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Comprehensive cytogenetic analysis of the human blastocyst stage
Monday, November 10, 2008 4:00 pm O-97 IMMUNOLOCALIZATION OF STEMNESS MARKERS IN TISSUE SECTIONS OF THE HUMAN PLACENTA. J. D. Lamb, T. Zdravkovic, M. J. Gormley, M. Kapidzic, O. Genbacev, S. J. Fisher. Dept. of ObGyn and Reproductive Sciences, University of California, San Francisco, San Francisco, CA; University of California, San Francisco, San Francisco, CA. OBJECTIVE: The trophoblast lineage is the first to differentiate during the early stages of embryonic development. Trophoblast stem (TS) cells have been identified in the mouse but have yet to be described in humans. Currently there are no reported markers of human placental stem/progenitor cells that distinguish them from their mature progeny. Our objective was to characterize expression of human embryonic stem cell (hESC) and TS cell markers in the human placenta across a wide spectrum of gestational ages using immunolocalization techniques. DESIGN: Longitudinal analysis of the expression of stem cell markers in tissue sections of human placenta. MATERIALS AND METHODS: A human placental tissue library was developed by fixing and embedding first and second trimester samples. Immunohistochemistry was performed on serial frozen sections across a wide spectrum of gestational ages using standard techniques. Stem cell antigens that were localized included markers of pluripotency (Oct4, SSEA4 and Gememin) and factors that are functionally linked to generation of the trophoblast lineage (Cdx2, hCG, and EOMES). Vimentin and Cytokeritin 7 expression were used to identify epithelial and mesenchymal derivatives respectively. RESULTS: A subset of chorionic villous cytotrophoblasts (CTBs) in tissue sections prepared from first trimester placentas co-expressed markers of both the hESC and TS lineages. Villous CTBs stained with antibodies that specifically reacted with Oct4, EOMES and Gememin. A small subset also co-expressed Cdx2, SSEA4, and Ki67, a marker of proliferation. Additional studies showed that expression of these markers was downregulated in the second trimester. Cells that made up anchoring villous columns also co-expressed markers of both lineages. Work to determine if invasive CTBs express these markers is on-going. A population of cells in the villous core expressed markers of stemness and stained for human chorionic gonadotropin (hCG), a marker of trophoblast differentiation. These cells also co-expressed markers of the hematopoetic/macrophage lineage (CD45 and CD14) and disappeared with advancing gestational age. CONCLUSIONS: The expression patterns of hESC and TS cell markers in the placenta suggest the possible presence of stem cells among the populations that were examined. This study identified candidate markers worthy of further exploration for use in isolation of various stem cell populations, with analysis of their functional roles another possibility. Supported by: NIH 5 R01 HD046744-04.
Monday, November 10, 2008 4:15 pm O-98 A NOVEL PROFILE OF CUMULUS CELL TRANSCRIPTS IS ASSOCIATED WITH OOCYTE ANEUPLOIDY. M. Katz-Jaffe, B. R. McCallie, N. R. Treff, K. A. Miller, R. T. Scott, W. B. Schoolcraft. Colorado Center for Reproductive Medicine, Lone Tree, CO; Reproductive Medicine Associates of New Jersey, Morristown. OBJECTIVE: Molecular cytogenetic studies have estimated that greater than 20% of human oocytes are aneuploid. Preimplantation genetic testing of biopsied polar bodies has allowed for oocyte aneuploidy screening. However, biopsy procedures are invasive to the oocyte with potential impact on outcome. Recently, it has been shown that oocyte developmental competence may be determined by specific cumulus cell (CC) gene expression, a non-invasive approach. This study therefore investigated the potential association between the CC transcriptome and oocyte aneuploidy. DESIGN: Genomic analysis of human CCs. MATERIALS AND METHODS: CCs were donated with patient consent immediately after oocyte retrieval. CCs were sharply trimmed, washed in PBS and resuspended in lysis buffer. Comprehensive chromosome aneuploidy screening of both biopsied polar bodies was determined after whole
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genome amplification and hybridization to the Affymetrix 250K SNP microarray. Upon identification of aneuploidy status, total RNA was isolated from CCs belonging to 6 individual euploid oocytes and 6 individual aneuploid oocytes. In vitro transcription was performed followed by hybridization to Codelink Whole Genome Human Bioarray (n¼12) (Applied Microrrays) and analysis with Genespring software (Agilent). RESULTS: Comprehensive human CC transcriptome analysis revealed expression of around 31,000 genes. Previously identified CC marker genes (n¼24) were verified on this data set including RBL2, CYP19, CCND2 and PTGS2. Two novel gene sets were identified with greater than 2-fold differential expression that separated CCs originating from euploid oocytes with those from aneuploid oocytes. The first set contained 234 genes that were downregulated in CCs belonging to aneuploid oocytes; mainly involved in cell cycle, cell communication, chromosome organization, transport and metabolism. The second set contained 179 genes that were upregulated in CCs belonging to aneuploid oocytes; primarily involved in signal transduction, transport, cell communication and differentiation. CONCLUSIONS: This study has shown that oocyte aneuploidy could be associated with the expression profile of a novel set of CC genes. This form of analysis may potentially allow for non-invasive selection of euploid human oocytes based on the respective specific CC transcriptome profile. Improvements to oocyte selection are anticipated to improve embryo developmental competence, implantation rates and the success of single embryo transfer. Supported by: None.
Monday, November 10, 2008 4:30 pm O-99 COMPREHENSIVE CYTOGENETIC ANALYSIS OF THE HUMAN BLASTOCYST STAGE. E. Fragouli, M. Katz-Jaffe, W. B. Schoolcraft, R. Ross, S. Munne, D. Wells. Reprogenetics UK, Oxford, United Kingdom; Colorado Center for Reproductive Medicine, Lone Tree, CO; La Jolla IVF, La Jolla, CA; Reprogenetics LLC, Livingston, NJ; Department of Obstetrics and Gynaecology, University of Oxford, Oxford, United Kingdom. OBJECTIVE: While much is known concerning the aneuploidy rates of cleavage stage embryos and fetuses in early pregnancy, the chromosomal status of the blastocyst stage has been little investigated. In order for an embryo to reach this stage, it must activate its own genome and successfully undertake the first cellular differentiation (trophectoderm- TE and inner cell massICM). Embryos that achieve these developmental milestones are considered to be of the highest quality, with superior implantation potential. For this reason culture to the blastocyst stage is favored by an increasing number of ART clinics. However, the capacity of blastocyst culture to eliminate aneuploid embryos remains poorly defined. We aimed to catalogue all chromosome errors present in good quality blastocysts using a comprehensive cytogenetic method. DESIGN: Investigation of all 46 chromosomes in human blastocysts using whole genome amplification and comparative genomic hybridization (CGH). MATERIALS AND METHODS: 187 blastocysts from 42 couples (mean maternal age: 36 years) were examined using CGH. Parallel FISH analysis of one or more cells from 12 blastocysts was also carried out. Additionally, the ICM and TE were separated from 10 embryos and tested to reveal any cytogenetic divergence between the two cell lineages. RESULTS: CGH was highly efficient, with results obtained from 96% of embryos. 59.4% of blastocysts were normal, while various anomalies were seen in the remaining 40.6%. CGH analysis of TE and ICM samples from the same blastocysts yielded identical results in all cases. A maternal age affect was observed; women >36 had 3x the aneuploidy rate of younger patients. Preimplantation genetic screening using 9-chromosome FISH would have failed to detect 18% of abnormal embryos. CONCLUSIONS: The aneuploidy rate was found to be significantly lower at the blastocyst stage, compared to the cleavage stage. However, most errors, including monosomy, imbalance affecting the larger chromosomes and complex aneuploidy can persist. In developmental terms, this suggests that this final stage of preimplantation development does not represent an absolute selective barrier to aneuploidy, and that most chromosomally abnormal embryos are not lost until after day-6, either at the time of implantation or shortly thereafter. The high aneuploidy rates observed suggest that chromosome screening at the blastocyst stage may be beneficial, particularly for women >36. Supported by: None.
Vol. 90, Suppl 1, September 2008
Monday, November 10, 2008 4:15 pm O-98 A NOVEL PROFILE OF CUMULUS CELL TRANSCRIPTS IS ASSOCIATED WITH OOCYTE ANEUPLOIDY. M. Katz-Jaffe, B. R. McCallie, N. R. Treff, K. A. Miller, R. T. Scott, W. B. Schoolcraft. Colorado Center for Reproductive Medicine, Lone Tree, CO; Reproductive Medicine Associates of New Jersey, Morristown. OBJECTIVE: Molecular cytogenetic studies have estimated that greater than 20% of human oocytes are aneuploid. Preimplantation genetic testing of biopsied polar bodies has allowed for oocyte aneuploidy screening. However, biopsy procedures are invasive to the oocyte with potential impact on outcome. Recently, it has been shown that oocyte developmental competence may be determined by specific cumulus cell (CC) gene expression, a non-invasive approach. This study therefore investigated the potential association between the CC transcriptome and oocyte aneuploidy. DESIGN: Genomic analysis of human CCs. MATERIALS AND METHODS: CCs were donated with patient consent immediately after oocyte retrieval. CCs were sharply trimmed, washed in PBS and resuspended in lysis buffer. Comprehensive chromosome aneuploidy screening of both biopsied polar bodies was determined after whole
S36
Abstracts
genome amplification and hybridization to the Affymetrix 250K SNP microarray. Upon identification of aneuploidy status, total RNA was isolated from CCs belonging to 6 individual euploid oocytes and 6 individual aneuploid oocytes. In vitro transcription was performed followed by hybridization to Codelink Whole Genome Human Bioarray (n¼12) (Applied Microrrays) and analysis with Genespring software (Agilent). RESULTS: Comprehensive human CC transcriptome analysis revealed expression of around 31,000 genes. Previously identified CC marker genes (n¼24) were verified on this data set including RBL2, CYP19, CCND2 and PTGS2. Two novel gene sets were identified with greater than 2-fold differential expression that separated CCs originating from euploid oocytes with those from aneuploid oocytes. The first set contained 234 genes that were downregulated in CCs belonging to aneuploid oocytes; mainly involved in cell cycle, cell communication, chromosome organization, transport and metabolism. The second set contained 179 genes that were upregulated in CCs belonging to aneuploid oocytes; primarily involved in signal transduction, transport, cell communication and differentiation. CONCLUSIONS: This study has shown that oocyte aneuploidy could be associated with the expression profile of a novel set of CC genes. This form of analysis may potentially allow for non-invasive selection of euploid human oocytes based on the respective specific CC transcriptome profile. Improvements to oocyte selection are anticipated to improve embryo developmental competence, implantation rates and the success of single embryo transfer. Supported by: None.
Monday, November 10, 2008 4:30 pm O-99 COMPREHENSIVE CYTOGENETIC ANALYSIS OF THE HUMAN BLASTOCYST STAGE. E. Fragouli, M. Katz-Jaffe, W. B. Schoolcraft, R. Ross, S. Munne, D. Wells. Reprogenetics UK, Oxford, United Kingdom; Colorado Center for Reproductive Medicine, Lone Tree, CO; La Jolla IVF, La Jolla, CA; Reprogenetics LLC, Livingston, NJ; Department of Obstetrics and Gynaecology, University of Oxford, Oxford, United Kingdom. OBJECTIVE: While much is known concerning the aneuploidy rates of cleavage stage embryos and fetuses in early pregnancy, the chromosomal status of the blastocyst stage has been little investigated. In order for an embryo to reach this stage, it must activate its own genome and successfully undertake the first cellular differentiation (trophectoderm- TE and inner cell massICM). Embryos that achieve these developmental milestones are considered to be of the highest quality, with superior implantation potential. For this reason culture to the blastocyst stage is favored by an increasing number of ART clinics. However, the capacity of blastocyst culture to eliminate aneuploid embryos remains poorly defined. We aimed to catalogue all chromosome errors present in good quality blastocysts using a comprehensive cytogenetic method. DESIGN: Investigation of all 46 chromosomes in human blastocysts using whole genome amplification and comparative genomic hybridization (CGH). MATERIALS AND METHODS: 187 blastocysts from 42 couples (mean maternal age: 36 years) were examined using CGH. Parallel FISH analysis of one or more cells from 12 blastocysts was also carried out. Additionally, the ICM and TE were separated from 10 embryos and tested to reveal any cytogenetic divergence between the two cell lineages. RESULTS: CGH was highly efficient, with results obtained from 96% of embryos. 59.4% of blastocysts were normal, while various anomalies were seen in the remaining 40.6%. CGH analysis of TE and ICM samples from the same blastocysts yielded identical results in all cases. A maternal age affect was observed; women >36 had 3x the aneuploidy rate of younger patients. Preimplantation genetic screening using 9-chromosome FISH would have failed to detect 18% of abnormal embryos. CONCLUSIONS: The aneuploidy rate was found to be significantly lower at the blastocyst stage, compared to the cleavage stage. However, most errors, including monosomy, imbalance affecting the larger chromosomes and complex aneuploidy can persist. In developmental terms, this suggests that this final stage of preimplantation development does not represent an absolute selective barrier to aneuploidy, and that most chromosomally abnormal embryos are not lost until after day-6, either at the time of implantation or shortly thereafter. The high aneuploidy rates observed suggest that chromosome screening at the blastocyst stage may be beneficial, particularly for women >36. Supported by: None.
Vol. 90, Suppl 1, September 2008