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Generation and characterization of disease-specific human embryonic stem cells from genetically abnormal embryos
O-99 Monday, October 25, 2010 06:00 PM IMPACT OF EXOGENOUS GONADOTROPIN STIMULATION ON FOLLICULAR FLUID CYTOKINE PROFILES. E. N. Baskind, V. Sharma, N. Orsi, S. Barber. The Leeds Centre for Reproductive Medicine, Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom; Perinatal Research Group, Leeds Institute of Molecular Medicine, Leeds, United Kingdom; Department of Statistics, University of Leeds, Leeds, United Kingdom. OBJECTIVE: To assess the impact of gonadotrophin (Gn) stimulation in in-vitro fertilization (IVF) cycles by comparing the cytokine profile in follicular fluid (FF) of natural cycles (NC) with that of individually aspirated follicles following controlled ovarian hyperstimulation (COH). DESIGN: Observational clinical study. MATERIALS AND METHODS: Women aged <39 years, with normal BMI, ovarian reserve and pelvis undergoing NC IVF or ICSI (n¼10) were recruited alongside matched women undergoing conventional IVF with/ without intracytoplasmic sperm injection (ICSI) after COH (n¼10) for male factor or unexplained subfertility. FF from dominant follicle in NC and clean catch FF from individually aspirated follicles yielding oocytes and best embryos for transfer after COH was analysed by fluid-phase multiplex immunoassay for 41 cytokines. Demographic/cycle data were compared using Wilcoxon’s test and binomial test of proportions; while correlations between cytokines were computed using Kendall’s tau (Holm’s correction applied for multiple testing). RESULTS: When comparing patient demographics and cycle data (including outcome), there were no significant differences between COH and NC groups. However, median cytokine levels were higher in the NC group for 33/41 cytokines (p<0.001). Specifically leukaemia inhibitory factor (LIF), monocyte chemotactic protein (MCP)-3, macrophage-colony stimulating factor (M-CSF), stromal cell-derived factor (SDF)-1a and tumour necrosis factor (TNF)-b were significantly higher in NC-FF when compared to that in FF of best embryo generating COH follicles. Furthermore correlations between cytokines in the NC group were significantly stronger than those for the COH group. CONCLUSION: These findings suggest that Gn stimulation perturbs intra-follicular cytokine networks; overall cytokine levels as well as their interrelationships may be significantly altered and this may explain the alteration in the developmental competence of oocytes after COH.
PROCEDURES AND TECHNIQUES-LABORATORY: ART
activation status, and aberrant early growth could be assessed in these hESCs. Furthermore, the colony cells could be differentiated into a variety of cell types, including the tissues most affected by the conditions. CONCLUSION: Our detailed analysis showed that we could establish hESCs carrying specific genetic abnormalities and utilize them as disease models. Those hESCs will provide new tools to study diseases as well as to develop new therapeutic approaches.
O-101 Monday, October 25, 2010 04:30 PM ANALYSIS OF mRNA IN HUMAN POLAR BODIES. P. C. Klatsky, G. M. Wessel, J. Robins, J. Wittmyer, S. A. Carson. Center for Reproduction & Infertility, Alpert Medical School of Brown University, Women & Infants Hospital, Providence, RI; Molecular Biology, Cell Biology, & Biochemistry, Brown University, Providence, RI. OBJECTIVE: To detect the presence of and measure mRNA in the human polar body. DESIGN: Descriptive Lab study. MATERIALS AND METHODS: Immature human oocytes retrieved from patients undergoing assisted reproduction with planned intracytoplasmic sperm injection (ICSI) were donated for research. GV and MI oocytes were cultured overnight in IVM media and inspected the following day for polar body extrusion. If an oocyte completed meiosis I the following day, it was eligible for inclusion in the study. Polar bodies were removed by partial zonal dissection, aspirated into a micropipette and transferred to a lysis solution containing DNase. Sibling oocytes were similarly transferred to a separate tube with the same reagents and processed identically. The cDNA was reverse transcribed using Poly T primers and random hexamers from the cell lysate in a single step reaction. The cDNA was pre-amplified using 12 specific candidate transcripts or whole transcriptome amplification. Candidate gene expression was quantified in triplicate by qPCR. RESULTS: RNA transcripts were quantified in oocytes for all 12 candidate genes, however detection in the majority of polar body samples was limited to six of these genes: bcl2l10, ddx4, dppa3, padi6, dicer and odc1. Individual RNA transcripts were more likely to be detected in a polar body if its gene was highly expressed in the sibling oocyte. Similar results were found after whole transcriptome amplification of RNA from a single oocyte and polar body. CONCLUSION: This is the first report to detect the presence of mRNA in a human polar body. The presence of mRNA in the polar body may reflect the abundance of gene expression in its parent/sibling oocyte. Supported by: Brown University Provost seed grant.
O-100 Monday, October 25, 2010 04:15 PM GENERATION AND CHARACTERIZATION OF DISEASESPECIFIC HUMAN EMBRYONIC STEM CELLS FROM GENETICALLY ABNORMAL EMBRYOS. C. Hansis, C. E. Rice, R. Lehmann, J. A. Grifo. Obstetrics and Gynecology/REI, NYU School of Medicine, New York, NY; Cell Biology (Skirball), NYU School of Medicine, New York, NY. OBJECTIVE: Since many frequent, fatal, and incurable diseases do not have an appropriate disease model for therapeutic research and drug discovery, with IRB approval we generated novel disease-specific human embryonic stem cells (hESCs) with new efficient protocols and performed in-depth characterization of their molecular and cellular features. DESIGN: Experimental. MATERIALS AND METHODS: Zona-free human blastocysts (n¼42) previously assessed by preimplantation genetic diagnosis (PGD) for genetic conditions were transferred onto feeder cells and cultured in DMEM-based media. Differentiation of hESCs was achieved by colony overgrowth or embryoid body formation. Embryonic and control cells were subjected to marker gene, protein, and chromosome analysis for pluripotency, differentiation, and mutation examination by reverse transcription PCR, immunofluorescence, and fluorescence in situ hybridization (FISH), respectively. RESULTS: Colonies (n¼17; 40.5% of transferred blastocysts) could be established and maintained which showed the typical morphological features of hESCs such as compact colony formation. Colonies were derived from affected embryos (one each of cystic fibrosis, Tay-Sachs disease, Zellweger syndrome, trisomy 16, trisomy X, 18, 21, 22 and two each of alpha-thalassemia X-linked mental retardation syndrome, unbalanced translocation long arm of chromosomes 8, 15), from embryos tested inconclusively (one each of cystic fibrosis and BRCA1 and three of Tay-Sachs disease), and from three normal control embryos. Genetic abnormalities, gender, X chromosome
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Abstracts
O-102 Monday, October 25, 2010 04:45 PM EFFECT OF ARTIFICIAL COLLAPSE AND EQUILIBRATION TIMES ON SURVIVAL OF HUMAN BLASTOCYSTS FOLLOWING VITRIFICATION USING A CLOSED DOUBLE STRAW SYSTEM. C. A. Guerrero, O. Perez, J. M. D. Goldstein, S. Chantilis, K. Lee, D. Hammitt. ARTS Program, Texas Health Presbyterian Hospital Dallas, Dallas, TX; ARTS Program, Texas Health Presbyterian Hospital Plano, Plano, TX; ARTS Program, Texas Health Harris Methodist Fort Worth, Fort Worth, TX. OBJECTIVE: The objective of this study was to evaluate the effect of artificial collapse and varying equilibration times prior to vitrification of blastocysts on survival rates post-warming. DESIGN: Blastocysts were randomly allocated into artificial collapse or no collapse with varying equilibration times (2, 5, 10 and 15 minutes) prior to vitrification. Survival rates post-warming were assessed after overnight culture. End points were analyzed using Fisher’s exact test. MATERIALS AND METHODS: Late day 6 expanded and hatching blastocysts (n¼208) not eligible for cryopreservation were used in this study. Artificial collapse was accomplished by aspirating the blastocoelic fluid using an ICSI needle. Vitrification was performed using a closed double-straw device after two-step loading of cryoprotectants. Blastocysts were suspended in equilibration medium containing 7.5% (v/v) DMSO + 7.5% ethylene glycol (EG) for either 2, 5, 10 or 15 min, then transferred to vitrification medium containing 15% (v/v) DMSO + 15% EG + 0.5 M sucrose for < 90 seconds. Warming and removal of cryoprotectants was performed in a two-step sucrose protocol. RESULTS: There was a significantly higher survival of artificially collapsed blastocysts as compared to intact blastocysts for all equilibration times
Vol. 94., No. 4, Supplement, September 2010
PROCEDURES AND TECHNIQUES-LABORATORY: ART
activation status, and aberrant early growth could be assessed in these hESCs. Furthermore, the colony cells could be differentiated into a variety of cell types, including the tissues most affected by the conditions. CONCLUSION: Our detailed analysis showed that we could establish hESCs carrying specific genetic abnormalities and utilize them as disease models. Those hESCs will provide new tools to study diseases as well as to develop new therapeutic approaches.
O-101 Monday, October 25, 2010 04:30 PM ANALYSIS OF mRNA IN HUMAN POLAR BODIES. P. C. Klatsky, G. M. Wessel, J. Robins, J. Wittmyer, S. A. Carson. Center for Reproduction & Infertility, Alpert Medical School of Brown University, Women & Infants Hospital, Providence, RI; Molecular Biology, Cell Biology, & Biochemistry, Brown University, Providence, RI. OBJECTIVE: To detect the presence of and measure mRNA in the human polar body. DESIGN: Descriptive Lab study. MATERIALS AND METHODS: Immature human oocytes retrieved from patients undergoing assisted reproduction with planned intracytoplasmic sperm injection (ICSI) were donated for research. GV and MI oocytes were cultured overnight in IVM media and inspected the following day for polar body extrusion. If an oocyte completed meiosis I the following day, it was eligible for inclusion in the study. Polar bodies were removed by partial zonal dissection, aspirated into a micropipette and transferred to a lysis solution containing DNase. Sibling oocytes were similarly transferred to a separate tube with the same reagents and processed identically. The cDNA was reverse transcribed using Poly T primers and random hexamers from the cell lysate in a single step reaction. The cDNA was pre-amplified using 12 specific candidate transcripts or whole transcriptome amplification. Candidate gene expression was quantified in triplicate by qPCR. RESULTS: RNA transcripts were quantified in oocytes for all 12 candidate genes, however detection in the majority of polar body samples was limited to six of these genes: bcl2l10, ddx4, dppa3, padi6, dicer and odc1. Individual RNA transcripts were more likely to be detected in a polar body if its gene was highly expressed in the sibling oocyte. Similar results were found after whole transcriptome amplification of RNA from a single oocyte and polar body. CONCLUSION: This is the first report to detect the presence of mRNA in a human polar body. The presence of mRNA in the polar body may reflect the abundance of gene expression in its parent/sibling oocyte. Supported by: Brown University Provost seed grant.
O-100 Monday, October 25, 2010 04:15 PM GENERATION AND CHARACTERIZATION OF DISEASESPECIFIC HUMAN EMBRYONIC STEM CELLS FROM GENETICALLY ABNORMAL EMBRYOS. C. Hansis, C. E. Rice, R. Lehmann, J. A. Grifo. Obstetrics and Gynecology/REI, NYU School of Medicine, New York, NY; Cell Biology (Skirball), NYU School of Medicine, New York, NY. OBJECTIVE: Since many frequent, fatal, and incurable diseases do not have an appropriate disease model for therapeutic research and drug discovery, with IRB approval we generated novel disease-specific human embryonic stem cells (hESCs) with new efficient protocols and performed in-depth characterization of their molecular and cellular features. DESIGN: Experimental. MATERIALS AND METHODS: Zona-free human blastocysts (n¼42) previously assessed by preimplantation genetic diagnosis (PGD) for genetic conditions were transferred onto feeder cells and cultured in DMEM-based media. Differentiation of hESCs was achieved by colony overgrowth or embryoid body formation. Embryonic and control cells were subjected to marker gene, protein, and chromosome analysis for pluripotency, differentiation, and mutation examination by reverse transcription PCR, immunofluorescence, and fluorescence in situ hybridization (FISH), respectively. RESULTS: Colonies (n¼17; 40.5% of transferred blastocysts) could be established and maintained which showed the typical morphological features of hESCs such as compact colony formation. Colonies were derived from affected embryos (one each of cystic fibrosis, Tay-Sachs disease, Zellweger syndrome, trisomy 16, trisomy X, 18, 21, 22 and two each of alpha-thalassemia X-linked mental retardation syndrome, unbalanced translocation long arm of chromosomes 8, 15), from embryos tested inconclusively (one each of cystic fibrosis and BRCA1 and three of Tay-Sachs disease), and from three normal control embryos. Genetic abnormalities, gender, X chromosome
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Abstracts
O-102 Monday, October 25, 2010 04:45 PM EFFECT OF ARTIFICIAL COLLAPSE AND EQUILIBRATION TIMES ON SURVIVAL OF HUMAN BLASTOCYSTS FOLLOWING VITRIFICATION USING A CLOSED DOUBLE STRAW SYSTEM. C. A. Guerrero, O. Perez, J. M. D. Goldstein, S. Chantilis, K. Lee, D. Hammitt. ARTS Program, Texas Health Presbyterian Hospital Dallas, Dallas, TX; ARTS Program, Texas Health Presbyterian Hospital Plano, Plano, TX; ARTS Program, Texas Health Harris Methodist Fort Worth, Fort Worth, TX. OBJECTIVE: The objective of this study was to evaluate the effect of artificial collapse and varying equilibration times prior to vitrification of blastocysts on survival rates post-warming. DESIGN: Blastocysts were randomly allocated into artificial collapse or no collapse with varying equilibration times (2, 5, 10 and 15 minutes) prior to vitrification. Survival rates post-warming were assessed after overnight culture. End points were analyzed using Fisher’s exact test. MATERIALS AND METHODS: Late day 6 expanded and hatching blastocysts (n¼208) not eligible for cryopreservation were used in this study. Artificial collapse was accomplished by aspirating the blastocoelic fluid using an ICSI needle. Vitrification was performed using a closed double-straw device after two-step loading of cryoprotectants. Blastocysts were suspended in equilibration medium containing 7.5% (v/v) DMSO + 7.5% ethylene glycol (EG) for either 2, 5, 10 or 15 min, then transferred to vitrification medium containing 15% (v/v) DMSO + 15% EG + 0.5 M sucrose for < 90 seconds. Warming and removal of cryoprotectants was performed in a two-step sucrose protocol. RESULTS: There was a significantly higher survival of artificially collapsed blastocysts as compared to intact blastocysts for all equilibration times
Vol. 94., No. 4, Supplement, September 2010