Cell atlas of the human female reproductive system across the lifespan

Sexual reproduction depends on the fusion of gametes (sperm and eggs) during fertilisation followed by implantation of the resulting embryo in the lining of the womb (the endometrium). Gametes halve their genome in preparation for fertilisation during a specialized cell division known as meiosis. The process by which meiosis is co-ordinated with gamete formation in females is poorly understood, principally because it commences in utero and is not completed until decades later when the mature egg is ovulated and fertilised.

From 3-5 weeks after fertilisation primordial germ cells (PGCs) begin to populate the ovary. Work on mice indicates that the PGCs form oogonia which undergo multiple rounds of mitotic cell division, remaining linked by cytoplasmic bridges as a consequence of incomplete cell division. Towards the end of the first trimester, these interlinked nests of cells begin to transition from mitosis to meiosis asynchronously across the ovary. The pre-meiotic round of DNA replication is followed by entry into meiotic prophase, which is marked by meiotic recombination following which pairs of homologous chromosomes remain linked at the sites of reciprocal exchange of DNA. This is co-ordinated with breakdown of cytoplasmic bridges to form individual oocytes, which in turn become surrounded by a small number of nurse cells (granulosa cells) to form a primordial follicle. Our knowledge of these events in the developing human ovary is fragmentary, and there appears be key differences between mouse and human.

The pool of primordial follicles present at birth contain the life-time supply of oocytes. Little is known about how oocytes maintain cellular homeostasis during decades of arrest in the non-growing phase. Of particular interest are the mechanisms employed to maintain their nuclear and mitochondrial genomes in pristine condition for transmission to the next generation. Cohorts of primordial follicles are recruited from the pool on an ongoing basis. Initiation of oocyte growth is accompanied by morphological changes and reorganisation of the surrounding granulosa cells and it is not known whether this is a cause or a consequence of the initiation of oocyte growth, or indeed whether other surrounding cells, known as stromal cells, have a role in initiating oocyte growth.

Following the onset of puberty, ovarian follicles can develop to the pro-ovulatory stage in response to hormonal triggers. Differentiation of the granulosa cells surrounding the oocyte enables fully grown oocytes to exit meiotic prophase and undergo the first meiotic division shortly before ovulation. In addition, hormones produced by the granulosa cells prepare the endometrium for implantation. While we have a broad understanding of the endocrine control of ovulation and endometrial priming, less is known about the molecular regulation of these transitions at the cellular level in the follicle and in the endometrium.

To address the many gaps in our knowledge of the development of the female germline and reproductive system in humans, we propose to generate a comprehensive cell atlas of the human ovary and endometrium from fetal development until advanced reproductive age. We will combine gene-expression profiling at the single-cell level with new methods of gene-expression detection directly on the tissue to study the cellular environment at an unprecedented resolution and coverage. Information about the expression of the myriad of cells and cell states in the human ovary and endometrium will greatly advance our understanding of the development of the female reproductive system and the emergence and maintenance of the female germline in humans. The work will provide new insights into the causes of infertility and will inform strategies on how to grow oocytes in vitro.

Sexual reproduction depends on the fusion of haploid gametes during fertilisation to form a diploid embryo, and on the endocrine regulation of the endometrium by the ovary to provide conditions for successful implantation. Despite their intimate cellular interactions and functional interdependence, holistic approaches to studying the female germline, the ovary, and the endometrium have been lacking. Indeed, in the context of human development, there is a dearth of systematic studies involving any of the tissues to be investigated here. Gametes halve their genome in preparation for fertilisation during a specialized cell division known as meiosis. However, the process by which meiosis is co-ordinated with gamete formation in females is poorly understood, principally because it commences in utero and is not completed until decades later when the mature egg is ovulated and fertilised.

In the present work, we will study the cell types and states, their spatial location and cellular interactions during the development of the fetal ovaries and the adult monthly remodelling of the ovaries and endometrium. We will combine single-cell and spatial transcriptomics methods to create a molecular spatial atlas of the female reproductive tissue in the fetal and adult life. In addition, by combining spatial transcriptomics methods with high-resolution microscopy we will be able to map the female germ cell proliferation and differentiation. Finally, we will use our cell-cell communication pipeline (www.cellphonedb.org) to map the cell-cell communication events in the female reproductive tissue across the menstrual cycle.

Because our experimental approach enables us to give biological context to gene expression data, the work proposed here will hugely advance our understanding of the basic biology of human female reproduction. It will also give foundation to a better understanding of the causes of female infertility.

The work outlined in the current project will enable us to maintain our position as world leaders in this field. The immediate benefit of our comprehensive analysis of the female reproductive tissue is the advancement of scientific knowledge. In the long- term, the findings will have the potential to how inform new strategies on female reproductive health and ageing and may underpin new treatments for female infertility.

Advancement of scientific knowledge and training: The use of novel single cell technologies to analyse the valuable samples of the ovaries and endometrium during development and adult maturation will push the boundaries of our current knowledge about female reproduction. By generating a comprehensive cell atlas of the human ovary and endometrium from fetal development until advanced reproductive we will greatly advance our understanding of the development of the female reproductive system. As the first systematic analysis of human ovarian development, the work will have a considerable impact on our knowledge of the development of female germ cells in humans. The work will also provide insight into developmental determinants of female reproductive ageing in the context of the size of reserve of oocytes in the ovary and their propensity to missegregate chromosomes during the meiotic divisions. Importantly, the work will provide an invaluable resource for future studies on female reproduction and fertility. The insights generated will be disseminated through presentations at scientific conferences and in international peer-reviewed journals. In addition, the data will be deposited in publicly accessible databases like the data coordination platform (DCP).

Health and well-being: Our vision is that the work proposed here will contribute to (i) prevention of infertility by giving foundation to the identification of new biomarkers to enable better management of female reproductive health (ii) development of new treatments for infertility. We will build on our strengths in translational research to exploit the clinical potential of relevant findings.