Making a good egg: human oocyte health, aging, and in vitro development.

Mammalian eggs (oocytes) are formed during fetal life and establish associations with somatic cells to form primordial follicles that create a store of germ cells (the primordial pool). The size of this pool is influenced by key events during the formation of germ cells and by factors that influence the subsequent activation of follicle growth. These regulatory pathways must ensure that the reserve of oocytes within primordial follicles in humans lasts for up to 50 years, yet only approximately 0.1% will ever be ovulated with the rest undergoing degeneration. This review outlines the mechanisms and regulatory pathways that govern the processes of oocyte and follicle formation and later growth, within the ovarian stroma, through to ovulation with particular reference to human oocytes/follicles. In addition, the effects of aging on female reproductive capacity through changes in oocyte number and quality are emphasized, with both the cellular mechanisms and clinical implications discussed. Finally, the details of current developments in culture systems that support all stages of follicle growth to generate mature oocytes in vitro and emerging prospects for making new oocytes from stem cells are outlined.

Spatio-temporal remodelling of the composition and architecture of the human ovarian cortical extracellular matrix during in vitro culture.

STUDY QUESTION: How does in vitro culture alter the human ovarian cortical extracellular matrix (ECM) network structure? SUMMARY ANSWER: The ECM composition and architecture vary in the different layers of the ovarian cortex and are remodelled during in vitro culture. WHAT IS KNOWN ALREADY: The ovarian ECM is the scaffold within which follicles and stromal cells are organized. Its composition and structural properties constantly evolve to accommodate follicle development and expansion. Tissue preparation for culture of primordial follicles within the native ECM involves mechanical loosening; this induces undefined modifications in the ECM network and alters cell-cell contact, leading to spontaneous follicle activation. STUDY DESIGN, SIZE, DURATION: Fresh ovarian cortical biopsies were obtained from six women aged 28-38 years (mean ± SD: 32.7 ± 4.1 years) at elective caesarean section. Biopsies were cut into fragments of ∼4 × 1 × 1 mm and cultured for 0, 2, 4, or 6 days (D). PARTICIPANTS/MATERIALS, SETTING, METHODS: Primordial follicle activation, stromal cell density, and ECM-related protein (collagen, elastin, fibronectin, laminin) positive area in the entire cortex were quantified at each time point using histological and immunohistological analysis. Collagen and elastin content, collagen fibre characteristics, and follicle distribution within the tissue were further quantified within each layer of the human ovarian cortex, namely the outer cortex, the mid-cortex, and the cortex-medulla junction regions. MAIN RESULTS AND THE ROLE OF CHANCE: Primordial follicle activation occurred concomitantly with a loosening of the ovarian cortex during culture, characterized by an early decrease in stromal cell density from 3.6 ± 0.2 × 106 at day 0 (D0) to 2.8 ± 0.1 × 106 cells/mm3 at D2 (P = 0.033) and a dynamic remodelling of the ECM. Notably, collagen content gradually fell from 55.5 ± 1.7% positive area at D0 to 42.3 ± 1.1% at D6 (P = 0.001), while elastin increased from 1.1 ± 0.2% at D0 to 1.9 ± 0.1% at D6 (P = 0.001). Fibronectin and laminin content remained stable. Moreover, collagen and elastin distribution were uneven throughout the cortex and during culture. Analysis at the sub-region level showed that collagen deposition was maximal in the outer cortex and the lowest in the mid-cortex (69.4 ± 1.2% versus 53.8 ± 0.8% positive area, respectively, P < 0.0001), and cortical collagen staining overall decreased from D0 to D2 (65.2 ± 2.4% versus 60.6 ± 1.8%, P = 0.033) then stabilized. Elastin showed the converse distribution, being most concentrated at the cortex-medulla junction (3.7 ± 0.6% versus 0.9 ± 0.2% in the outer cortex, P < 0.0001), and cortical elastin peaked at D6 compared to D0 (3.1 ± 0.5% versus 1.3 ± 0.2%, P < 0.0001). This was corroborated by a specific signature of the collagen fibre type across the cortex, indicating a distinct phenotype of the ovarian cortical ECM depending on region and culture period that might be responsible for the spatio-temporal and developmental pattern of follicular distribution observed within the cortex. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ovarian cortical biopsies were obtained from women undergoing caesarean sections. As such, the data obtained may not accurately reflect the ECM distribution and structure of non-pregnant women. WIDER IMPLICATIONS OF THE FINDINGS: Clarifying the composition and architecture signature of the human ovarian cortical ECM provides a foundation for further exploration of ovarian microenvironments. It is also critical for understanding the ECM-follicle interactions regulating follicle quiescence and awakening, leading to improvements in both in vitro activation and in vitro growth techniques. STUDY FUNDING/COMPETING INTEREST(S): Medical Research Council grant MR/R003246/1 and Wellcome Trust Collaborative Award in Science: 215625/Z/19/Z. The authors have no conflicts to declare. TRIAL REGISTRATION NUMBER: N/A.

Implications of Nonphysiological Ovarian Primordial Follicle Activation for Fertility Preservation.

In recent years, ovarian tissue cryopreservation has rapidly developed as a successful method for preserving the fertility of girls and young women with cancer or benign conditions requiring gonadotoxic therapy, and is now becoming widely recognized as an effective alternative to oocyte and embryo freezing when not feasible. Primordial follicles are the most abundant population of follicles in the ovary, and their relatively quiescent metabolism makes them more resistant to cryoinjury. This dormant pool represents a key target for fertility preservation strategies as a resource for generating high-quality oocytes. However, development of mature, competent oocytes derived from primordial follicles is challenging, particularly in larger mammals. One of the main barriers is the substantial knowledge gap regarding the regulation of the balance between dormancy and activation of primordial follicles to initiate their growing phase. In addition, experimental and clinical factors also affect dormant follicle demise, while the mechanisms involved remain largely to be elucidated. Moreover, most of our basic knowledge of these processes comes from rodent studies and should be extrapolated to humans with caution, considering the differences between species in the reproductive field. Overcoming these obstacles is essential to improving both the quantity and the quality of mature oocytes available for further fertilization, and may have valuable biological and clinical applications, especially in fertility preservation procedures. This review provides an update on current knowledge of mammalian primordial follicle activation under both physiological and nonphysiological conditions, and discusses implications for fertility preservation and priorities for future research.

Interaction between PI3K/AKT and Hippo pathways during in vitro follicular activation and response to fragmentation and chemotherapy exposure using a mouse immature ovary model.

Understanding and control of the massive and accelerated follicular growth that occurs during in vitro culture of ovarian tissue is a crucial step toward the development of efficient culture systems that offer an attractive alternative to ovarian tissue transplantation for fertility restoration in cancer survivors. One outstanding question focuses on processes that occur prior to cryopreservation, such as tissue sectioning or chemotherapeutic treatment, might exacerbate this follicular activation. Although the PI3K/AKT/mTOR pathway is well known as a major trigger of physiological and chemotherapy-induced follicular activation, studies have shown that disruption of Hippo pathway due to ovarian fragmentation acts as an additional stimulator. This study aimed to characterize the possible interactions between these pathways using post-natal day 3 mouse ovaries cultured for 4 or 48 h. Morphology, gene transcription, and protein levels were assessed to investigate the impact of sectioning or chemotherapy exposure (4-hydroperoxycyclophosphamide [4HC], 3 and 20 µM). The effect of an mTORC1 inhibitor, Everolimus, alone or as a 4HC co-treatment to prevent follicle activation was evaluated. The results showed that organ removal from its physiological environment was as effective as sectioning for disruption of Hippo pathway and induction of follicle activation. Both PI3K/AKT/mTOR and Hippo pathways were involved in chemotherapy-induced follicular activation and responded to fragmentation. Surprisingly, Everolimus was able to prevent the activation of both pathways during chemotherapy exposure, suggesting cross-talk between them. This study underscores the major involvement of PI3K/AKT/mTOR and Hippo pathways in in vitro follicle activation and provides evidence that both can be regulated using mTORC1 inhibitor.