Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing.

The reproductive and endocrine functions of the ovary involve spatially defined interactions among specialized cell populations. Despite the ovary's importance in fertility and endocrine health, functional attributes of ovarian cells are largely uncharacterized. Here, we profiled >18,000 genes in 257 regions from the ovaries of two premenopausal donors to examine the functional units in the ovary. We also generated single-cell RNA sequencing data for 21,198 cells from three additional donors and identified four major cell types and four immune cell subtypes. Custom selection of sampling areas revealed distinct gene activities for oocytes, theca, and granulosa cells. These data contributed panels of oocyte-, theca-, and granulosa-specific genes, thus expanding the knowledge of molecular programs driving follicle development. Serial samples around oocytes and across the cortex and medulla uncovered previously unappreciated variation of hormone and extracellular matrix remodeling activities. This combined spatial and single-cell atlas serves as a resource for future studies of rare cells and pathological states in the ovary.

Ovarian tissue cryopreservation and novel bioengineering approaches for fertility preservation.

PURPOSE OF REVIEW: Breast cancer patients who cannot delay treatment or for whom hormone stimulation and egg retrieval are contraindicated require alternative methods of fertility preservation prior to gonadotoxic treatment. Ovarian tissue cryopreservation is an alternative approach that may offer patients the opportunity to preserve fertility and carry biologically-related children later in life. Various experimental approaches are being explored to obtain mature gametes from cryopreserved and thawed ovarian tissue for fertilization and implantation using biomimetic tissue culture in vitro. Here we review the most recent developments in ovarian tissue cryopreservation and exciting advances in bioengineering approaches to in vitro tissue and ovarian follicle culture. RECENT FINDINGS: Slow freezing is the most widely accepted method for ovarian tissue cryopreservation, but efforts have been made to modify vitrification for this application as well. Numerous approaches to in vitro tissue and follicle culture are in development, most prominently two-step culture systems for ovarian cortical tissue and encapsulation of ovarian follicles in biomimetic matrices for in vitro culture. SUMMARY: Refinements to slow freeze and vitrification protocols continue to address challenges associated with cryopreservation, such as ice crystal formation and damage to the stroma. Similarly, improvements to in vitro tissue and follicle culture show promise for utilizing patients' cryopreserved tissues to obtain mature gametes after disease treatment and remission. Development of an effective and reproducible culture system for human ovarian follicles will serve as a broad assisted reproductive technology for cancer survivors who cryopreserved tissue prior to treatment.

Follicle development as an orchestrated signaling network in a 3D organoid.

The ovarian follicle is the structural and functional unit of the ovary, composed of the female gamete (the oocyte) and supportive somatic cells. Follicles are not only the source of a female's germ cell supply, but also secrete important hormones necessary for proper endocrine function. Folliculogenesis, the growth and maturation of the follicular unit, is a complex process governed by both intrafollicular crosstalk and pituitary-secreted hormones. While the later stages of this process are gonadotropin-dependent, early folliculogenesis appears to be controlled by the ovarian microenvironment and intrafollicular paracrine and autocrine signaling. In vitro follicle culture remains challenging because of the limited knowledge of growth factors and other cytokines influencing early follicle growth. Here we discuss the current state of knowledge on paracrine and autocrine signaling influencing primary follicles as they develop into the antral stage. Given the importance of intrafollicular signaling and the ovarian microenvironment, we reviewed the current engineering approaches for in vitro follicle culture, including 3D systems using natural hydrogels such as alginate and synthetic hydrogels such as poly(ethylene glycol). Our discussion is focused on what drives the proliferation of granulosa cells, development of the thecal layer, and antrum formation-three processes integral to follicle growth up to the antral stage. Further research in this area may reveal the mechanisms behind these complex signaling relationships within the follicle, leading to more successful and physiologically-relevant in vitro culture methods that will translate well to clinical applications.