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Dissecting the spatio-temporal development of the human reproductive tract https://doi.org/10.17863/CAM.122352 Lorenzi, Valentina Marioni, John Vento-Tormo, Roser The human reproductive tract plays a crucial role in fertility and overall health due to the systemic effect of sex hormones. Its development depends on tightly regulated processes of sex differentiation, spatial patterning, and morphogenesis, which, when disrupted, can lead to congenital alterations and long-term reproductive disorders. In this thesis, I generated the first comprehensive single-cell and spatial transcriptomic atlas of the human reproductive tract during prenatal development by computationally integrating data from 89 fetal samples spanning 6 to 21 post-conception weeks. This large-scale, multi-organ dataset enabled the investigation of key developmental processes at a resolution not previously achievable in human tissue. Using this atlas, I identified molecular regulators of early reproductive development, including epithelial transcriptional signatures associated with Müllerian duct emergence and elongation, and novel male-biased genes expressed in the Müllerian mesenchyme likely involved in duct regression. I also identified potential regulators of sexual dimorphism in the external genitalia, pinpointing candidate genes involved in urethral canalisation of the penis, with direct relevance to the developmental origins of hypospadias. By integrating histological features with gene expression and chromatin accessibility data, I defined the transcription factors and cell signalling events likely involved in the regionalisation of the Müllerian and Wolffian ducts. This analysis led to a refined model of how the HOX code is established across distinct reproductive organs, including the unexpected enrichment of thoracic HOX gene expression in the rostral mesenchyme of the fallopian tube and epididymis. The atlas further revealed that epithelial regionalisation of the fallopian tube and epididymis, likely supporting functions such as sperm maturation in adulthood, is established early in development. In contrast, the uterocervical canal epithelium remains transcriptionally uniform during mid-gestation, suggesting that its adult architecture depends on hormonal cues that emerge later in fetal life or postnatally. Finally, using this single-cell and spatially-resolved dataset, I predicted that the fetal uterine epithelium would be responsive to estrogen-mimicking endocrine disrupting chemicals, and confirmed this prediction experimentally using fetal-derived epithelial organoids. Exposure to compounds such as bisphenol A and butyl benzyl phthalate led to the upregulation of estrogen-responsive genes, confirming that fetal tissues can mount transcriptional responses to environmental estrogens. 3 By mapping the sex-specific regionalisation and differentiation of the reproductive tract at single-cell resolution, this thesis provides a foundational resource and reveals previously uncharacterised cellular trajectories and putative molecular mechanisms that advance our understanding of human reproductive development. These findings establish a new framework for investigating the developmental origins of reproductive disorders and open new avenues for translational research in diagnostics, toxicology, and regenerative medicine. University of Cambridge 2025-06-13 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/391097 https://www.repository.cam.ac.uk/bitstreams/2ac4f7df-c585-440d-8d90-6e4cc8e024c6/download fca65442b7780638e20475bd9c612648 https://www.repository.cam.ac.uk/bitstreams/7754f294-9973-43e1-90a2-ec2a4ce0e410/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved developmental biology reproductive biology computational biology spatial transcriptomics single-cell genomics data analysis reproductive development