Motivation and approach Women are increasingly having children at later ages, which has been attributed to lifestyle choices such as the pursuit of higher education, establishment of careers or postponement of marriage. However, the ability to have children declines with age, with infertility becoming more pronounced after the age of 35. Even with assisted reproductive technologies such as in vitro fertilization (IVF), the pregnancy rate declines with advancing female age [1]. The percentage of pregnancies that failed to result in a live birth increases with age (Table 1) [8-10]. This decline in fecundity and increased risk of spontaneous abortion has primarily been attributed to issues associated with the oocyte. However, the molecular mechanisms underlying ineffective oocyte development remain unclear, as the process of follicle development is difficult to in vivo. We have developed an approach to mature follicles in vitro, which provides the means to investigate nearly all aspects of follicle maturation. Applying this system to the growth and development of aged and juvenile follicles can identify the mechanisms underlying abnormal follicle maturation and develop a predictive algorithm that correlates follicle growth with oocyte quality, and could provide novel reproductive options for women with idiopathic infertility. We propose to employ synthetic scaffolds for ovarian follicle growth and maturation to investigate the underlying cellular and molecular mechanisms of age-related infertility and aneuploidy. A synthetic scaffold can serve as a stroma that creates a cellular environment designed to provide the factors that stimulate follicle maturation, but lacks the factors found in the native stroma that inhibit follicle maturation. Synthetic scaffolds can be created which maintain the appropriate size, shape and architecture of the tissue while providing the necessary signals to direct cellular responses [11]. Importantly, synthetic threedimensional scaffolds function to maintain the intimate physiological connections between the oocytes and somatic cells, which are essential for normal development. We will initially develop the hydrogel for primary and two-layer secondary follicles, and subsequently investigate growth of follicles from juvenile, young adult, and aged mice, and for follicles treated with bisphenol A (BPA). In addition to the traditional endpoints of follicle growth and oocyte quality, we will also characterize the patterns of gene expression and activated signaling pathways. These studies will identify the components of the microenvironment that stimulate growth and development, and also the intrinsic growth potential of the follicle. The objective is to develop an environment that mimics the native stroma and also a molecular signature that can predict oocyte quality. Follicle development Follicle maturation is a complex, multi-stage process that involves multiple cell types, cell-cell and cell-substrate interactions, and a variety of soluble stimuli (e.g., hormones, growth factors) (Fig. 1) [12-14]. During the maturation of primary and secondary follicles, the oocyte increases in volume and the granulosa cells multiply to form several layers. To complete the follicle unit, theca cells from the surrounding stroma differentiate to form a cell layer outside the granulosa cells. Oocyte growth is dependent upon gap junction mediated communication between the oocyte and its companion granulosa cells and the rate of growth is related to the number of granulosa cells coupled to the oocyte [15]. Later in development, follicles are stimulated by growth and differentiation factors and pituitary hormones, such as follicle stimulating hormone (FSH) and luteinizing hormone (LH). FSH acts on a subset of follicles, causing them to begin explosive growth leading to a fully mature follicle. At the end of maturation, the gonadotrophin surges stimulate many events, including oocyte maturation, cumulus expansion, degradation of the surface epithelial cells and ovulation. Oocyte maturation involves progression from prophase of the first meiotic division to metaphase of the second meiotic division. Throughout the life cycle of the follicle, growth factors, hormones, and environmental cues (O2, matrix, cell-cell contacts) change to orchestrate the developmental process. If the oocyte is not fertilized, new follicles are recruited, and the cycle of follicular maturation and hormone activation continues. Within the ovary, the process of follicle maturation is highly regulated, with inhibitory factors that restrict follicle recruitment and maturation. Isolating follicles from the ovary removes these inhibitory stimuli, and can allow follicle development given the appropriate environment.
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