Currently, 12% of the reproductive age population (aged 15–44 years) experience difficulty getting pregnant or carrying a baby to term. Infertility is caused by a multitude of health problems; however, an absence or paucity of germline cells guarantees infertility because this population of cells is the sole provider of genetic and epigenetic information passing from parent to offspring. The germ cell lineage eventually forms the gametes (eggs and sperm) and thus if germline cell development is compromised or lost, the outcome is infertility. One form of female infertility caused by errors in the germline is primary ovarian insufficiency (POI). When there are errors in germ cell specification or differentiation into oocytes, the size or quality of the female's oocyte pool is compromised leading to POI. With this research project, I intend to elucidate developmental mechanisms that control the specification of germ cells on their journey to become oocytes'. Through the work proposed under this F32 application, I plan to use hiPSCs derived from monozygotic (MZ) twins with discordant POI to improve our understanding of human germ cell development, and to determine whether loss of germ cells can be reversed in infertile women through induced reprogramming followed by germ cell differentiation. The MZ twins employed in this study are discordant for the disease POI and have been consented to donate skin bunch biopsies. These discordant MZ twins provide a powerful form of case-control study for assessing the epigenome independent of major underlying genomic variation that can confound comparisons with un-related individuals. I propose three aims to discern whether it is possible to regenerate oocytes in the infertile twin sister and therefore theoretically restore fertility. The first aim will address whether germline competency can be resorted to the infertile twin using epigenetic reprogramming of skin. My preliminary data using one pair of twins shows that hiPSC lines derived from the twin pair can be differentiated into primordial germ cell-like cells (hPGCLCs), this will be evaluated in additional twin pairs. In aim 2, we will use stem cell models of embryonic-like sac structures to identify whether hPGCLCs first emerge in the amnion of each twin. In aim 3, we will generate ovarian organoids using the twins' hiPSCs to determine whether each twin has the ability to differentiate oocytes. These investigations will produce novel research that could lead to methods of eventual fertility restoration through the study of differentiating stem cells to the germ cell lineage in vitro.