Expression of apolipoprotein A-1 (ApoA1) in human preimplantation embryos

Abstract

OBJECTIVE: Identify mRNA and protein expression patterns which correlate with embryo quality.DESIGN: 2-dimension (2D) gel electrophoresis coupled with tandem mass spectrometry was used to identify protein differences in spent culture media.MATERIALS AND METHODS: Human embryos frozen at the pronuclear stage donated for research under a University of Iowa IRB-approved protocol were thawed. Embryos were group cultured in InVitroCare (IVC)-1 medium supplemented with 20% SPSS for d1-2 and then individually cultured in 15 ul drops (IVC-1, 10% SPSS on d3 and IVC-3, 10% SPSS on d4-5). The spent culture media from d3-4 and d4-5 was collected and used for 2D gel electrophoresis and mass spectrometry (Applied Biomics) and ELISA (Cayman Chemical Company). Spent media was also collected on individually cultured blastocysts from fresh IVF cycles and analyzed for protein. Quantitative RT-PCR was used to determine ApoA1 mRNA expression levels from embryos.RESULTS: Pooled culture media from day 5 good/excellent blastocysts and from day 5 embryos arrested at cleavage stage were labeled with Cy3 and Cy5. Media concentration of ApoA1, one of the enhanced proteins, was 25.1% greater in good/excellent blastocysts (n=30, p=0.0036) when compared to arrested embryos (n=30) by ELISA. Pregnancy outcome did not correlate with media ApoA1 levels (p=0.13). Quantitative RT-PCR confirmed the presence of ApoA1 mRNA transcripts in human blastocysts.CONCLUSIONS: ApoA1 is produced by human preimplantation embryos, and increased levels are associated with embryos of higher grade. Our initial results examining ApoA1 as a clinical predictor of pregnancy outcome were not favorable. Nonetheless, these results support the possibility that analysis of embryonic conditioned media protein levels may provide a mechanism in the future for objective assessment of embryo quality. A developmental role for lipoproteins in early embryologic development is supported by the elevated synthesis of this protein by high quality embryos, and further investigation is warranted. OBJECTIVE: Identify mRNA and protein expression patterns which correlate with embryo quality. DESIGN: 2-dimension (2D) gel electrophoresis coupled with tandem mass spectrometry was used to identify protein differences in spent culture media. MATERIALS AND METHODS: Human embryos frozen at the pronuclear stage donated for research under a University of Iowa IRB-approved protocol were thawed. Embryos were group cultured in InVitroCare (IVC)-1 medium supplemented with 20% SPSS for d1-2 and then individually cultured in 15 ul drops (IVC-1, 10% SPSS on d3 and IVC-3, 10% SPSS on d4-5). The spent culture media from d3-4 and d4-5 was collected and used for 2D gel electrophoresis and mass spectrometry (Applied Biomics) and ELISA (Cayman Chemical Company). Spent media was also collected on individually cultured blastocysts from fresh IVF cycles and analyzed for protein. Quantitative RT-PCR was used to determine ApoA1 mRNA expression levels from embryos. RESULTS: Pooled culture media from day 5 good/excellent blastocysts and from day 5 embryos arrested at cleavage stage were labeled with Cy3 and Cy5. Media concentration of ApoA1, one of the enhanced proteins, was 25.1% greater in good/excellent blastocysts (n=30, p=0.0036) when compared to arrested embryos (n=30) by ELISA. Pregnancy outcome did not correlate with media ApoA1 levels (p=0.13). Quantitative RT-PCR confirmed the presence of ApoA1 mRNA transcripts in human blastocysts. CONCLUSIONS: ApoA1 is produced by human preimplantation embryos, and increased levels are associated with embryos of higher grade. Our initial results examining ApoA1 as a clinical predictor of pregnancy outcome were not favorable. Nonetheless, these results support the possibility that analysis of embryonic conditioned media protein levels may provide a mechanism in the future for objective assessment of embryo quality. A developmental role for lipoproteins in early embryologic development is supported by the elevated synthesis of this protein by high quality embryos, and further investigation is warranted.