Abstract
BackgroundGrowing concerns about safety of ART on human gametes, embryos, clinical outcomes and long-term health of offspring require improved methods of risk assessment to provide functionally relevant assays for quality control testing and pre-clinical studies prior to clinical implementation. The one-cell mouse embryo assay (MEA) is the most widely used for development and quality testing of human ART products; however, concerns exist due to the insensitivity/variability of this bioassay which lacks standardization and involves subjective analysis by morphology alone rather than functional analysis of the developing embryos. We hypothesized that improvements to MEA by the use of functional molecular biomarkers could enhance sensitivity and improve detection of suboptimal materials/conditions.ResultsFresh one-cell transgenic mouse embryos with green fluorescent protein (GFP) expression driven by Pou6f1 or Cdx2 control elements were harvested and cultured to blastocysts in varied test and control conditions to compare assessment by standard morphology alone versus the added dynamic expression of GFP for screening and selection of critical raw materials and detection of suboptimal culture conditions. Transgenic mouse embryos expressing functionally relevant biomarkers of normal early embryo development can be used to monitor the developmental impact of culture conditions.ConclusionsThis novel approach provides a superior MEA that is more meaningful and sensitive for detection of embryotoxicity than morphological assessment alone.
Highlights
Growing concerns about safety of assisted reproduction technology (ART) on human gametes, embryos, clinical outcomes and longterm health of offspring require improved methods of risk assessment to provide functionally relevant assays for quality control testing and pre-clinical studies prior to clinical implementation
We hypothesized that Pou5f1-green fluorescent protein (GFP) zygotes with 2 pronuclei (2PN) produced by mating transgenic mice strain B6; CBA-Tg (Pou5f1-enhanced GFP (EGFP)) 2Mnn/J with B6D2F1/J can be cultured in vitro and monitored at 48 h and 96 h to assess their development (Fig. 2)
POU5F1-GFP was expressed to varying degrees among embryos at 48 h, and those embryos showing a high early fluorescence intensity (EFI) were shown to correlate with successive development to the advanced blastocyst stages, while embryos with a low EFI (Fig. 1a & b, arrows) exhibited arrested or delayed development
Summary
Growing concerns about safety of ART on human gametes, embryos, clinical outcomes and longterm health of offspring require improved methods of risk assessment to provide functionally relevant assays for quality control testing and pre-clinical studies prior to clinical implementation. Growing concerns about detrimental effects of ART on human gametes, embryos, clinical outcomes and long-term health of offspring require improved methods of risk assessment to provide scientifically robust and Gilbert et al Reproductive Biology and Endocrinology (2016) 14:13 functionally relevant assays for pre-clinical studies prior to clinical implementation [7,8,9,10]. POU5F1 and CDX2 are initially co-expressed in pre-implantation mouse embryos and form a complex for the reciprocal repression of their target genes in embryonic stem cells [20]. A regulatory complex formed with CDX2 suppresses expression of Pou5f1 in the TE, while the expression of Pou5f1 is maintained within the ICM in the absence of CDX2, thereby establishing localized expression patterns of POU5F1 and CDX2 [21]
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