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In vitro fertilization and culture is associated with decreased phosphorylation of AktSer473 in mouse embryos
EMBRYO BIOLOGY P-188 Tuesday, October 26, 2010 HUMAN EMBRYO SPLITTING: PROOF OF HOMOZYGOSITY. K. Illmensee, M. Levanduski, C. Konialis, C. Pangalos, A. Vithoulkas, V. T. Goudas. International Fertility Services, RiverVale, NJ; Genesis Fertility Center, Patras, Greece; Intergenetics-Diagnostic Genetic Center, Athens, Greece. OBJECTIVE: To document that human embryos split at cleavage stage develop to twin blastocysts and prove monozygosity at the DNA level. DESIGN: Descriptive study. MATERIALS AND METHODS: Couples undergoing IVF donated triploid embryos (with IRB approval). Embryos at cleavage stages (6 - 8 cells) were split to twin embryos. Half the number of blastomeres were biopsied and inserted into empty zonae pellucidae and both donor and recipient embryos (D & R) were cultured to blastocysts. Twin embryos were washed and placed in PCR tubes with Alkaline Lysis Buffer (200mM KOH, 50mM DTT) covered with mineral oil. As controls, both non-split (dizygotic) embryos and blank samples were treated similarly. Analysis of selected polymorphic STR markers within the human HLA locus on chromosome 6 (Ring3-CA, G51152, D6S273, MIB, D6S1624, MOGCA) was performed following nested multiplex PCR reactions with fluorescently labeled primers. Samples were analyzed on ABI 3730xl DNA sequencer (Applied Biosystems U.S.A) with GeneMapper software. RESULTS: Of 11 twin embryos that developed to blastocysts and were analyzed, 5 sets of twin embryos provided detectable profiles and were used for interpretation. In all 5 samples, the obtained genotype was identical between the two twin embryos, thereby documenting monozygocity with more than 95% probability. (see table 1, + ¼ signal detected, nd ¼ no detectable signal). For the remaining 6 twin embryos, the profiles exhibited poor expression and were not suitable for interpretation. STR marker expression in monozygotic twin embryos
Six highly polymorphic STR markers Embryos
RING3-CA
G51152
D6S273
MIB
D6S1624
MOGCA
6D 6R 7D 7R 8D 8R 10D 10R 11D 11R
nd nd + + + + nd nd + +
+ + + + + + + + + +
+ + + + + + + + nd nd
+ + + + + + + + + +
+ + nd nd + + nd nd + +
nd nd + + + + + + + +
CONCLUSION: This is the first report on twinned human embryos proving monozygosity at the DNA level. Embryo splitting exhibits the potential for novel applications in human assisted reproduction. Supported by: Internal Institutional Support.
P-189 Tuesday, October 26, 2010 ALTERATIONS OF MITOTIC CHECKPOINT GENES PLAY A ROLE IN EMBRYONIC CHROMOSOME MOSAICISM. R. Loper, N. R. Treff, J. C. Parks, R. T. Scott, W. B. Schoolcraft, M. G. Katz-Jaffe. Colorado Center for Reproductive Medicine, Lone Tree, CO; Colorado Foundation for Fertility Research, Lone Tree, CO; Reproductive Medicine Associates of NJ, Morristown, NJ; UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: Mitotic cell division errors during early human development can result in the generation of chromosomal mosaic embryos. It is believed that the fate of mosaic embryos includes implantation failure and early pregnancy loss. Altered levels of mitotic checkpoint genes have been shown to cause chromosome instability in human cancer progression. The aim of this study was to investigate the association between a defective mitotic checkpoint and chromosomal instability in human blastocysts.
S148
Abstracts
DESIGN: Experimental study. MATERIALS AND METHODS: Human cryopreserved blastocysts of transfer quality (RGrade 3BB) (n¼21) were donated with IRB consent. Following thawing, blastocysts were allowed to recover for 2 hours prior to dissection into 3-4 sections. Comprehensive aneuploidy screening of all 23 pairs of chromosomes was performed on 2-3 sections by real-time PCR. Total RNA was isolated from one section for gene expression analysis of the major mitotic checkpoint genes: BUB1, BUB3, BUBR1, CDC20, MAD1L1, MAD2L2 and TTK by quantitative real-time PCR. RESULTS: Comprehensive aneuploidy screening defined blastocysts in the following groups: Group A, n¼8 euploid, Group B, n¼8 uniform aneuploidy (meiotic error) and Group C, n¼5 mosaic (mitotic error with more than one chromosome constitution). Gene expression levels of the mitotic checkpoint genes MAD2L2 and TTK were significantly increased in Group C compared to Groups A and B, relative to the internal housekeeping gene, PPIA (P<0.05). No significant differences were observed between Group A and B for any of the 7 major mitotic checkpoint genes. CONCLUSION: An increase in mitotic checkpoint gene expression appears to play a role in mitotic chromosome instability and the generation of blastocyst chromosomal mosaicism. High expression of mitotic checkpoint genes has also been indicated as a causative factor for human cancer chromosome instability. A potential mechanism for this alteration in gene expression could originate with the inaccurate activation of the embryonic genome. P-190 Tuesday, October 26, 2010 IN VITRO FERTILIZATION AND CULTURE IS ASSOCIATED WITH DECREASED PHOSPHORYLATION OF AKTSEr473 IN MOUSE EMBRYOS. X. Liu, A. Donjacour, W. Lin, P. Rinaudo. Obsetrics Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA. OBJECTIVE: The serine/threonine kinase Akt is functional in the preimplantation embryos and plays a central role in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Increase and decrease of Akt activation has been documented in an array of different diseases, including type-2 diabetes and cancer. Inhibition of Akt activity results in a significant delay in blastocyst hatching and implantation potential. Importantly, it is unknown if this pathway is altered after in vitro culture. DESIGN: mouse model of IVF. MATERIALS AND METHODS: Presence of total Akt, Akt2/b and phosphorilation of Akt at Ser473 and Ser308 was assessed in IVF and in vivo mouse blastocysts. IVF was performed in CF1 x B6D2F1/J mice. Fertilized eggs were cultured to the blastocyst stage in KSOM with amino acids and 5% O2. Control blastocysts were flushed out of the uterus 96h after mating. Western blot were performed extracting proteins from 15 blastocysts. Briefly blastocysts were suspended in cold lysis buffer, resolved on a 12% SDS-PAGE, transferred to a membrane, incubated overnight with total Akt, Akt2, pAktSer473 and pAktSer308 antibodies (Cell Signaling). After washing, the membrane was incubated with goat anti-Rabbit IgG antibody (Santa Cruz Biotechnology). The signal was detected with the SuperSignal West Dura Extended Duration Substrate (Therma). RESULTS: Both in vivo and IVF mouse blastocysts had detectable levels of total Akt, Akt2/b, pAktSer473 and pAktSer308. Protein levels could be detected in as low as 15 pooled blastocysts. IVF blastocysts showed a 60% decline in pAktSer473 phosphorilation. CONCLUSION: IVF is associated with alteration in pAktSer473 phosphorilation, indicating reduced growth and altered survival signals. Activation of pAktSer473 could be used as a biological marker to compare different culture media. Supported by: Bayer Award to PR.
P-191 Tuesday, October 26, 2010 THE RELEVANCE OF REACTIVE OXYGEN SPECIES LEVELS IN FOLLICULAR FLUID AND CULTURE MEDIA TO PREIMPLANTATION EMBRYO QUALITY. T.-H. Lee, C.-H. Liu, C.-C. Huang, M.-S. Lee. Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung, Taiwan, Taiwan; Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, Taiwan; Department of Infertility Clinic, Lee Women’s Hospital, Taichung, Taiwan, Taiwan. OBJECTIVE: This study was designed to determine the relevance of the levels of reactive oxygen species (ROS) in follicular fluid and culture media to the early development of human embryos.
Vol. 94., No. 4, Supplement, September 2010
Six highly polymorphic STR markers Embryos
RING3-CA
G51152
D6S273
MIB
D6S1624
MOGCA
6D 6R 7D 7R 8D 8R 10D 10R 11D 11R
nd nd + + + + nd nd + +
+ + + + + + + + + +
+ + + + + + + + nd nd
+ + + + + + + + + +
+ + nd nd + + nd nd + +
nd nd + + + + + + + +
CONCLUSION: This is the first report on twinned human embryos proving monozygosity at the DNA level. Embryo splitting exhibits the potential for novel applications in human assisted reproduction. Supported by: Internal Institutional Support.
P-189 Tuesday, October 26, 2010 ALTERATIONS OF MITOTIC CHECKPOINT GENES PLAY A ROLE IN EMBRYONIC CHROMOSOME MOSAICISM. R. Loper, N. R. Treff, J. C. Parks, R. T. Scott, W. B. Schoolcraft, M. G. Katz-Jaffe. Colorado Center for Reproductive Medicine, Lone Tree, CO; Colorado Foundation for Fertility Research, Lone Tree, CO; Reproductive Medicine Associates of NJ, Morristown, NJ; UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: Mitotic cell division errors during early human development can result in the generation of chromosomal mosaic embryos. It is believed that the fate of mosaic embryos includes implantation failure and early pregnancy loss. Altered levels of mitotic checkpoint genes have been shown to cause chromosome instability in human cancer progression. The aim of this study was to investigate the association between a defective mitotic checkpoint and chromosomal instability in human blastocysts.
S148
Abstracts
DESIGN: Experimental study. MATERIALS AND METHODS: Human cryopreserved blastocysts of transfer quality (RGrade 3BB) (n¼21) were donated with IRB consent. Following thawing, blastocysts were allowed to recover for 2 hours prior to dissection into 3-4 sections. Comprehensive aneuploidy screening of all 23 pairs of chromosomes was performed on 2-3 sections by real-time PCR. Total RNA was isolated from one section for gene expression analysis of the major mitotic checkpoint genes: BUB1, BUB3, BUBR1, CDC20, MAD1L1, MAD2L2 and TTK by quantitative real-time PCR. RESULTS: Comprehensive aneuploidy screening defined blastocysts in the following groups: Group A, n¼8 euploid, Group B, n¼8 uniform aneuploidy (meiotic error) and Group C, n¼5 mosaic (mitotic error with more than one chromosome constitution). Gene expression levels of the mitotic checkpoint genes MAD2L2 and TTK were significantly increased in Group C compared to Groups A and B, relative to the internal housekeeping gene, PPIA (P<0.05). No significant differences were observed between Group A and B for any of the 7 major mitotic checkpoint genes. CONCLUSION: An increase in mitotic checkpoint gene expression appears to play a role in mitotic chromosome instability and the generation of blastocyst chromosomal mosaicism. High expression of mitotic checkpoint genes has also been indicated as a causative factor for human cancer chromosome instability. A potential mechanism for this alteration in gene expression could originate with the inaccurate activation of the embryonic genome. P-190 Tuesday, October 26, 2010 IN VITRO FERTILIZATION AND CULTURE IS ASSOCIATED WITH DECREASED PHOSPHORYLATION OF AKTSEr473 IN MOUSE EMBRYOS. X. Liu, A. Donjacour, W. Lin, P. Rinaudo. Obsetrics Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA. OBJECTIVE: The serine/threonine kinase Akt is functional in the preimplantation embryos and plays a central role in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Increase and decrease of Akt activation has been documented in an array of different diseases, including type-2 diabetes and cancer. Inhibition of Akt activity results in a significant delay in blastocyst hatching and implantation potential. Importantly, it is unknown if this pathway is altered after in vitro culture. DESIGN: mouse model of IVF. MATERIALS AND METHODS: Presence of total Akt, Akt2/b and phosphorilation of Akt at Ser473 and Ser308 was assessed in IVF and in vivo mouse blastocysts. IVF was performed in CF1 x B6D2F1/J mice. Fertilized eggs were cultured to the blastocyst stage in KSOM with amino acids and 5% O2. Control blastocysts were flushed out of the uterus 96h after mating. Western blot were performed extracting proteins from 15 blastocysts. Briefly blastocysts were suspended in cold lysis buffer, resolved on a 12% SDS-PAGE, transferred to a membrane, incubated overnight with total Akt, Akt2, pAktSer473 and pAktSer308 antibodies (Cell Signaling). After washing, the membrane was incubated with goat anti-Rabbit IgG antibody (Santa Cruz Biotechnology). The signal was detected with the SuperSignal West Dura Extended Duration Substrate (Therma). RESULTS: Both in vivo and IVF mouse blastocysts had detectable levels of total Akt, Akt2/b, pAktSer473 and pAktSer308. Protein levels could be detected in as low as 15 pooled blastocysts. IVF blastocysts showed a 60% decline in pAktSer473 phosphorilation. CONCLUSION: IVF is associated with alteration in pAktSer473 phosphorilation, indicating reduced growth and altered survival signals. Activation of pAktSer473 could be used as a biological marker to compare different culture media. Supported by: Bayer Award to PR.
P-191 Tuesday, October 26, 2010 THE RELEVANCE OF REACTIVE OXYGEN SPECIES LEVELS IN FOLLICULAR FLUID AND CULTURE MEDIA TO PREIMPLANTATION EMBRYO QUALITY. T.-H. Lee, C.-H. Liu, C.-C. Huang, M.-S. Lee. Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung, Taiwan, Taiwan; Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, Taiwan; Department of Infertility Clinic, Lee Women’s Hospital, Taichung, Taiwan, Taiwan. OBJECTIVE: This study was designed to determine the relevance of the levels of reactive oxygen species (ROS) in follicular fluid and culture media to the early development of human embryos.
Vol. 94., No. 4, Supplement, September 2010