SUMMARY The placenta, uterus, and fallopian tubes act in concert to achieve successful pregnancy outcomes. They undergo marked changes in structure and function during pregnancy, coordinating with one another and other maternal organs via direct and distant cell-cell interactions. Here, we propose to generate and integrate data from in vivo imaging and ex vivo histopathologic and multi-omic bulk, single-nucleus, and spatial profiling data, in order to generate reference multiscale 3D maps of healthy human pregnant female reproductive organs across pregnancy. These will serve as key frames of reference for future studies aiming to discover how perturbation of tissue structure and function leads to organ dysfunction and disease. Our team is well-equipped to achieve this goal, with expertise in clinical Obstetrics & Gynecology and Placental/Perinatal Pathology, imaging, spatial molecular profiling, single-cell omics, extracellular matrix (ECM) biology, data management, and computational biology. We will leverage our well-established translational research infrastructure to recruit diverse cohorts of healthy pregnant women. We will then perform in vivo imaging using MRI and ultrasound to delineate tissue structure, perfusion, inflammation, calcifications, and other features. After delivery/surgery, tissues will be rapidly sampled, processed, and stored in multiple ways (fresh, flash frozen, preserved in solutions for optimal nucleic acid analysis, and FFPE) to enable generation of high-quality data using a variety of molecular approaches, with additional samples retained for future studies. Detailed review of clinical data and formal histopathologic evaluation of adjacent tissue sections will be performed to confirm that normal tissue has been obtained from subjects with normal outcomes. Initial ex vivo analysis includes extracellular matrix proteomics, and bulk and single-nucleus RNAseq and ATACseq. The results of these studies will be analyzed to identify targets for the subsequent imaging mass cytometry (IMC) and spatial transcriptomic studies. These data types will be integrated to enable mapping of the relationships among different cell types and between cells and the surrounding extracellular matrix on the microscopic level. We note that generation of multiple data types from adjacent samples, and the use of the Multi-Ome assay (which performs RNAseq and ATACseq data indexed to the same nuclei) and spatial transcriptomic methods with immunohistochemistry-based pre-imaging, will enable us to bridge between diverse datasets. The unique populations of endothelial cells in the placenta and the endothelial mimicry of the extravillous trophoblast will provide unique opportunities to integrate with other endothelial-centric HuBMAP projects (e.g. lymphatic system, endothelial atlas, and kidney atlas). Close interactions with other HuBMAP Centers will be established to enable harmonization of data and metadata standards, sharing of resources (e.g. antibodies with HuBMAP Centers that are developing/using CODEX and MIBI-TOF), rapid adoption of new technologies, collaborative data analysis, and rapid sharing of data with the HIVE and the broader scientific community.

NARRATIVE The goals of the Pregnant Female Reproductive Tissue Mapping Center are to develop three-dimensional multiscale maps of the placenta, pregnant uterine endomyometrium and fallopian tubes, to explore signaling between the placenta and maternal tissues, and to trace the trajectories of cellular differentiation and migration in the placenta across gestation. To do this, we will apply a range of imaging and molecular profiling modalities, including in vivo MRI and ultrasound imaging, and ex vivo extracellular matrix proteomics, formal histopathologic evaluation, imaging mass cytometry, single-nucleus multiomics, spatial transcriptomics and deep morphologic and hyperplex spatial immunoprofiling. The resulting data will be analyzed and distributed to the broader research community in collaboration with the HuBMAP Integration, Visualization, and Engagement (HIVE) Collaboratory. This project is relevant to public health because the resulting 3D multiscale maps of normal pregnant female reproductive tissues will serve as key frames of reference for future studies aiming to discover how perturbation of tissue structure and/or function leads to organ dysfunction in common, yet poorly understood, pregnancy complications, such as preeclampsia and preterm labor.