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Apoptotic signaling pathways during early embryonic development: comparison between human and mouse
P1, P2 and P3 in Group 1 were 0.2 0.4 h, 9 1 hr and 2 1 h, respectively. Group 2 were 0.8 0.3 h, 4 1 h and 4 1 h, respectively. Developmental parameters were compared using a one way ANOVA and the difference between the values for P2 in Groups 1 and 2 was significantly different (p¼0.0007). CONCLUSION: Our data demonstrate that ICSI produced rhesus embryos complete the first 3 cell divisions within specific time periods correlating to blastocyst development by day 6. Early events in the first 1-2 days post-fertilization including cytokinesis and mitosis accurately predict embryos that are likely to progress to blastocyst stage. This suggests that rhesus embryo development closely models that of human and may serve as an accurate model system to study human development. Supported by: A grant from NIH (R01RR016581), FollistimÒ Supported by a grant from Merck Inc. P-331 Tuesday, October 15, 2013 METABOLIC DIFFERENCES BETWEEN IN VIVO AND IN VITRO PRODUCED EMBRYOS SUGGEST CRITICAL METABOLITES RELATED TO VIABILITY. R. L. Krisher,a A. L. Heuberger,b W. B. Schoolcraft.c aNational Foundation for Fertility Research, Lone Tree, CO; bProteomics and Metabolomics Facility, Colorado State University, Fort Collins, CO; cColorado Center for Reproductive Medicine, Lone Tree, CO. OBJECTIVE: Noninvasive examination of embryo metabolism can be accomplished using metabolomics technology to analyze media following in vitro culture. The objective of this study was to characterize embryo metabolites that are related to blastocyst viability. DESIGN: Research study. MATERIALS AND METHODS: Compact morulae (B6D2F1 mice) were recovered on d2.5 following ovarian stimulation and mating (in vivo), or were produced following IVM/IVF/IVC (78 hpi; in vitro). Embryos were cultured individually in 7 mL medium for 24 hr to expanded blastocyst. Media collected from drops that did and did not contain embryos was analyzed using GC-MS and MALDI-MS (n¼23). RESULTS: Of the 30 identified metabolites detected in culture media, 13 were significantly different between media in which in vivo versus in vitro embryos had been cultured. In vitro produced embryos were more metabolically active, depleting greater amounts of asparagine, aspartate, cystine, glucitol, glucose, isoleucine, lactate, leucine, lysine, ornithine, phenylalanine, phosphoric acid and tyrosine from the media during culture. In vitro embryos with low Oct4 expression took up significantly more aspartate than in vivo embryos or in vitro embryos with high Oct4 expression. In contrast to published reports, lactate was not produced but tended to be taken up by in vitro embryos and was unchanged by in vivo embryos. Glucose was not significantly taken up by in vivo embryos, although it was by in vitro embryos. Two contaminants of culture medium were also discovered (ditertbutylphenol and an unknown compound). CONCLUSION: Reduced metabolite use is associated with higher quality (in vivo) embryos. This study demonstrates that metabolomic technology can non-invasively detect differences in metabolism between embryos of different quality, and provides a novel look at embryo metabolism in a complex media environment. In particular, aspartate may be a useful biomarker to predict embryo viability.
P-332 Tuesday, October 15, 2013 APOPTOTIC SIGNALING PATHWAYS DURING EARLY EMBRYONIC DEVELOPMENT: COMPARISON BETWEEN HUMAN AND MOUSE. I. Boumela, D. Haouzi, S. Assou, S. Traver, S. Hamamah. CHU Montpellier, IRB-INSERM U1040, Montpellier, Languedoc Roussillon, France. OBJECTIVE: Apoptotic signaling pathways controlling the first week of embryonic development are poorly characterized. The objective of this study, is to understand mechanisms controlling the early embryonic development in term of survival and apoptotic signals. DESIGN: Human unfertilized MII oocytes, day 3 embryos and day 5/6 blastocysts were included after informed consent of the patients and authority approval. MII oocytes, day 1.5 embryos and day 3/4 blastocysts were collected from mice in vivo. We compared gene expression profiles between MII and day 3 embryos, MII and blastocyts, day 3 embryos and blastocyts in human as well as between MII and day1.5 embryos, MII and blastocysts, day 1.5 embryos and blastocysts in mouse.
FERTILITY & STERILITYÒ
MATERIALS AND METHODS: The mRNAs extracted from each embryonic stage were analyzed on GeneChip Human Genome U133 Plus 2.0 or Mouse Genome 430 2.0 Arrays. Significant Analysis of Microarrays was used to identify genes differentially between groups. RESULTS: Gene expression profiles between embryonic stages diverge in human compared with the mouse. Indeed, we identified 1801, 1031, 2023 over-expressed genes exclusive to the MII, day 3 embryos and blastocyst stage respectively in human versus 869, 3 and 299 genes in mouse. In addition, only 439 over-expressed genes were in common between embryonic stages in human versus 2439 genes in mouse. Consequently, survival signals differ between embryonic stages in human (over-expression of IGFBP1, HDGF2 in MII oocytes versus FGFR2 and IGFR1 in blastocysts) as for apoptotic signals (over-expression of CASP6 in oocytes versus CASP2 in blastocysts). Inversely, survival and apoptotic signals were stable between mouse embryonic stages. Moreover, signaling pathways related to oxidative stress, hypoxia and DNA damage were over-represented in the human. CONCLUSION: This study opens new perspectives for understanding the molecular regulation of oocyte and normal and degenerative embryo survival and death. Supported by: Partially Supported by a grant from Ferring Pharmaceuticals. P-333 Tuesday, October 15, 2013 THE HUMAN FIRST CELL CYCLE; IMPACT ON IMPLANTATION. J. Aguilar,a Y. Motato,b M. Ojeda,a M. J. Escriba,b E. Mu~noz,c M. Meseguer.b aLaboratory of IVF, IVI Vigo, Vigo, Pontevedra, Spain; bLaboratory of IVF, IVI Valencia, Valencia, Spain; cGyneacology, IVI Vigo, Vigo, Pontevedra, Spain. OBJECTIVE: To relate successful implantation of embryos to the exact timing of fertilization events by using a time-lapse system. DESIGN: Retrospective cohort study, whereas the timings of the second polar body extrusion (2PB), the appearance of two pronuclei (2PN), the pronuclear (PN) abuttal and the PN fading, and the categorical variables PN Score, PN Symmetry and PN movements in implanted and non-implanted embryos where recorded and analyzed. MATERIALS AND METHODS: Two-year cohort retrospective study comparing embryos according with known implantation. Participants attended a University-affiliated private clinic where ICSI was perfomed. Using an IVF incubator with a built.in camera designed to automatically acquire images at defined time-points, we monitored individual embryos from 842 patients:only embryos from treatments where the number of gestaional sacs matched the number of trasnferred embryos (n¼212) and embryos from treatments where no biochemical pregnancy was achieved (n¼687) were included in the study. The chronological pattern of fertilization events as well as of other morphologic features (cell size and nucleation) were registered. RESULTS: Timings of the pronuclei events in implanted and non-implanted embryos were respectively; 2PB 3.37h v.s 3.21h; 2PN 9.55h vs 9.42h; PN abuttal 12.23h vs 12.84h; PN fading 23.89h vs 24.41h. We found no differences in the proportion of implanted embryos for the categorical events analyzed. CONCLUSION: The timing of PN Fading could be linked to successful embryo implantation; the other parameters studied were not apparently related as determined by image acquisition and time-lapse analysis.
P-334 Tuesday, October 15, 2013 METABOLOMIC PROFILING OF HUMAN EMBRYOS DURING IN VITRO CULTURE: WHICH METABOLITES MATTER A. L. Heuberger,b M. Paczkowski,a WHEN? R. L. Krisher,a J. Stevens,c M. Rawlins,c W. B. Schoolcraft.d aNational Foundation for Fertility Research, Lone Tree, CO; bProteomics and Metabolomics Facility, Colorado State University, Fort Collins, CO; cFertility Laboratories of Colorado, Lone Tree, CO; dColorado Center for Reproductive Medicine, Lone Tree, CO. OBJECTIVE: To characterize the consumption and production of metabolites by human embryos during preimplantation development, and determine if these metabolomic profiles are influenced by culture media. DESIGN: Research study. MATERIALS AND METHODS: Media was collected from drops that did and did not contain embryos on D3 or D5 following culture of patient embryos in either Sage Blastocyst Medium (BM) or in house (IH) sequential
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P-332 Tuesday, October 15, 2013 APOPTOTIC SIGNALING PATHWAYS DURING EARLY EMBRYONIC DEVELOPMENT: COMPARISON BETWEEN HUMAN AND MOUSE. I. Boumela, D. Haouzi, S. Assou, S. Traver, S. Hamamah. CHU Montpellier, IRB-INSERM U1040, Montpellier, Languedoc Roussillon, France. OBJECTIVE: Apoptotic signaling pathways controlling the first week of embryonic development are poorly characterized. The objective of this study, is to understand mechanisms controlling the early embryonic development in term of survival and apoptotic signals. DESIGN: Human unfertilized MII oocytes, day 3 embryos and day 5/6 blastocysts were included after informed consent of the patients and authority approval. MII oocytes, day 1.5 embryos and day 3/4 blastocysts were collected from mice in vivo. We compared gene expression profiles between MII and day 3 embryos, MII and blastocyts, day 3 embryos and blastocyts in human as well as between MII and day1.5 embryos, MII and blastocysts, day 1.5 embryos and blastocysts in mouse.
FERTILITY & STERILITYÒ
MATERIALS AND METHODS: The mRNAs extracted from each embryonic stage were analyzed on GeneChip Human Genome U133 Plus 2.0 or Mouse Genome 430 2.0 Arrays. Significant Analysis of Microarrays was used to identify genes differentially between groups. RESULTS: Gene expression profiles between embryonic stages diverge in human compared with the mouse. Indeed, we identified 1801, 1031, 2023 over-expressed genes exclusive to the MII, day 3 embryos and blastocyst stage respectively in human versus 869, 3 and 299 genes in mouse. In addition, only 439 over-expressed genes were in common between embryonic stages in human versus 2439 genes in mouse. Consequently, survival signals differ between embryonic stages in human (over-expression of IGFBP1, HDGF2 in MII oocytes versus FGFR2 and IGFR1 in blastocysts) as for apoptotic signals (over-expression of CASP6 in oocytes versus CASP2 in blastocysts). Inversely, survival and apoptotic signals were stable between mouse embryonic stages. Moreover, signaling pathways related to oxidative stress, hypoxia and DNA damage were over-represented in the human. CONCLUSION: This study opens new perspectives for understanding the molecular regulation of oocyte and normal and degenerative embryo survival and death. Supported by: Partially Supported by a grant from Ferring Pharmaceuticals. P-333 Tuesday, October 15, 2013 THE HUMAN FIRST CELL CYCLE; IMPACT ON IMPLANTATION. J. Aguilar,a Y. Motato,b M. Ojeda,a M. J. Escriba,b E. Mu~noz,c M. Meseguer.b aLaboratory of IVF, IVI Vigo, Vigo, Pontevedra, Spain; bLaboratory of IVF, IVI Valencia, Valencia, Spain; cGyneacology, IVI Vigo, Vigo, Pontevedra, Spain. OBJECTIVE: To relate successful implantation of embryos to the exact timing of fertilization events by using a time-lapse system. DESIGN: Retrospective cohort study, whereas the timings of the second polar body extrusion (2PB), the appearance of two pronuclei (2PN), the pronuclear (PN) abuttal and the PN fading, and the categorical variables PN Score, PN Symmetry and PN movements in implanted and non-implanted embryos where recorded and analyzed. MATERIALS AND METHODS: Two-year cohort retrospective study comparing embryos according with known implantation. Participants attended a University-affiliated private clinic where ICSI was perfomed. Using an IVF incubator with a built.in camera designed to automatically acquire images at defined time-points, we monitored individual embryos from 842 patients:only embryos from treatments where the number of gestaional sacs matched the number of trasnferred embryos (n¼212) and embryos from treatments where no biochemical pregnancy was achieved (n¼687) were included in the study. The chronological pattern of fertilization events as well as of other morphologic features (cell size and nucleation) were registered. RESULTS: Timings of the pronuclei events in implanted and non-implanted embryos were respectively; 2PB 3.37h v.s 3.21h; 2PN 9.55h vs 9.42h; PN abuttal 12.23h vs 12.84h; PN fading 23.89h vs 24.41h. We found no differences in the proportion of implanted embryos for the categorical events analyzed. CONCLUSION: The timing of PN Fading could be linked to successful embryo implantation; the other parameters studied were not apparently related as determined by image acquisition and time-lapse analysis.
P-334 Tuesday, October 15, 2013 METABOLOMIC PROFILING OF HUMAN EMBRYOS DURING IN VITRO CULTURE: WHICH METABOLITES MATTER A. L. Heuberger,b M. Paczkowski,a WHEN? R. L. Krisher,a J. Stevens,c M. Rawlins,c W. B. Schoolcraft.d aNational Foundation for Fertility Research, Lone Tree, CO; bProteomics and Metabolomics Facility, Colorado State University, Fort Collins, CO; cFertility Laboratories of Colorado, Lone Tree, CO; dColorado Center for Reproductive Medicine, Lone Tree, CO. OBJECTIVE: To characterize the consumption and production of metabolites by human embryos during preimplantation development, and determine if these metabolomic profiles are influenced by culture media. DESIGN: Research study. MATERIALS AND METHODS: Media was collected from drops that did and did not contain embryos on D3 or D5 following culture of patient embryos in either Sage Blastocyst Medium (BM) or in house (IH) sequential
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