Comprehensive characterization of the human blastocyst's miRNome from the Inner Cell Mass, the Trophectoderm and their related IVF spent culture media

Study question: Can we consistently profile and characterize miRNAs from blastocoel cavity? Summary answer: MicroRNAs can be consistently profiled in human blastocoel cavity and the population of miRNAs detected within the blastocoel is mostly heterogeneous with respect to the cellular counterparts of the blastocyst, with some miRNAs significantly up-regulated or preferentially expressed in the former with respect to human ICM and TE. What is known already: Only proteomic and metabolomic analyses have been published up to date characterizing human blastocoel, but no data have been ever reported profi ling miRNAs within the inner cavity of a blastocyst. There are evidences of miRNAs secretion by donor cells in order to exert a gene regulatory effect upon recipient ones, highly suggesting the possibility of a miRNAs-mediated autocrine communication within the blastocyst via the blastocoel fluid. Study design, size, duration: Five good quality expanded human blastocysts underwent blastocoel collection according to a previously published method between November and December 2014. Blastocoel samples were screened for miRNA content and compared to a reference database previously built on human ICM and TE miRNA expression profiles. Participants/materials, setting, methods: MiRNA expression was evaluated using TLDA Cards A (Applied Biosystems) containing primer sets for 381 human miRNA sequences. Ct values at a level 37 cycles and miRNAs expressed in <60% biological replicates were excluded from the analysis. The mean expression level of expressed miRNAs was used for normalization. Main results and the role of chance: Pearson’s correlation of raw Ct values among blastocoel samples ranged between 0.69 and 0.78. It was instead comprised between 0.33 and 0.45 when comparing them to ICM, and between 0.29 and 0.39 to TE. Minimum and maximum number of miRNAs detected in the blastocoel were 50 and 51. Thirty-three were expressed in at least 60% of samples run. Twenty-one out of them were also common to ICM (63.6%), 24 to TE (72.7%), and 19 to both (57.5%). Normalized Ct hierarchical clustering, built on complete linkage and Euclidean distance, highlighted a sharp division between ICM, TE and blastocoel. The volcano plot built on the parametric test Limma and Benjamini-Hochberg correction showed 6 and 7 miRNAs up-regulated (p < 0.001) in blastocoel versus ICM and TE, respectively. One and 2 were instead respectively down-regulated (p < 0.001). Limitations, reason for caution: Since miRNAs secretion is a dynamic process throughout blastocyst expansion and only fresh good quality expanded blastocysts were included, we need data on blastocoels from thawed, bad quality and/or blastocysts of different expansion grades. Prospective studies are also needed to investigate correlations between blastocoel miRNA profi ling and blastocyst implantation. Wider implications of the findings: This is the first profiling of miRNAs from blastocoel. It can mirror important parameters such as blastocyst morphological quality, metabolic or degenerative processes, and reproductive competence. To this regard, we still lack methods to boost our predictive power upon blastocyst implantation potential beyond current limits, and blastocoel miRNA evaluation is an unexplored strategy in this scenario. Study funding/competing interest(s): Funding by hospital/clinic(s) – G.EN.E.R.A. centres for reproductive medicine. Trial registration number: None.