Characterization and quantification of proteins secreted by single human embryos prior to implantation.
Bottom Line: By using targeted proteomics, we demonstrate the feasibility of quantifying multiple proteins in samples derived from single blastocoels and that such measurements correlate with aspects of embryo viability, such as chromosomal (ploidy) status.This study illustrates the potential of high-sensitivity proteomics to measure clinically relevant biomarkers in minute samples and, more specifically, suggests that key aspects of embryo competence could be measured using a proteomic-based strategy, with negligible risk of harm to the living embryo.Our work paves the way for the development of "next-generation" embryo competence assessment strategies, based on functional proteomics.
Affiliation: Nuffield Department of Obstetrics and Gynaecology, Institute of Reproductive Sciences University of Oxford, Oxford, UK Oxford Fertility Unit, Institute of Reproductive Sciences, Oxford, UK Reprogenetics UK, Institute of Reproductive Sciences, Oxford, UK.Show MeSH
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Mentions: Blastosol samples were collected using blastocentesis, allowing the collection of 4–6 nL from each blastocyst cavity (Materials and Methods) (Movie EV1). Samples were initially processed using a standard procedure involving urea for protein solubilization and peptide desalting following enzymatic digestion. However, this approach was only compatible with pooled samples containing at least 20 blastocoels. For smaller samples, the majority of the peptides were lost during the multiple steps of the procedure. Therefore, a previously described nano-scale protocol was adapted for the analysis of individual blastocoel samples (Wang et al, 2005). This methodology utilized volatile buffers and organic solvents and had no requirement for peptide desalting prior to MS analysis (referred to as MonoPrep). In preliminary experiments, the MonoPrep procedure allowed the detection of more than 2,000 peptides comprising as little as 80 ng of proteins (cytoplasmic extract obtained from ∼1,000 HeLa cells) (FigEV1A). We performed shotgun proteomics analysis on 80 blastocoels sampled from human embryos 5 or 6 days after fertilization. The samples were divided into four sets each composed of 20 pooled specimens. Two sets were processed using the standard urea-based procedure and two with MonoPrep. For both procedures, blank samples obtained from plated sterile PBS microdrops swiped with a microneedle of the same type used for blastosol collection were analyzed in parallel and treated as controls for the identification of contaminant proteins.
Affiliation: Nuffield Department of Obstetrics and Gynaecology, Institute of Reproductive Sciences University of Oxford, Oxford, UK Oxford Fertility Unit, Institute of Reproductive Sciences, Oxford, UK Reprogenetics UK, Institute of Reproductive Sciences, Oxford, UK.