A total of 59 uterus samples (whole uterus and endometrial biopsies) from healthy donors and 25 endometrial biopsies from patients diagnosed with severe preeclampsia (sPE patients) and their healthy controls have been collected (Reproductive Medicine Collection, registered under reference C.0005866 in the national registry of biobanks in the ISCIII Health institute. Spain). During HUTER execution, samples processing has provide 117 cDNA libraries for sc-RNA sequencing, 37 paraffin blocks for Protein Atlas, 40 OCT blocks for Spatial Transcriptomics, 12 myometrial samples and 18 sPE & control endometrium sorted plates for sc-epigenomics and 53 snap frozen samples to ehole genomic sequencing (WSG).
HUTER partners have gained expertise in conducting the 10X Genomics Spatial Gene Expression protocols on uterine samples and profiling more than 1 million human uterine cells with single-cell or single-nuclei RNA-seq. Generated data has allowed a better characterisation of uterine tissues and helped to better define uterine cell subpopulations and cell-to-cell interactions. This information has revealed how cells organize into uterine tissues and allow the regulation of important processes such the menstrual cycle or embryo implantation, and how deregulation of those processes could contribute to infertility and disease.
Using the optimised scEM&T-seq protocol we generated single-cell transcriptomes and methylomes for the primary endometrial and myometrial samples. Processing of SMART-seq2 scRNA-seq libraries revealed that 56-89% of samples had worked efficiently. This project has generated single cell and in silico merged methylation atlases for endometrium and myometrium biopsies. Multi-omics protocols have been used to deconvolute samples into their major cell types using specific marker expression and determine their unique methylation profiles. Also cell-specific endometrial methylation signatures linked with aberrant profiles associated with sPE.
HUTER have also gained further expertise in all the technologies and pipelines that have been used for generation of high-resolution spatial maps. We generated Digital high-resolution images corresponding to all HUTER samples IHC stained, comprising in total 2,261 images. The spatial characterization of cell types in endometrium and myometrium was enhanced by the use of an immunofluorescence multiplex approach developed. Therefore, we have located in the tissue the previously uncharacterised cell states uncovered. One of the main focus has been on the novel epithelial progenitors previously defined in our single-cell transcriptomics dataset, as well as the microenvironment essential to maintain these progenitors.
The HUTER project also implemented an open-source platform making the first comprehensive single-cell and spatial atlas of the uterus across the lifespan accessible to the scientific community. The platform enabled data uploading and data query for raw data and analytical results from our single cell molecular characterisation, epigenomics, transcriptomics and spatial resolution images. The platform includes advanced visualization tools, providing intuitive visualization of data with high dimensionality, and facilitating comparative assessments. The HUTER platform performance has been validated by used to identify cell types, states and transcriptional fingerprints related to endometrial decidualization resistance in sPE patients. Using scRNA-seq results from scEM&T-seq dataset generated, we observed that the most robust differential expressed genes were located in within stromal-associated clusters.
Finally, HUTER jointly with other EU funded Consortia (pilot actions to build the foundations of the human cell atlas) have stablished alignment with different working groups of the Human Cell Atlas (HCA) initiative and HCA-DCP managers that will facilitate current and further progress in the field.
