- Email: [email protected]
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are crucial in order to create psychological supports and interventions aimed at assisting families with the complexities of third party reproduction. Supported by: None
PREIMPLANTATION GENETIC DIAGNOSIS SPECIAL INTEREST GROUP Tuesday, October 24, 2006 3:30 pm O-134 RELATIONSHIP BETWEEN EMBRYONIC SECRETOME AND CHROMOSOMAL ABNORMALITIES IN HUMAN IVF. M. G. KatzJaffe, J. Stevens, W. G. Kearns, D. K. Gardner, W. B. Schoolcraft. Colorado Center for Reproductive Medicine, Englewood, CO; Shady Grove Center for Preimplantation Genetics, Rockville, MD. OBJECTIVE: Preimplantation genetic diagnosis (PGD) has become an established and successful IVF procedure screening for specific chromosomal abnormalities in human embryos. PGD involves the biopsy of polar bodies or embryonic cells for genetic analysis. Though successful, biopsy procedures are invasive to the embryo, possibly compromising development, and time consuming for the embryology laboratory. With recent advances in the field of proteomics it has become possible to analyze the proteins produced and secreted by human embryos into the surrounding medium (secretome). The objective of this study was to investigate whether embryos with chromosomal abnormalities could be distinguished from normal embryos by their respective secretomes DESIGN: Prospective Study MATERIALS AND METHODS: Cryopreserved day 1 embryos and aneuploid human blastocysts from PGD cycles were donated with consent for research. Day 1 embryos were thawed and subsequently cultured in sequential media (G1/G2) supplemented with recombinant human albumin at 37oC, 5% O2 & 6% CO2 up to the hatching blastocyst stage. Trophectoderm biopsy was performed removing up to 12 cells for FISH (chromosomes 13, 14, 15, 16, 17, 18, 21, 22, X and Y). Aneuploid blastocysts (abnormalities involving any of the analyzed chromosomes) from PGD cycles were cultured under these same conditions for 24 hrs. Drops of media cultured in the same dishes without blastocysts were used as controls. All spent media (aneuploid n⫽18, normal n⫽6) and controls (n⫽12) were subsequently processed and analyzed by time-of-flight mass spectrometry. RESULTS: The secretome profiles from hatching blastocysts identified as normal for the 10 chromosomes tested exhibited notably different secretome profiles to hatching blastocysts identified as aneuploid. Significantly, five proteins/biomarkers were observed to be in higher abundance in the secretome of hatching aneuploid blastocysts (P ⬍ 0.05). In addition, aneuploid embryos arrested in culture prior to the blastocyst stage also showed higher abundance of several proteins/biomarkers in their secretome profiles compared with hatching aneuploid blastocysts (P ⬍ 0.05). CONCLUSION: This is the first study to describe the differences in the secretome between human blastocysts identified as either normal or aneuploid for 10 chromosomes tested. This study has also shown that degenerating aneuploid embryos exhibit a significantly different secretome profile to hatching aneuploid blastocysts. This approach of protein analysis will further increase our understanding of human blastocyst physiology and its association with chromosomal composition. This work could lead to the development of a non-invasive assay to identify human embryos with chromosomal abnormalities. Supported by: None.
Tuesday, October 24, 2006 3:45 pm O-135 THE EFFICACY OF INTERPHASE-NUCLEAR-CONVERSION INDUCED BY PARTHENOGENETIC ACTIVATED FRESH MOUSE OOCYTES. L. Jiawei Sr., F. Cong, X. Yanwen, Z. Canquan, L. Tao, Z. Guanglun. the First Affiliated Hospital of Sun Yat-Sen Univ, Guangzhou, China.
FERTILITY & STERILITY威
OBJECTIVE: The protocol of interphase-nuclear-conversion induced by fusion of a blastomere with a mouse zygote has been set up as a method of preimplantation genetic diagnosis(PGD) to detect the chromosomal imbalance in the embryos of translocation carriers. But it was found that metaphase chromosome could not be obtained in some cells. The reasons maybe the fertilization of a mouse oocyte with an abnormal sperm, or self activation occuring in aged oocytes, all of which may compromise the cleavage potential of the heterokaryons and then lead to the failure of the conversion. In this study, we used fresh mouse oocytes activated by SrCl2 to induce the nuclear conversion, and compared the efficacy with that of mouse zygotes used. DESIGN: A randomized, prospective analysis. MATERIALS AND METHODS: 227 blastomeres from 61 untransfered human embryos in our reproductive center were used in the study and divided into two groups randomly, one was fused with mouse zygotes(103 blastomeres, Z-group) and the other with fresh mouse oocytes parthenogenetic activated by SrCl2(124 blastomeres, P-group). When the nuclear envelop of the heterokaryon disappeared, the cell was fixed and observed under the microscope for chromosome. In the end, the fusion rate, chromosome conversion rate and time required for conversion were compared between these two groups.The use of the untransfered embryos has got consent from every patient, and the study has been approved by the Ethical Review Board of our hospital. RESULTS: 1.There is no significant difference in fusion rate between these two groups.(P⬎0.05)
2. The chromosome conversion rate of the P-group is significantly higher than that of the Z-group.(P⬍0.01)
3. There is no significant difference between these two groups in the mean time required for chromosome conversion(P⬎0.05), which is 9.27⫾2.33 hours in P-group and 11.50⫾5.21 hours in Z-group. But it seems that the range of time is broader in the Z-group. CONCLUSION: There is no significant difference in fusion rate and time required for chromosome conversion between the parthenogenetic activation group and the zygote group, while the chromosome conversion rate is higher in the former group and the range of mean time required is broader in the latter, which implies that parthenogenetic activated fresh mouse oocytes could be a better alternative for induction of premature condensed chromosome, especially for PGD. Supported by: This work was supported by the NSFC(No. 30200301) and the Guangdong Natural Science Foundation(No. 5001672).
Tuesday, October 24, 2006 4:00 pm O-136 PREIMPLANTATION GENETIC DIAGNOSIS OF CHROMOSOME REARRANGEMENTS BY ANALYSIS OF METAPHASE SPREADS OBTAINED AFTER SELECTIVE EMBRYO BIOPSY ON DAY 3. A. Shkumatov, J. Cieslak-Janzen, V. Kuznyetsov, Y. Ilkevitch, Y. Verlinsky. Reproductive Genetics Institute, Chicago, IL. OBJECTIVE: The purpose of the study was to develop a method to obtain metaphase chromosomes from single blastomeres on Day 3 of embryo development for preimplantation genetic diagnosis (PGD) of chro-
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PREIMPLANTATION GENETIC DIAGNOSIS SPECIAL INTEREST GROUP Tuesday, October 24, 2006 3:30 pm O-134 RELATIONSHIP BETWEEN EMBRYONIC SECRETOME AND CHROMOSOMAL ABNORMALITIES IN HUMAN IVF. M. G. KatzJaffe, J. Stevens, W. G. Kearns, D. K. Gardner, W. B. Schoolcraft. Colorado Center for Reproductive Medicine, Englewood, CO; Shady Grove Center for Preimplantation Genetics, Rockville, MD. OBJECTIVE: Preimplantation genetic diagnosis (PGD) has become an established and successful IVF procedure screening for specific chromosomal abnormalities in human embryos. PGD involves the biopsy of polar bodies or embryonic cells for genetic analysis. Though successful, biopsy procedures are invasive to the embryo, possibly compromising development, and time consuming for the embryology laboratory. With recent advances in the field of proteomics it has become possible to analyze the proteins produced and secreted by human embryos into the surrounding medium (secretome). The objective of this study was to investigate whether embryos with chromosomal abnormalities could be distinguished from normal embryos by their respective secretomes DESIGN: Prospective Study MATERIALS AND METHODS: Cryopreserved day 1 embryos and aneuploid human blastocysts from PGD cycles were donated with consent for research. Day 1 embryos were thawed and subsequently cultured in sequential media (G1/G2) supplemented with recombinant human albumin at 37oC, 5% O2 & 6% CO2 up to the hatching blastocyst stage. Trophectoderm biopsy was performed removing up to 12 cells for FISH (chromosomes 13, 14, 15, 16, 17, 18, 21, 22, X and Y). Aneuploid blastocysts (abnormalities involving any of the analyzed chromosomes) from PGD cycles were cultured under these same conditions for 24 hrs. Drops of media cultured in the same dishes without blastocysts were used as controls. All spent media (aneuploid n⫽18, normal n⫽6) and controls (n⫽12) were subsequently processed and analyzed by time-of-flight mass spectrometry. RESULTS: The secretome profiles from hatching blastocysts identified as normal for the 10 chromosomes tested exhibited notably different secretome profiles to hatching blastocysts identified as aneuploid. Significantly, five proteins/biomarkers were observed to be in higher abundance in the secretome of hatching aneuploid blastocysts (P ⬍ 0.05). In addition, aneuploid embryos arrested in culture prior to the blastocyst stage also showed higher abundance of several proteins/biomarkers in their secretome profiles compared with hatching aneuploid blastocysts (P ⬍ 0.05). CONCLUSION: This is the first study to describe the differences in the secretome between human blastocysts identified as either normal or aneuploid for 10 chromosomes tested. This study has also shown that degenerating aneuploid embryos exhibit a significantly different secretome profile to hatching aneuploid blastocysts. This approach of protein analysis will further increase our understanding of human blastocyst physiology and its association with chromosomal composition. This work could lead to the development of a non-invasive assay to identify human embryos with chromosomal abnormalities. Supported by: None.
Tuesday, October 24, 2006 3:45 pm O-135 THE EFFICACY OF INTERPHASE-NUCLEAR-CONVERSION INDUCED BY PARTHENOGENETIC ACTIVATED FRESH MOUSE OOCYTES. L. Jiawei Sr., F. Cong, X. Yanwen, Z. Canquan, L. Tao, Z. Guanglun. the First Affiliated Hospital of Sun Yat-Sen Univ, Guangzhou, China.
FERTILITY & STERILITY威
OBJECTIVE: The protocol of interphase-nuclear-conversion induced by fusion of a blastomere with a mouse zygote has been set up as a method of preimplantation genetic diagnosis(PGD) to detect the chromosomal imbalance in the embryos of translocation carriers. But it was found that metaphase chromosome could not be obtained in some cells. The reasons maybe the fertilization of a mouse oocyte with an abnormal sperm, or self activation occuring in aged oocytes, all of which may compromise the cleavage potential of the heterokaryons and then lead to the failure of the conversion. In this study, we used fresh mouse oocytes activated by SrCl2 to induce the nuclear conversion, and compared the efficacy with that of mouse zygotes used. DESIGN: A randomized, prospective analysis. MATERIALS AND METHODS: 227 blastomeres from 61 untransfered human embryos in our reproductive center were used in the study and divided into two groups randomly, one was fused with mouse zygotes(103 blastomeres, Z-group) and the other with fresh mouse oocytes parthenogenetic activated by SrCl2(124 blastomeres, P-group). When the nuclear envelop of the heterokaryon disappeared, the cell was fixed and observed under the microscope for chromosome. In the end, the fusion rate, chromosome conversion rate and time required for conversion were compared between these two groups.The use of the untransfered embryos has got consent from every patient, and the study has been approved by the Ethical Review Board of our hospital. RESULTS: 1.There is no significant difference in fusion rate between these two groups.(P⬎0.05)
2. The chromosome conversion rate of the P-group is significantly higher than that of the Z-group.(P⬍0.01)
3. There is no significant difference between these two groups in the mean time required for chromosome conversion(P⬎0.05), which is 9.27⫾2.33 hours in P-group and 11.50⫾5.21 hours in Z-group. But it seems that the range of time is broader in the Z-group. CONCLUSION: There is no significant difference in fusion rate and time required for chromosome conversion between the parthenogenetic activation group and the zygote group, while the chromosome conversion rate is higher in the former group and the range of mean time required is broader in the latter, which implies that parthenogenetic activated fresh mouse oocytes could be a better alternative for induction of premature condensed chromosome, especially for PGD. Supported by: This work was supported by the NSFC(No. 30200301) and the Guangdong Natural Science Foundation(No. 5001672).
Tuesday, October 24, 2006 4:00 pm O-136 PREIMPLANTATION GENETIC DIAGNOSIS OF CHROMOSOME REARRANGEMENTS BY ANALYSIS OF METAPHASE SPREADS OBTAINED AFTER SELECTIVE EMBRYO BIOPSY ON DAY 3. A. Shkumatov, J. Cieslak-Janzen, V. Kuznyetsov, Y. Ilkevitch, Y. Verlinsky. Reproductive Genetics Institute, Chicago, IL. OBJECTIVE: The purpose of the study was to develop a method to obtain metaphase chromosomes from single blastomeres on Day 3 of embryo development for preimplantation genetic diagnosis (PGD) of chro-
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