SUMMARY The placenta, uterus, ovary, and fallopian tube act in concert to achieve successful pregnancy outcomes. These organs undergo marked changes in structure and function throughout the lifespan, coordinating with one another via direct and distant cell-cell interactions. Here, we propose to generate and integrate data from in vivo imaging and ex vivo histopathologic, biomechanical, and multi-omic spatial profiling data, in order to generate reference multiscale 3D maps of healthy human female reproductive organs representing a range of normal physiologic states that exist at different points in a woman’s life. 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 and Gynecologic Pathology, imaging, spatial molecular profiling, single- cell omics, extracellular matrix (ECM) biology, biomechanical testing, data management, and computational biology. We will leverage our well-established translational research infrastructure to recruit diverse cohorts of healthy pregnant women and non-pregnant premenopausal and postmenopausal women undergoing gynecologic surgery for benign indications. 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 extra 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 will include biomechanical testing, extracellular matrix proteomics, and bulk and single-cell 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, and between tissue architecture and the biomechanical properties and imaging features on a macroscopic level. 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,...