Unknown,Transcriptomics,Genomics,Proteomics

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Single-cell RNA-seq reveal lineage formation and X-chromosome dosage compensation in human preimplantation embryos

ABSTRACT: Single-cell RNA-seq of 1,529 cells obtained from 88 human preimplantation embryos ranging from embryonic day 3 to 7.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Homo sapiens

SUBMITTER: Sophie Petropoulos

PROVIDER: E-MTAB-3929 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Study question: Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? Summary answer: A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable. What is known already: There are no data in the literature on what non-declared proteins are present in un-conditioned (fresh media in which no embryos have been cultured) commercial embryo media. Study design, size, duration: The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analysed for each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analysed. Participants/materials, setting, methods: For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulphate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. Main results and the role of chance: Using advanced mass spectrometry and high confidence criteria for accepting proteins (p< 0.01), a total of 110 proteins other than HSA were identified. The average serum albumin content was found to be 94% (92% - 97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins. Limitations, reasons for caution: For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions. Wider implications of the findings: The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analysing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in un-conditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring.

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Single-cell RNA-seq reveal lineage formation and X-chromosome dosage compensation in human preimplantation embryos

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