Fig. 1. Human ovary and study design.

a A representative ovary cross-section of a 26-year-old GRP showing the thin cortex layer (∼1 mm, used for fertility preservation) harboring clusters of small primordial follicles that form the ovarian reserve. Beneath the cortex is the inner part of the ovary, medulla, with looser connective tissue, denser vasculature, and larger growing follicles. Yellow rectangle is enlarged to highlight the ovarian reserve composed of primordial follicles resting in the cortex. One early growing (primary) follicle is indicated (arrowhead). Red rectangle is enlarged to highlight the more prominent vasculature in the medulla. Scale bars: 2 mm (overview), 250 µm (zoom-in). b Schematic representation of the study. In total, ovarian cortical tissue from 16 GRPs (mean age 26y) and five C-sec patients (mean age 34y) were used. Ovaries were trimmed upon tissue receival to separate the cortex. Cortex was cut into pieces, and the quality was assessed via general histology and viability of GRP follicles was further confirmed with neutral red staining. The samples were cryopreserved. Thawed ovarian cortex was enzymatically digested into single-cell suspension, filtered, and subjected either uncultured to single-cell transcriptome and surface proteome profiling using 10xGenomics technology and cell surface marker screening panel or cultured under oogonial stem cell (OSC) conditions and subjected to single-cell transcriptome analysis using Smart-seq2. In all assays, cells were stained with DDX4 Ab in order to relate the findings to DDX4 Ab+ (suggested OSCs) and Ab− ovarian cell populations. In the final data analysis, cortical cell populations were identified, related to cultured DDX4 Ab+ and Ab− cells, and compared with cells in the inner part of human ovaries²¹ and fetal ovarian cell populations²². Expression of key markers was validated with immunostainings, qPCR and RNA-FISH. Ab antibody, C-sec cesarean section, GRP gender reassignment patient, qPCR quantitative real-time PCR, RNA-FISH RNA-fluorescent in situ hybridisation, scRNA-seq single-cell RNA-sequencing.