrification and warming. DESIGN: Prospective randomized clinical study MATERIALS AND METHODS: Couples undergoing ICSI elected to participate in this IRB-approved study. Sibling MII oocytes were randomly used in a fresh cycle (NoCryo) or vitrified (Vit). Oocytes were vitrified with dimethyl sulfoxide/ethylene glycol/sucrose with a Cryotip (Irvine Sci). After warming oocytes were observed for survival, incubated for 4h, and inseminated by ICSI. No/normal/abnormal fertilization and oocyte lysis were assessed. Zygotes were placed in G1 media (VitroLife) for growth and assessed for cleavage and embryo development. Statistical analyses were performed by paired Student’s t-test. RESULTS: Sixty-nine patients (age: 34 1; ave sem) with an average of 18 MII oocytes participated in this study. Post-warming survival was 83 3%. Following ICSI, no fertilization (14 2% vs 11 2%) or abnormal fertilization (1 0.2% vs 2 0.6%) were not significantly different between NoCryo and Vit, respectively. Normal fertilization was significantly higher in the NoCryo group (82 2%) compared to Vit (76 2%; P<0.05), due to increased incidence of post-ICSI oocyte lysis (4 1% vs 9 2%, respectively; P<0.01). Cleavage rate was similar between groups. Day 3 embryos had significantly more cells (6.4 0.1 vs 6.1 0.1; P<0.05) in NoCryo compared to Vit, respectively. Degree of embryo fragmentation was similar between groups. CONCLUSION: In an infertile population, vitrification of oocytes resulted in acceptable fertilization and embryo development, yet not equivalent to non-cryopreserved oocytes. Knowledge of these oocyte functional and embryo developmental differences will assist in future planning for efficient donor oocyte cryo-banking.
O-207 Tuesday, October 18, 2011 05:15 PM MORPHOKINETIC ASSESSMENT OF THE EARLY EMBRYO DEVELOPMENT. L. Escrich, N. Grau, C. Albert, P. Gamiz, J. L. Romero, M. J. Escriba. Instituto Valenciano de Infertilidad, University of Valencia, Valencia, Spain. OBJECTIVE: To analyze the morphokinetics of the initial cleavages of in vitro embryos which would shed light on the early development of human embryos and to explore the significance of these events in the development of in vitro embryos into blastocysts. DESIGN: We obtained images of 187 embryos cultured. Blastomere diameter and volume of them were measured to determine if there are differences between the rate of embryos reaching the blastocyst stage. MATERIALS AND METHODS: This is a study monitoring a total of 187 embryos from 28 couples enrolled in our ovum donation programme and undergoing ICSI for the first time. Embryos were cultured for at least 120 hrs and images of 6 different focal planes were automatically obtained every 15 minutes for each embryo. Blastomere diameter Q was measured retrospectively in 2-cell embryos and their volume (4/3 r3) was calculated after acquiring a spherical shape. The following were then defined: 1) volume of large (LB) and small (SB) blastomere, 2) blastomere symmetry (LB/SB) 3) order of cleavage according to volume and, 4) ability to progress to the blastocyst stage. Statistical significance of variables was calculated using the Student’s t test. RESULTS: 120 blastocysts (64.2%) were obtained from 187 embryos. Significant variations were observed in the blastomere volume of each embryo (LB: 431.7 1.8mm3; 95CI:405.6-457.8mm3 vs. SB:275.6 97.2mm3; 95CI:261.6-289.6mm3; P<0.01). The SB was the first to cleave in 81.3% of embryos (n ¼ 152), whereas the LB was first in 35 embryos (18.7%; P<0.05), providing different symmetry ratios (1.7 0.6 vs. 1.4 0.4, respectively; P¼0.01) but comparable blastocyst rates (62.5% and 71.4%; P¼0.32) CONCLUSION: Time-lapse observations provide a novel morphometric perspective of 2-cell embryos and reveal previously undiscovered aspects
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Abstracts
of early human embryo development; namely that there is a small blastomere that usually cleaves first but which is unrelated to in vitro embryo competence.
O-208 Tuesday, October 18, 2011 05:30 PM MATERNAL SMOKING HASTENS TELOMERE SHORTENING IN NEONATAL UMBILICAL CORD BLOOD LEUKOCYTES. J. Rodriguez, E. McQueen, K. Downes, S. Plosker, D. Keefe, C. Silva. Obstetrics and Gynecology - Division of Reproductive Endocrinology and Infertility, University of South Florida, Tampa, FL; Obstetrics and Gynecology, NYU Langone Medical Center, New York, NY. OBJECTIVE: Smoking during pregnancy is associated with adverse obstetrical outcomes. Smoking exerts its toxicity in part by generating reactive oxygen species(ROS). Telomeres preserve genomic integrity, and are susceptible to oxidative stress. Telomere length(TL) is a marker of cellular damage, and shorter telomeres are present in placentas of patients with pre-eclampsia and diabetes. We evaluated telomere attrition in leukocytes from human umbilical cord blood(UCB) in patients exposed to tobacco during pregnancy. DESIGN: Prospective cohort. MATERIALS AND METHODS: UCB was obtained after expulsion of the placenta from subjects with and without exposure to tobacco during pregnancy. Exposure was defined as: 1)current first hand; 2)current second hand; 3)past first hand; and 4)past second hand. Cotinine levels were assessed with an Elisa assay, and used to confirm and quantify cigarette exposure. UCB leukocytes were extracted using Ficoll separation. TL was assessed by QPCR using the Cawthon method. Linear regression analysis was used to model mean TL as a function of tobacco exposure, controlling for maternal and gestational age, obstetrical and medical conditions. RESULTS: Linear regression analysis showed that the adjusted mean cord blood TL was significantly different between patients with tobacco exposure and controls (beta ¼ 705.7, P¼0.034). Differential cord blood TL was detected according to type of smoking exposure. Baseline characteristics were comparable in terms of race, mean maternal age, and mean gestational age. CONCLUSION: Maternal smoking exposure negatively impacts on neonatal TL. In addition, TL was negatively impacted in neonates of past firsthand smokers, despite maternal smoking cessation during pregnancy. These findings support previous in vitro studies that show that tobacco exposure negatively impacts on TL. Further studies on the association between shorter TL in UCB and neonatal outcomes may determine if TL in these tissues could be used as marker of neonatal health. Supported by: Departmental
O-209 Tuesday, October 18, 2011 05:45 PM THE PROGRESS OF CHROMOSOME ABNORMALITIES FROM MEIOSIS TO THE BLASTOCYST STAGE. E. Fragouli, S. Alfarawati, M. Konstantinidis, S. Jaroudi, D. Wells. Reprogenetics UK, Oxford, Oxfordshire, United Kingdom; Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, Oxfordshire, United Kingdom. OBJECTIVE: Despite the clinical importance of aneuploidy, little is known concerning the ultimate fates of specific types of cytogenetic anomaly. This study aimed to shed light on the genesis, progression and survival of different forms of aneuploidy during transition from gamete to blastocyst. The most appropriate stage for preimplantation genetic screening (PGS) was also considered. DESIGN: Comprehensive cytogenetic evaluation of oocytes/embryos. MATERIALS AND METHODS: A total of 2,055 samples (polar bodies, blastomeres and trophectoderm biopsies) derived from 319 couples were examined using microarray-CGH. RESULTS: Progression from day-0 to 5 was associated with an increase in the proportion of embryos that were normal or affected by a single chromosome anomaly (65% to 76%; P<0.001). Concurrently, a decrease in the incidence of embryos with >3 abnormal chromosomes was observed (14% to 4%; P<0.001). Although the trend from day-0 to day-5 was towards reduced abnormalities, a transient increase in aneuploidy was seen at the cleavage stage, the incidence of highly abnormal embryos doubling, matched by a decline in the proportion of normal embryos. CONCLUSION: This study confirms that mitotic errors occur frequently at the cleavage stage, but that many of the affected embryos either arrest or succeed in eliminating their abnormal cells. No selection against
Vol. 96., No. 3, Supplement, September 2011
O-207 Tuesday, October 18, 2011 05:15 PM MORPHOKINETIC ASSESSMENT OF THE EARLY EMBRYO DEVELOPMENT. L. Escrich, N. Grau, C. Albert, P. Gamiz, J. L. Romero, M. J. Escriba. Instituto Valenciano de Infertilidad, University of Valencia, Valencia, Spain. OBJECTIVE: To analyze the morphokinetics of the initial cleavages of in vitro embryos which would shed light on the early development of human embryos and to explore the significance of these events in the development of in vitro embryos into blastocysts. DESIGN: We obtained images of 187 embryos cultured. Blastomere diameter and volume of them were measured to determine if there are differences between the rate of embryos reaching the blastocyst stage. MATERIALS AND METHODS: This is a study monitoring a total of 187 embryos from 28 couples enrolled in our ovum donation programme and undergoing ICSI for the first time. Embryos were cultured for at least 120 hrs and images of 6 different focal planes were automatically obtained every 15 minutes for each embryo. Blastomere diameter Q was measured retrospectively in 2-cell embryos and their volume (4/3 r3) was calculated after acquiring a spherical shape. The following were then defined: 1) volume of large (LB) and small (SB) blastomere, 2) blastomere symmetry (LB/SB) 3) order of cleavage according to volume and, 4) ability to progress to the blastocyst stage. Statistical significance of variables was calculated using the Student’s t test. RESULTS: 120 blastocysts (64.2%) were obtained from 187 embryos. Significant variations were observed in the blastomere volume of each embryo (LB: 431.7 1.8mm3; 95CI:405.6-457.8mm3 vs. SB:275.6 97.2mm3; 95CI:261.6-289.6mm3; P<0.01). The SB was the first to cleave in 81.3% of embryos (n ¼ 152), whereas the LB was first in 35 embryos (18.7%; P<0.05), providing different symmetry ratios (1.7 0.6 vs. 1.4 0.4, respectively; P¼0.01) but comparable blastocyst rates (62.5% and 71.4%; P¼0.32) CONCLUSION: Time-lapse observations provide a novel morphometric perspective of 2-cell embryos and reveal previously undiscovered aspects
S62
Abstracts
of early human embryo development; namely that there is a small blastomere that usually cleaves first but which is unrelated to in vitro embryo competence.
O-208 Tuesday, October 18, 2011 05:30 PM MATERNAL SMOKING HASTENS TELOMERE SHORTENING IN NEONATAL UMBILICAL CORD BLOOD LEUKOCYTES. J. Rodriguez, E. McQueen, K. Downes, S. Plosker, D. Keefe, C. Silva. Obstetrics and Gynecology - Division of Reproductive Endocrinology and Infertility, University of South Florida, Tampa, FL; Obstetrics and Gynecology, NYU Langone Medical Center, New York, NY. OBJECTIVE: Smoking during pregnancy is associated with adverse obstetrical outcomes. Smoking exerts its toxicity in part by generating reactive oxygen species(ROS). Telomeres preserve genomic integrity, and are susceptible to oxidative stress. Telomere length(TL) is a marker of cellular damage, and shorter telomeres are present in placentas of patients with pre-eclampsia and diabetes. We evaluated telomere attrition in leukocytes from human umbilical cord blood(UCB) in patients exposed to tobacco during pregnancy. DESIGN: Prospective cohort. MATERIALS AND METHODS: UCB was obtained after expulsion of the placenta from subjects with and without exposure to tobacco during pregnancy. Exposure was defined as: 1)current first hand; 2)current second hand; 3)past first hand; and 4)past second hand. Cotinine levels were assessed with an Elisa assay, and used to confirm and quantify cigarette exposure. UCB leukocytes were extracted using Ficoll separation. TL was assessed by QPCR using the Cawthon method. Linear regression analysis was used to model mean TL as a function of tobacco exposure, controlling for maternal and gestational age, obstetrical and medical conditions. RESULTS: Linear regression analysis showed that the adjusted mean cord blood TL was significantly different between patients with tobacco exposure and controls (beta ¼ 705.7, P¼0.034). Differential cord blood TL was detected according to type of smoking exposure. Baseline characteristics were comparable in terms of race, mean maternal age, and mean gestational age. CONCLUSION: Maternal smoking exposure negatively impacts on neonatal TL. In addition, TL was negatively impacted in neonates of past firsthand smokers, despite maternal smoking cessation during pregnancy. These findings support previous in vitro studies that show that tobacco exposure negatively impacts on TL. Further studies on the association between shorter TL in UCB and neonatal outcomes may determine if TL in these tissues could be used as marker of neonatal health. Supported by: Departmental
O-209 Tuesday, October 18, 2011 05:45 PM THE PROGRESS OF CHROMOSOME ABNORMALITIES FROM MEIOSIS TO THE BLASTOCYST STAGE. E. Fragouli, S. Alfarawati, M. Konstantinidis, S. Jaroudi, D. Wells. Reprogenetics UK, Oxford, Oxfordshire, United Kingdom; Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, Oxfordshire, United Kingdom. OBJECTIVE: Despite the clinical importance of aneuploidy, little is known concerning the ultimate fates of specific types of cytogenetic anomaly. This study aimed to shed light on the genesis, progression and survival of different forms of aneuploidy during transition from gamete to blastocyst. The most appropriate stage for preimplantation genetic screening (PGS) was also considered. DESIGN: Comprehensive cytogenetic evaluation of oocytes/embryos. MATERIALS AND METHODS: A total of 2,055 samples (polar bodies, blastomeres and trophectoderm biopsies) derived from 319 couples were examined using microarray-CGH. RESULTS: Progression from day-0 to 5 was associated with an increase in the proportion of embryos that were normal or affected by a single chromosome anomaly (65% to 76%; P<0.001). Concurrently, a decrease in the incidence of embryos with >3 abnormal chromosomes was observed (14% to 4%; P<0.001). Although the trend from day-0 to day-5 was towards reduced abnormalities, a transient increase in aneuploidy was seen at the cleavage stage, the incidence of highly abnormal embryos doubling, matched by a decline in the proportion of normal embryos. CONCLUSION: This study confirms that mitotic errors occur frequently at the cleavage stage, but that many of the affected embryos either arrest or succeed in eliminating their abnormal cells. No selection against
Vol. 96., No. 3, Supplement, September 2011