22 23 KEY WORDS 24 Adult human ovary, Oogonial stem cells, DDX4 antibody, Germline stem cells, Single cell RNA 25 sequencing, Cell surface marker. 26 27 2 ABSTRACT 28 The human ovary orchestrates sex hormone production and undergoes monthly 29 structural changes to release mature oocytes. The outer lining of the ovary (cortex) has 30 a key role in defining fertility in women as it harbors the ovarian reserve. It has been 31 postulated that putative oogonial stem cells exist in the ovarian cortex and that these 32 can be captured by DDX4 antibody isolation. We analysed on a single cell level the 33 transcriptome and cell surface antigen profiles of over 24,000 cells from high quality 34 ovarian cortex samples from 21 patients. Our single cell mapping reveals transcriptional 35 profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, 36 perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular 37 cells, not oogonial stem cells. Our data does not support the existence of germline stem 38 cells in adult human ovaries thereby reinforcing the dogma of a limited ovarian reserve. 39 40 65 in a xeno-transplantation model [9]. The biological significance of these cells is poorly 66 understood. In addition, many laboratories have not been able to repeat these findings and 67 confirm the existence of OSCs [14-16].68 4 Recently, somatic cells from the inner part of human ovaries have been characterized 69 by single cell sequencing [17]. Here, we complete the map of cell types in human ovaries by 70 providing the first extensive characterization of germ cells and somatic cells in the outer layer 71 of human ovaries, i.e. in the cortex. We used tissue samples provided by 21 patients with 72 proven healthy follicles. Our results demonstrate six main cell types, but cannot provide 73 support to the existence of OSCs. This dataset will be a valuable tool for studying the role of 74 specific cell populations in ovarian biology, dissecting causes of infertility, and developing 75 novel assisted reproductive technologies or even contraceptives. 76 77 78 79 5 RESULTS 80 Study setup 81 We used single cell RNA-sequencing (scRNA-seq) and surface marker screening to determine 82 transcriptomes and cell surface proteomes of cells present in ovarian cortex (Fig. 1). We used 83 tissue from altogether 21 Caesarean section (C-sec, N=5) and gender reassignment surgery 84 patients (GRPs, N=16) to have a comprehensive coverage of individuals and commonly used 85 donor types. All tissue samples were validated to contain healthy follicles (Fig. 1). In order to 86 relate the findings to OSCs, we marked the cells with DDX4 antibodies (Ab) in all experiments; 87 the scRNA-seq experiments were done on uncultured, unsorted cells and sorted DDX4 Ab+ 88 and Ab-cell fractions as well as on cultured, sorted DDX4 Ab+ and Ab-cells. The surface 89 marker screen included DDX4 Ab as an additional marker. Data analysis included validation 90 of markers by immunostaining of ovarian samples, and mergi...