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Protein expression in cumulus cells as indicator of pregnancy success
P-275 Tuesday, October 20, 2015 NON-INVASIVE PREDICTION OF BLASTOCYST FORMATION BY DAY THREE EMBRYO CULTURE MEDIUM MASS SPECTROMETRY LIPID FINGERPRINTING. D. P. Braga,a,b,c A. S. Setti,a,b,d E. C. Cabral,e M. N. Eberlin,e E. G. Lo Turco,c A. Iaconelli, Jr.,f,b E. Borges, Jr.,f,b aScientific Department, Fertility Medical Group, Sao Paulo, Brazil; bScientific Department, Instituto Sapientiae - Centro de Estudos e Pesquisa em Reproduc¸~ao Assistida, Sao Paulo, Brazil; cUrology Deparment, Universidade Federal de S~ao Paulo (UNIFESP), Sao Paulo, Brazil; dFaculdade de Ciencias Medicas da Santa Casa de Sao Paulo, Sao Paulo, Brazil; e Chemistry Institute - ThoMSon Mass Spectrometry Laboratory, Universidade Estadual de Campinas, Campinas, Brazil; fClinical Department, Fertility Medical Group, Sao Paulo, Brazil. OBJECTIVE: To identify lipid markers of blastocyst formation by day three culture medium mass spectrometry (MS) fingerprinting. DESIGN: Case-control study. MATERIALS AND METHODS: For this study 50 embryo culture media samples were harvested on the day three, from patients undergoing day five embryo transfers. Embryos were split into groups based on their degree of expansion and hatching status on day five (Complete-Blastocysts, n¼25 and No-Blastocysts, n¼25) and its secretomes were analysed by MS. Mass spectra fingerprinting was acquired using a Q-Tof spectrometer (LC-MS, Agilent 6550 iFunnel Q-TOF) equipped with an automated injector. Data were analysed using the principal component analysis (PCA) followed by a partial least square discrimination analysis (PLS-DA), combined with variable influence in the projection (VIP) scores. The statistical analysis was performed using Metabo-Analyst 2.0 (http://www.metaboanalyst.ca). RESULTS: Overall, 1657 ions were observed. When the univariate analysis was performed, 165 ions were observed to be differentially expressed between the groups, with a fold chance R 4x and p<0.001, in the t test. The PLS-DA showed a clear separation between the groups and among 15 VIPs selected by the program, 13 of them were high-expressed in the Complete-Blastocysts Group and 2 in the No-Blastocysts Group. The lipids suggested to be high-expressed in the Complete-Blastocysts Group includes Isoprenoids, Diradylglycerols, Sterols, Fatty Esters, Secosteroids, Phosphosphingolipids, Glycerophosphates and Diacylglycerophosphates, while Fatty Amides were suggested to be high expressed in the No-Blastocysts Group. CONCLUSIONS: The day three culture medium MS may identify possible lipid biomarkers of embryos which are able to develop into blastocysts, therefore this may be a promising approach for the identification of embryos that should be cultured until day five. P-276 Tuesday, October 20, 2015 PROTEIN EXPRESSION IN CUMULUS CELLS AS INDICATOR OF D. P. Braga,c,b,d PREGNANCY SUCCESS. A. Iaconelli, Jr.,a,b A. S. Setti,c,b,e E. C. Cabral,f M. N. Eberlin,f F. B. Cordeiro,d E. G. Lo Turco,d E. Borges, Jr.,a,b aClinical Department, Fertility Medical Group, Sao Paulo, Brazil; bScientific Department, Instituto Sapientiae - Centro de Estudos e Pesquisa em Reproduc¸~ao Assistida, Sao Paulo, Brazil; cScientific Department, Fertility Medical Group, Sao Paulo, Brazil; dUrology Deparment, Universidade Federal de S~ao Paulo (UNIFESP), Sao Paulo, Brazil; e Faculdade de Ciencias Medicas da Santa Casa de Sao Paulo, Sao Paulo, Brazil; fChemistry Institute - ThoMSon Mass Spectrometry Laboratory, Universidade Estadual de Campinas, Campinas, Brazil. OBJECTIVE: To utilize the analytical power of mass spectrometry to predict the pregnancy outcome by differential protein expression in cumulus cells (CC). DESIGN: Case-control study. MATERIALS AND METHODS: Cumulus cells samples were collected immediately after ovum pick up from patients undergoing intracytoplasmic sperm injection (ICSI), and split into two groups according to the pregnancy outcome (Positive-Group, n¼10 and Negative-Group, n¼10). To obtain the minimum amount of material for the study, CC samples from each group were pooled. Samples were submitted to protein extraction and injected into a liquid chromatography system (Agilent 1290 Infinity LC System) coupled to a mass spectrometer (LC-MS, Agilent 6550 iFunnel Q-TOF). Spectra were processed using Mascot Distiller, and results were submitted to database search using a reviewed SwissProt Human database. RESULTS: Overall, 72 different proteins were detected, of which 19 were expressed exclusively in the Positive-Group and 16 other were expressed exclusively in the Negative-Group. Thirty seven other proteins were expressed in both groups, and of these, 16 were equally expressed between
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the groups and 21 were differentially expressed between groups. Differentially expressed proteins included binding proteins, enzymes, activation proteins, and proteins in charge of cell development. CONCLUSIONS: From the results of this study, potential biomarkers for the pregnancy outcome have been suggested. In a next step these proteins may be individually identified and its frequency in subjects may be determined. In conclusion, CCs proteomics may be a useful tool for the prediction of ICSI cycles outcomes. Supported by: FAPESP Pesquisa Inovativa em Pequenas Empresas (PIPE). 2013/50052-7. P-277 Tuesday, October 20, 2015 CUMULUS CELLS PROTEOMICS AS A TOOL FOR SELECTION OF PATIENTS FOR EXTENDED EMBRYO CULTURE PROGRAMMES. A. S. Setti,a,b,c D. P. Braga,a,b,d E. C. Cabral,e M. N. Eberlin,e F. B. Cordeiro,d E. G. Lo Turco,d A. Iaconelli, Jr.,f,b E. Borges, Jr.,f,b aScientific Department, Fertility Medical Group, Sao Paulo, Brazil; bScientific Department, Instituto Sapientiae - Centro de Estudos e Pesquisa em Reproduc¸~ao Assistida, Sao Paulo, Brazil; cFaculdade de Ciencias Medicas da Santa Casa de Sao Paulo, Sao Paulo, Brazil; dUrology Deparment, Universidade Federal de S~ao Paulo (UNIFESP), Sao Paulo, Brazil; e Chemistry Institute - ThoMSon Mass Spectrometry Laboratory, Universidade Estadual de Campinas, Campinas, Brazil; fClinical Department, Fertility Medical Group, Sao Paulo, Brazil. OBJECTIVE: To identify patients that would benefit from extended embryo culture programmes and blastocyst-stage embryo transfers. DESIGN: Case-control study. MATERIALS AND METHODS: Immediately after ovum pick up, cumulus cells samples from 20 patients undergoing intracytoplasmic sperm injection (ICSI) were collected and storage under -20o C. Samples were split according to blastocyst formation rate: Blastocyts group (n¼10), including patients in which all embryos converted into blastocysts; and Non-blastocyst group, (n¼10), including patients in which none of the embryos reached the blastocyst stage. The inclusion criteria were: (i) ICSI cycle using ejaculated sperm with embryo transfer performed on day five; (ii) patient’s age % 35 years old and (iii) number of retrieved mature oocytes (MII) R 4. To obtain the minimum amount of material for the study, CC samples from each group were pooled. Samples were submitted to protein extraction and injected into a liquid chromatography system (Agilent 1290 Infinity LC System) coupled to a mass spectrometer (LC-MS, Agilent 6550 iFunnel Q-TOF). Spectra were processed using Mascot Distiller, and results were submitted to database search using a reviewed SwissProt Human database. RESULTS: Overall, 87 different proteins were detected, of which 17 were expressed exclusively in Non-blastocyst group and 30 other proteins were expressed exclusively in the Blastocyts group. The remaining 40 proteins were expressed in both groups, and from those, 6 proteins were equally expressed between the groups and another 34 were differentially expressed between groups. Differentially expressed proteins included binding proteins, enzymes, transport proteins, antioxidants, proteins in charge of cell development, catalysts and structural proteins. CONCLUSIONS: Potential biomarkers for blastocyst formation chance have been suggested. In a next step these proteins may be individually identified and its frequency in subjects may be determined, therefore, CCs proteomics may be useful for the identification of patients that should be included in extended embryo culture programmes or patients who would benefit from cleavage-stage embryo transfers. Supported by: FAPESP Pesquisa Inovativa em Pequenas Empresas (PIPE). 2013/50052-7. P-278 Tuesday, October 20, 2015 RNA SEQUENCING AND IMMUNO-FLUORESCENCE STAINING OF CUMULUS CELLS ASSOCIATED WITH HUMAN OOCYTE SENESCENCE PATHWAYS. E. Molinari,a A. Pyle,b H. Bar,c P. Patrizio.d aDept. of Obstetrics, Gynecology & Reproductive Sci, Yale University School of Medicine, New Haven, CT; bDept. of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT; cDept. of Statistics, University of Connecticut, Storrs, CT; dYale Fertility Center & Fertility Preservation, New Haven, CT. OBJECTIVE: To further characterize pathways leading to human oocyte aging using cumulus cells (CCs) RNA sequencing, immuno-fluorescent staining and weighted gene co-expression network analysis.
Vol. 104, No. 3, Supplement, September 2015
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the groups and 21 were differentially expressed between groups. Differentially expressed proteins included binding proteins, enzymes, activation proteins, and proteins in charge of cell development. CONCLUSIONS: From the results of this study, potential biomarkers for the pregnancy outcome have been suggested. In a next step these proteins may be individually identified and its frequency in subjects may be determined. In conclusion, CCs proteomics may be a useful tool for the prediction of ICSI cycles outcomes. Supported by: FAPESP Pesquisa Inovativa em Pequenas Empresas (PIPE). 2013/50052-7. P-277 Tuesday, October 20, 2015 CUMULUS CELLS PROTEOMICS AS A TOOL FOR SELECTION OF PATIENTS FOR EXTENDED EMBRYO CULTURE PROGRAMMES. A. S. Setti,a,b,c D. P. Braga,a,b,d E. C. Cabral,e M. N. Eberlin,e F. B. Cordeiro,d E. G. Lo Turco,d A. Iaconelli, Jr.,f,b E. Borges, Jr.,f,b aScientific Department, Fertility Medical Group, Sao Paulo, Brazil; bScientific Department, Instituto Sapientiae - Centro de Estudos e Pesquisa em Reproduc¸~ao Assistida, Sao Paulo, Brazil; cFaculdade de Ciencias Medicas da Santa Casa de Sao Paulo, Sao Paulo, Brazil; dUrology Deparment, Universidade Federal de S~ao Paulo (UNIFESP), Sao Paulo, Brazil; e Chemistry Institute - ThoMSon Mass Spectrometry Laboratory, Universidade Estadual de Campinas, Campinas, Brazil; fClinical Department, Fertility Medical Group, Sao Paulo, Brazil. OBJECTIVE: To identify patients that would benefit from extended embryo culture programmes and blastocyst-stage embryo transfers. DESIGN: Case-control study. MATERIALS AND METHODS: Immediately after ovum pick up, cumulus cells samples from 20 patients undergoing intracytoplasmic sperm injection (ICSI) were collected and storage under -20o C. Samples were split according to blastocyst formation rate: Blastocyts group (n¼10), including patients in which all embryos converted into blastocysts; and Non-blastocyst group, (n¼10), including patients in which none of the embryos reached the blastocyst stage. The inclusion criteria were: (i) ICSI cycle using ejaculated sperm with embryo transfer performed on day five; (ii) patient’s age % 35 years old and (iii) number of retrieved mature oocytes (MII) R 4. To obtain the minimum amount of material for the study, CC samples from each group were pooled. Samples were submitted to protein extraction and injected into a liquid chromatography system (Agilent 1290 Infinity LC System) coupled to a mass spectrometer (LC-MS, Agilent 6550 iFunnel Q-TOF). Spectra were processed using Mascot Distiller, and results were submitted to database search using a reviewed SwissProt Human database. RESULTS: Overall, 87 different proteins were detected, of which 17 were expressed exclusively in Non-blastocyst group and 30 other proteins were expressed exclusively in the Blastocyts group. The remaining 40 proteins were expressed in both groups, and from those, 6 proteins were equally expressed between the groups and another 34 were differentially expressed between groups. Differentially expressed proteins included binding proteins, enzymes, transport proteins, antioxidants, proteins in charge of cell development, catalysts and structural proteins. CONCLUSIONS: Potential biomarkers for blastocyst formation chance have been suggested. In a next step these proteins may be individually identified and its frequency in subjects may be determined, therefore, CCs proteomics may be useful for the identification of patients that should be included in extended embryo culture programmes or patients who would benefit from cleavage-stage embryo transfers. Supported by: FAPESP Pesquisa Inovativa em Pequenas Empresas (PIPE). 2013/50052-7. P-278 Tuesday, October 20, 2015 RNA SEQUENCING AND IMMUNO-FLUORESCENCE STAINING OF CUMULUS CELLS ASSOCIATED WITH HUMAN OOCYTE SENESCENCE PATHWAYS. E. Molinari,a A. Pyle,b H. Bar,c P. Patrizio.d aDept. of Obstetrics, Gynecology & Reproductive Sci, Yale University School of Medicine, New Haven, CT; bDept. of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT; cDept. of Statistics, University of Connecticut, Storrs, CT; dYale Fertility Center & Fertility Preservation, New Haven, CT. OBJECTIVE: To further characterize pathways leading to human oocyte aging using cumulus cells (CCs) RNA sequencing, immuno-fluorescent staining and weighted gene co-expression network analysis.
Vol. 104, No. 3, Supplement, September 2015