Assessment of a high fat diet on transcriptomic and epigenomic profiles of Immortalized Human Endometrial Stromal Cells

Infertility is a multifactorial pathological condition affecting approximately 10–15% of couples, especially in industrialized regions that could be influenced by many factors, such as improper eating habits. Diet, including hypercaloric nutrition due to a high fat diet (HFD), could promote an increment in endometrial epigenetic modifications and dysfunction improving the risk of infertility. Thereby, the aim of the project is to study in vitro the effect of an excessive intake of non-esterified fatty acids, mimicking the HFD lifestyle, on transcriptomic and epigenomic signatures of Immortalized Human Endometrial Stromal Cells (HESC) (Abmgood). Thus, the Affymetrix Microarray platform will be put into use to investigate gene and microRNA (miRNA) expression using the GeneChip Scanner 3000Dx v2. Moreover, for methylome analysis, NGS Illumina platforms will be used and processed by the NextSeq 550 and MiSeq detection systems. So far, the first part of the PhD course focused mainly on the study of the literature for the choice of the HESC cell line and on apprehending the main molecular biology techniques useful for its future development. The methods learned include: the validation of protocols for manual extraction of DNA and total RNA, including miRNAs, from single cell as well as pooled cells with consequent quantization of nucleic acids; the validation of bisulfite conversion/cleanup of converted DNA and PCR amplification for methylation analysis protocols for pyrosequencing, and of reverse transcription and Real-Time protocols (qRT-PCR) for target analysis of gene expression and miRNAs both by SYBR and TaqMan array panels. All the experiments were performed on different in vitro models. Therefore, the next step will be to extend the acquired knowledge and perform all the experiments through omics investigations to have a deeper knowledge from a molecular point of view that might help clarify the multifaceted elements of infertility and potentially curative treatments.