Bioengineering the 3D In Vitro Ovarian Follicle Mocroenvironment

Abstract

Follicles are the functional units of the ovary and are composed of a germ cell (the oocyte) and layers of somatic cells (granulosa and theca cells). The ovaries contain a limited number of immature follicles. Serving as the ovarian reserve, follicles have the potential to develop and produce mature oocytes capable of fertilization in a highly regulated process called folliculogenesis. Chemo- and radiation therapies used as cancer treatments can have unintended effects on patients such as detrimental effects on the non-replenishable ovarian reserve, resulting primary ovarian insufficiency (POI) and/or infertility. Clinically established fertility preservation methods, such as egg and embryo cryopreservation, are not applicable to all patients. This gap has motivated the development of new strategies to produce mature oocytes to restore fertility at a later date. Among the emerging technologies, in vitro ovarian follicle growth (IVFG) systems represent great translational potentials to harvest fertilizable eggs regardless of patients’ age or hormone stimulation. Isolated single follicles can be cultured in two-dimensional (2D) or three-dimensional (3D) systems. Specifically among 3D methods, encapsulated in vitro follicle growth (eIVFG) systems have been developed to mimic the native 3D physiological environment of the ovaries by maintaining a spherical morphology and the cell-cell and cell-matrix interactions between the oocyte and somatic cells of the follicle.

To better understand reflect various signals (growth factors, hormones, extracellular matrixes, mechanics, etc.) in the culture environment, we investigated multiple aspects of the eIVFG environment to provide guiding principles to facilitate the development of alternative fertility preservation options. We first started with secondary follicles that can autonomously grow in vitro and examined the effects of xenobiotics on developing follicles in vitro. This knowledge not only complemented our current understanding of the toxicity of chemotherapy drugs and environmental toxicants. More importantly, the application of such a high-throughput eIVFG system together with its dynamic culture environment provides a faster and cheaper alternative toxicity evaluation method compared to traditional animal models. Next, we examined the interactions between small, early stage (late primary and early secondary) follicles and supporting stromal cells. By allowing such interactions in our novel biomimetic matrix, we were able to harvest mature, fertilizable oocytes from small follicles. Additionally, by using supporting cells derived in a patient-specific way, this synthetic and xeno-free matrix represents great translational potentials for clinical application. Lastly, we examined the paracrine cues present in various culture environments by monitoring the dynamic activities of specific transcription factors (TFs) in real time. For the first time ever lentiviral transduction of the follicles, we delivered specific TF reporters. By quantifying bioluminescence intensity from the reporters, we investigated the underlying mechanism that results in a number-dependent manner of folliculogenesis. Additionally, this technique can serve as a powerful tool to probe potential TFs on a large scale. By quantifying in real time the dynamic activities of specific TFs, this technique can provide insightful knowledge to the causation between TF activities and phenotypical changes.

In conclusion, by examining multiple aspects of the in vitro ovarian follicle environment, this dissertation provides new understanding of the bioengineering principles in numerous in vitro follicle culture applications and in the field of reproductive biology more broadly. By providing novel solutions to ongoing clinical issues, the research described in this dissertation reveals a translational opportunity for combining biomaterials technology and the field of reproductive biology.