Abstract
OBJECTIVE: Chromosome aneuploidy is a common causative factor in infertility, early pregnancy loss and implantation failure. To date only invasive biopsy techniques which may compromise viability can identify aneuploid embryos. The aim of this study was to investigate the secretome of human blastocysts relative to comprehensive chromosome constitution in a non-invasive fashion.DESIGN: IRB approved study.MATERIALS AND METHODS: Spent culture media (8ul microdrops) was donated with consent from 74 couples undergoing IVF and blastocyst comprehensive chromosome screening (CCS) of all 23 pairs of chromosomes. CCS was performed by mCGH (Reprogenetics) or SNP based microarray (RMA-NJ). Blastocysts of quality adequate for transfer (>Grade 3BB) were divided into 3 groups; (A) euploid & ongoing pregnancy (n=40), (B) euploid & no pregnancy (n=40) and (C) aneuploid not transferred (n=40). Proteins were separated by 1D gel electrophoresis, in-gel digested and analyzed on a LTQ-FT Ultra Hybrid Mass Spectrometer (Thermo/Finnigan). Aneuploid biomarkers were identified using Mascot™ (Matrix Science) and analyzed comparing Groups A & B with Group C.RESULTS: The secretome of aneuploid blastocysts (Group C) exhibited a notably different protein profile to the secretome of euploid blastocysts (Groups A and B). Differentially expressed biomarkers characteristically classified aneuploid blastocysts. Perhaps the most significant biomarker was lipocalin-1, expressed by human blastocysts and increased in the aneuploid secretome. Lipocalins belong to a group of extracellular proteins that inhibit cystein proteinases, which play a key role in embryo hatching and invasion.CONCLUSION: Non-invasive secretome analysis clearly discriminated between euploid and aneuploid blastocysts. We believe that this is the first study to identify secreted proteins in association with chromosome aneuploidy. The ability to assay for CCS using a non-invasive platform may eliminate invasive biopsy techniques and provide a vital tool for embryo selection in human ART. OBJECTIVE: Chromosome aneuploidy is a common causative factor in infertility, early pregnancy loss and implantation failure. To date only invasive biopsy techniques which may compromise viability can identify aneuploid embryos. The aim of this study was to investigate the secretome of human blastocysts relative to comprehensive chromosome constitution in a non-invasive fashion. DESIGN: IRB approved study. MATERIALS AND METHODS: Spent culture media (8ul microdrops) was donated with consent from 74 couples undergoing IVF and blastocyst comprehensive chromosome screening (CCS) of all 23 pairs of chromosomes. CCS was performed by mCGH (Reprogenetics) or SNP based microarray (RMA-NJ). Blastocysts of quality adequate for transfer (>Grade 3BB) were divided into 3 groups; (A) euploid & ongoing pregnancy (n=40), (B) euploid & no pregnancy (n=40) and (C) aneuploid not transferred (n=40). Proteins were separated by 1D gel electrophoresis, in-gel digested and analyzed on a LTQ-FT Ultra Hybrid Mass Spectrometer (Thermo/Finnigan). Aneuploid biomarkers were identified using Mascot™ (Matrix Science) and analyzed comparing Groups A & B with Group C. RESULTS: The secretome of aneuploid blastocysts (Group C) exhibited a notably different protein profile to the secretome of euploid blastocysts (Groups A and B). Differentially expressed biomarkers characteristically classified aneuploid blastocysts. Perhaps the most significant biomarker was lipocalin-1, expressed by human blastocysts and increased in the aneuploid secretome. Lipocalins belong to a group of extracellular proteins that inhibit cystein proteinases, which play a key role in embryo hatching and invasion. CONCLUSION: Non-invasive secretome analysis clearly discriminated between euploid and aneuploid blastocysts. We believe that this is the first study to identify secreted proteins in association with chromosome aneuploidy. The ability to assay for CCS using a non-invasive platform may eliminate invasive biopsy techniques and provide a vital tool for embryo selection in human ART.
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