Investigating Follicle Quality and Activation Mechanisms in Cryopreserved Human Ovarian Cortex

Abstract

Over the last 30 years, cancer incidence has increased among children, adolescents and young adults. Encouragingly, advanced detection and treatment regimens have increased cancer survivorship. Unfortunately, many chemotherapy agents can severely damage the non-renewable pool of ovarian follicles that are the only source of the patient’s future fertility and ovarian endocrine function, leaving more cancer survivors facing quality-of-life issues caused by the gonadotoxicity of their anti-cancer therapies. A decrease in the follicular reserve may lead to an inability to enter pubertal development or an earlier onset of menopause, a condition known as primary ovarian insufficiency (POI). For these reasons, prepubertal patients are offered fertility preservation procedures prior to receiving anti-cancer therapy. Currently, the only available option for these patients is ovarian tissue cryopreservation and auto-transplantation (OTCT). During this process, the ovarian cortex containing the follicular reserve is resected and cryopreserved prior to treatment, and after cancer survival, this tissue can be auto-transplanted back into the patient. Unfortunately, this process raises the risk of reintroducing malignant cells potentially harbored in the tissue, highlighting an urgent need for alternative fertility preservation methods. One such experimental method is in vitro activation (IVA) in which the cryopreserved ovarian cortex pieces, or isolated immature follicles, are grown in a lab to eventually obtain mature fertilizable eggs. Successful IVA depends on the good quality of the frozen ovarian cortex tissue and follicles within it and an in-depth understanding of the mechanisms that drive follicle activation. This thesis focused on analyzing the quality and transcriptomic profiles of cryopreserved oocytes in human ovarian cortex tissue and characterizing a mechanically sensitive signaling cascade in human follicles transitioning from dormant to active stages. Our approach involved studying oocytes and somatic cells from early-stage follicles within cryopreserved human ovarian cortex tissue. Through our partnership with the International Institute for the Advancement of Medicine (IIAM), this dissertation research exclusively utilized the donation of human ovarian tissue from deceased donors which we have meticulously characterized for our repository of 50 donors. In Chapter 2 we aimed to understand transcriptomic changes that may arise due the freeze-thaw process. We performed single-oocyte RNA-sequencing on fresh and previously cryopreserved early-stage oocytes and discovered that aside from an expected level of cytoskeletal stress, the transcriptional profiles of fresh and frozen oocytes were similar reassuring the safety of tissue cryopreservation for fertility preservation. In Chapter 3, we investigated the potential transcriptome divergence among oocytes from early-stage follicles to help elucidate changes that arise during the critical period of follicle activation. Our results showed the oocyte transcriptomes formed two interconnected clusters, with differentially expressed genes (DEGs) that represented an early-stage ‘dormant’ profile and a later-stage ‘active’ profile. This novel transcriptomic characterization of oocytes within the human ovarian cortex complements the recent atlasing efforts of the human ovary. Finally, in Chapter 4, we investigated the mechanically sensitive Hippo signaling cascade in human oocytes and granulosa cells (GCs), which has been implicated in murine follicle activation, but never validated in humans. Indeed, our findings confirmed the conserved role of the Hippo cascade in human follicle activation. In conclusion, the work presented here provides in-depth analysis and characterization of human early-stage follicles to further inform the development of alternative fertility preservation strategies for patients in need.