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Gene expression profiles of human cumulus cells and pregnancy outcome: identification of molecular biomarkers of embryo competence
morphologies (P < 0.01). In particular, the CC proteomic fingerprint of developing blastocysts displayed higher expression of all five proteins. Oocytes that did not fertilize exhibited the lowest levels of expression, whilst embryos that arrested during the cleavage stage showed higher expression than unfertilized oocytes but lower compared to developing blastocysts (P < 0.01). A potential candidate ID for the 16.5kDa biomarker is presenilin-2, involved in cell division, differentiation and modulation of intracellular calcium signaling. CONCLUSIONS: This study has shown that like certain aspects of the CC genome, its proteome also correlates to morphological characteristics of resulting embryos. Oocytes which subsequently developed to blastocyst exhibit a unique CC proteomic fingerprint with significantly higher expression of a novel panel of proteins that could form the basis of a non-invasive assay to determine oocyte and embryo developmental competence. Additionally, this approach of protein analysis could improve our understanding of oogenesis. Supported by: None.
Tuesday, November 11, 2008 4:00 pm O-198 GENE EXPRESSION PROFILES OF HUMAN CUMULUS CELLS AND PREGNANCY OUTCOME: IDENTIFICATION OF MOLECULAR BIOMARKERS OF EMBRYO COMPETENCE. M. Fourar, D. Haouzi, A. Aouacheria, H. Dechaud, K. Bendhaou, S. Hamamah. Institut de Recherche en Biotherapie — INSERM, Montpellier, France; Hoˆpital Arnaud de Villeneuve, CHU Montpellie, Montpellier, France; Centre de Fecondation In Vitro, Tunis, Tunisia. OBJECTIVE: Identification of new criteria for embryo quality is required to improve the clinical outcome of in vitro fertilization (IVF). In order to identify news markers of good embryonic quality as well as successful pregnancy, we performed the gene expression profiles between cumulus cells from oocytes giving rise to different embryo outcomes from normo-responders patients undergoing controlled ovarian hyperstimulation (COS) for ICSI (Intra Cytoplasmic Sperm Injection). DESIGN: Individual cumulus cell samples were analyzed by DNA chip microarray to evaluate gene expression profiles associated with good embryonic quality and pregnancy outcome. The differential gene expression profile was evaluated with bioinformatics methods. MATERIALS AND METHODS: The mRNA extracted from cumulus cells (CC) from single oocytes (n¼35) were analysed on GeneChip oligonucleotide microarrays: 11 CC from grade 3/4 embryos without pregnancy (10 patients), 16 CC from grade 1/2 embryos without pregnancy (9 patients) and 18 CC from grade 1/2 embryos with positive pregnancy outcome (11 patients). A genetic imprint associated with a positive pregnancy outcome was identified, which permitted to select candidate genes for embryo competence. 11 candidate genes were selected, including 5 down-regulated genes in cumulus cells group associated to a pregnancy, which were evaluated by quantitative PCR. Gene modules over represented in selected gene lists were determined with Ingenuity software. RESULTS: We found that the expression of BCL2L11, CFLAR, MMP14, NFIB and PCK1 in cumulus cells is significantly associated with a successful pregnancy. These genes play a role in apoptosis, embryonic development, tissue remodeling and gluconeogenesis. Our results were validated by quantitative PCR in the original samples (n¼10) and in an independent series of cumulus samples (n¼8). BCL2L11 protein expression was also confirmed by immunofluorescence labelling and was correlated with a good embryonic quality and development potential. This group of genes shows the potential to be useful predictors for successful pregnancy. CONCLUSIONS: The pregnancy outcome can be inferred from the cumulus cells expression profile. Five selected genes have been independently validated and are proposed as new biomarkers for pregnancy success rate. As this test is available as early as the oocyte stage, these results offer new perspectives for competent embryo selection particularly for single embryo transfer. Supported by: A grant from Ferring Company.
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Abstracts
Tuesday, November 11, 2008 4:15 pm O-199 SUCCESSFUL VITRIFIED OOCYTE DONATION PROGRAM WITH BLASTOCYST TRANSFER. ONE YEAR OF FOLLOW UP STUDY IN MEXICO CITY. S. Cubillos, S. Sanchez, G. Charria, E. Cervantes, H. Aparicio, S. Cuneo. Assisted Reproduction Laboratory, Concibe Reproduccio´n Asistida, Mexico City, Mexico; Reproductive Medicine, Concibe Reproduccio´n Asistida - Cuernavaca, Mexico City, Mexico. OBJECTIVE: Up to now few clinical data are available about large series of vitrified egg donation and there are a lot of doubts if the vitrification method affect spindle morphology, aberrant chromatin alignment or premature cortical granule releases that can be affect embryo development and pregnancy, implantation and/or miscarriage rates. Blastocyst development stage allow self selection from those embryos capable of blastulation and self exclusion from those which have a lack of embryonic development or arrestment. Our objective is to show that oocyte vitrification and the sequential culture is a good combination to have a success in a oocyte donation program with an oocyte bank. DESIGN: Retrospective and descriptive study. MATERIALS AND METHODS: We included sixteen patient’s cycles receptors, done from May 2006 to December 2007. 30 oocytes donors were frozen by vitrificaction, and Sixten cycles were thawed. Donators were stimulated with conventional down regulation protocols. For this study a requirement was that all of the donors had a proven in vivo or in vitro fertility. Oocytes were retrieved by ultrasound-guided transvaginal aspiration and vitrified with the Cryotop method. 154 oocytes were thawed, and a mean of 7 oocytes MII were assigned for each recipient. The survived oocytes were inseminated by intracytoplasmic sperm injection. The zygotes were further cultured in vitro for up to 120 hours until time of embryo transfer, day 5 or 6 according to embryo development. RESULTS: The oocyte survival rate observed was 96.7%. Fertilization rate was 85,71%, day 2 cleavage (93,9%), day 3 cleavage (77,41%), and blastocyst development (41,6%). A total of 29 embryos were transferred on the 5th or 6th day. The media of embyos transferred were two. The refreezing rate by vitrification was (27,5 %) blastocyst, that are still frozen. Pregnancy, implantation and miscarriage rates, were 66,67%, 31,06%, and 22,22%, respectively. Our pregnancy, implantation and miscarriage rates in fresh egg donation are (70,55 %), (36,11 %) and (13,04 %) respectively. CONCLUSIONS: Excellent clinical outcome indicates the possibility to use of the egg donation program technology and establishes oocyte banking. The costs and time of this procedure are other benefits for patients waiting for oocytes. Sequential culture is a strategy to consider to selected embryos with a high potential implantation to transfer. More studies should be done to confirm these results. Supported by: None.
Tuesday, November 11, 2008 4:30 pm O-200 MULTI-NUCLEATION OF HUMAN EMBRYOS: CLINICAL AND LABORATORY FACTORS IN ITS OCCURRENCE AND ITS IMPACT ON DEVELOPMENTAL CAPACITY. D. A. Sheehan, M. C. Summers, J. C. Patel, K. J. Go. Reproductive Science Center of New England, Lexington, MA; University of Massachusetts Medical Center, Worcester, MA. OBJECTIVE: Multinucleation, the occurrence in embryos of blastomeres with R 2 nuclei per blastomere, can be a criterion for exclusion from transfer in In Vitro Fertilization (IVF). Clinical and laboratory factors that may influence the occurrence of multinucleation were considered. DESIGN: Retrospective review of clinical profiles of patients with embryos in which 0 % (control) or R50% multinucleated blastomeres (MNB-emb) were identified on Day 2 of development, and the prospective study of morphology and implantation of the embryos from these two groups. MATERIALS AND METHODS: Two groups of patients in a continuous interval from January to May 2007 were studied: those having no MNB-emb
Vol. 90, Suppl 1, September 2008
Tuesday, November 11, 2008 4:00 pm O-198 GENE EXPRESSION PROFILES OF HUMAN CUMULUS CELLS AND PREGNANCY OUTCOME: IDENTIFICATION OF MOLECULAR BIOMARKERS OF EMBRYO COMPETENCE. M. Fourar, D. Haouzi, A. Aouacheria, H. Dechaud, K. Bendhaou, S. Hamamah. Institut de Recherche en Biotherapie — INSERM, Montpellier, France; Hoˆpital Arnaud de Villeneuve, CHU Montpellie, Montpellier, France; Centre de Fecondation In Vitro, Tunis, Tunisia. OBJECTIVE: Identification of new criteria for embryo quality is required to improve the clinical outcome of in vitro fertilization (IVF). In order to identify news markers of good embryonic quality as well as successful pregnancy, we performed the gene expression profiles between cumulus cells from oocytes giving rise to different embryo outcomes from normo-responders patients undergoing controlled ovarian hyperstimulation (COS) for ICSI (Intra Cytoplasmic Sperm Injection). DESIGN: Individual cumulus cell samples were analyzed by DNA chip microarray to evaluate gene expression profiles associated with good embryonic quality and pregnancy outcome. The differential gene expression profile was evaluated with bioinformatics methods. MATERIALS AND METHODS: The mRNA extracted from cumulus cells (CC) from single oocytes (n¼35) were analysed on GeneChip oligonucleotide microarrays: 11 CC from grade 3/4 embryos without pregnancy (10 patients), 16 CC from grade 1/2 embryos without pregnancy (9 patients) and 18 CC from grade 1/2 embryos with positive pregnancy outcome (11 patients). A genetic imprint associated with a positive pregnancy outcome was identified, which permitted to select candidate genes for embryo competence. 11 candidate genes were selected, including 5 down-regulated genes in cumulus cells group associated to a pregnancy, which were evaluated by quantitative PCR. Gene modules over represented in selected gene lists were determined with Ingenuity software. RESULTS: We found that the expression of BCL2L11, CFLAR, MMP14, NFIB and PCK1 in cumulus cells is significantly associated with a successful pregnancy. These genes play a role in apoptosis, embryonic development, tissue remodeling and gluconeogenesis. Our results were validated by quantitative PCR in the original samples (n¼10) and in an independent series of cumulus samples (n¼8). BCL2L11 protein expression was also confirmed by immunofluorescence labelling and was correlated with a good embryonic quality and development potential. This group of genes shows the potential to be useful predictors for successful pregnancy. CONCLUSIONS: The pregnancy outcome can be inferred from the cumulus cells expression profile. Five selected genes have been independently validated and are proposed as new biomarkers for pregnancy success rate. As this test is available as early as the oocyte stage, these results offer new perspectives for competent embryo selection particularly for single embryo transfer. Supported by: A grant from Ferring Company.
S72
Abstracts
Tuesday, November 11, 2008 4:15 pm O-199 SUCCESSFUL VITRIFIED OOCYTE DONATION PROGRAM WITH BLASTOCYST TRANSFER. ONE YEAR OF FOLLOW UP STUDY IN MEXICO CITY. S. Cubillos, S. Sanchez, G. Charria, E. Cervantes, H. Aparicio, S. Cuneo. Assisted Reproduction Laboratory, Concibe Reproduccio´n Asistida, Mexico City, Mexico; Reproductive Medicine, Concibe Reproduccio´n Asistida - Cuernavaca, Mexico City, Mexico. OBJECTIVE: Up to now few clinical data are available about large series of vitrified egg donation and there are a lot of doubts if the vitrification method affect spindle morphology, aberrant chromatin alignment or premature cortical granule releases that can be affect embryo development and pregnancy, implantation and/or miscarriage rates. Blastocyst development stage allow self selection from those embryos capable of blastulation and self exclusion from those which have a lack of embryonic development or arrestment. Our objective is to show that oocyte vitrification and the sequential culture is a good combination to have a success in a oocyte donation program with an oocyte bank. DESIGN: Retrospective and descriptive study. MATERIALS AND METHODS: We included sixteen patient’s cycles receptors, done from May 2006 to December 2007. 30 oocytes donors were frozen by vitrificaction, and Sixten cycles were thawed. Donators were stimulated with conventional down regulation protocols. For this study a requirement was that all of the donors had a proven in vivo or in vitro fertility. Oocytes were retrieved by ultrasound-guided transvaginal aspiration and vitrified with the Cryotop method. 154 oocytes were thawed, and a mean of 7 oocytes MII were assigned for each recipient. The survived oocytes were inseminated by intracytoplasmic sperm injection. The zygotes were further cultured in vitro for up to 120 hours until time of embryo transfer, day 5 or 6 according to embryo development. RESULTS: The oocyte survival rate observed was 96.7%. Fertilization rate was 85,71%, day 2 cleavage (93,9%), day 3 cleavage (77,41%), and blastocyst development (41,6%). A total of 29 embryos were transferred on the 5th or 6th day. The media of embyos transferred were two. The refreezing rate by vitrification was (27,5 %) blastocyst, that are still frozen. Pregnancy, implantation and miscarriage rates, were 66,67%, 31,06%, and 22,22%, respectively. Our pregnancy, implantation and miscarriage rates in fresh egg donation are (70,55 %), (36,11 %) and (13,04 %) respectively. CONCLUSIONS: Excellent clinical outcome indicates the possibility to use of the egg donation program technology and establishes oocyte banking. The costs and time of this procedure are other benefits for patients waiting for oocytes. Sequential culture is a strategy to consider to selected embryos with a high potential implantation to transfer. More studies should be done to confirm these results. Supported by: None.
Tuesday, November 11, 2008 4:30 pm O-200 MULTI-NUCLEATION OF HUMAN EMBRYOS: CLINICAL AND LABORATORY FACTORS IN ITS OCCURRENCE AND ITS IMPACT ON DEVELOPMENTAL CAPACITY. D. A. Sheehan, M. C. Summers, J. C. Patel, K. J. Go. Reproductive Science Center of New England, Lexington, MA; University of Massachusetts Medical Center, Worcester, MA. OBJECTIVE: Multinucleation, the occurrence in embryos of blastomeres with R 2 nuclei per blastomere, can be a criterion for exclusion from transfer in In Vitro Fertilization (IVF). Clinical and laboratory factors that may influence the occurrence of multinucleation were considered. DESIGN: Retrospective review of clinical profiles of patients with embryos in which 0 % (control) or R50% multinucleated blastomeres (MNB-emb) were identified on Day 2 of development, and the prospective study of morphology and implantation of the embryos from these two groups. MATERIALS AND METHODS: Two groups of patients in a continuous interval from January to May 2007 were studied: those having no MNB-emb
Vol. 90, Suppl 1, September 2008