The toxic effects of chemotherapy and/or radiation leave many young cancer survivors unable to have children later on in life. Some women choose to undergo in vitro fertilization (IVF) and freeze the embryos before starting chemotherapy. This is not an option for women and girls who do not have a life partner, or for those who have an aggressive form of cancer that requires immediate therapy because IVF requires lengthy hormonal treatments to produce mature eggs (oocytes) in the ovaries, as well as availability of donor sperm. In such cases, the only possibility for long-term preservation of fertility is removal and freezing of ovaries or pieces of ovarian tissue. After the patient is cancer-free and ready to start a family, banked ovarian tissue can be transplanted back, but there is a risk of re-introducing malignant cells. To avoid this risk, follicles containing immature oocytes can be grown in the laboratory through a process termed in follicle maturation (IFM). Follicles are functional units within an ovary that contain a single oocyte surrounded by supporting cells called theca cells and granulosa cells. In IFM, follicles are grown and matured to the stage when the oocytes can be released from the follicles (as occurs naturally during ovulation) and fertilized in vitro for subsequent implantation to produce offspring.
3D Matrix Supports Follicle Maturation in Vitro
Follicular maturation is regulated by growth factors and hormones released by the ovary and the pituitary gland, as well as signals from the tissue surrounding the follicle and communication between the oocyte and its supporting cells. In traditional cell culture systems where follicles grow attached to a 2D surface, the connectivity between the supporting theca and granulosa cells and the oocyte are disturbed, leading to disruption of signals that direct oocyte development.
To address this challenge, Lonnie Shea, Professor of Chemical and Biological Engineering at Northwestern University, and Teresa Woodruff, Thomas J. Watkins Professor of Obstetrics and Gynecology and Professor of Biochemistry, Molecular Biology and Cell Biology at Northwestern University, have collaborated on developing 3D culture systems for maturation of fresh and frozen follicles. The 3D systems are based on an alginate hydrogel scaffold designed to mimic the environment of the native ovary. The hydrogel maintains the follicle architecture and interactions between follicular cells. With the addition of growth factors and hormones, individual follicles can be matured in vitro.
Although follicles are self-contained units, the absence of stromal cells in the culture system may alter the normal follicular development process. For example, the Shea and Woodruff research team found that the expression levels of several genes were different in vivo and in vitro, including a gene for a protein that regulates buildup of liquid in the follicle – a process that affects follicle size. This might explain why the follicles grown in vitro are smaller than in vivo. Differential gene expression in fresh and in frozen follicles might be caused by damage done through the freezing process. “There are two main challenges in freezing tissue – penetration of cryoprotectants throughout the tissue to the appropriate concentration for each cell type, and maintaining the organization and connection of cells within the tissue during freezing and thawing,” explains Shea. Rapid freezing (vitrification) is emerging as a promising alternative to slow freezing because it eliminates the risk of ice crystal formation, which can damage the cells.
Understanding Follicular Disease and Age-Associated Infertility
To further improve upon the 3D system, Shea examined how different hydrogel compositions affect follicle maturation. He noted that the stiffness of the hydrogel had an impact on follicle size, steroid hormone production, and oocyte quality. Numerous genes were differentially expressed in more rigid versus less rigid hydrogels. “We started to see that follicles were not developing the same way in a rigid gel. For example, the steroid levels differ as the gel becomes more rigid,” says Shea. Intriguingly, a similar problem with steroid hormone regulation occurs in women with Polycystic Ovarian Syndrome (PCOS), a metabolic disorder that impacts one in ten American women and is a common cause of infertility. This finding suggests that the mechanics of the ovary might be different in PCOS patients relative to healthy women.
Another common cause of infertility is advanced maternal age, as many couples postpone starting a family. “This in vitro system allows us to investigate the molecular mechanisms underlying follicle health, egg quality, aneuploidy (chromosomal number changes), and many of the natural consequences of aging that we see in the human but can’t unravel. It is still unknown what the relative contributions from the egg and the granulosa cells are to age-associated infertility,” explains Teresa Woodruff.
The Oncofertility Consortium
To facilitate the translation of IFM technologies into the clinic, Woodruff initiated the Oncofertility Consortium, a program that provides a range of thoughtful approaches for fertility management for young people with cancer. “The program includes ethicists, humanists, policy makers, economists, and people interested in communication and education. It provides the translational opportunity of taking methods that have been developed in the mouse and testing them in the monkey and to adapt from monkeys to humans,” explains Woodruff. “The environment of the ovary and the mechanical rigidity of the tissue differ between animal species. One reason that we’ve been able to adapt the system across animal platforms is having a tailored biomaterial solution to follicle maturation [e.g., various 3D alginate hydrogel scaffolds to support follicles from different species],” she adds. Shea’s collaborators at the National Primate Center in Oregon are already translating the culture system to primates. Shea's research uses human tissue donated by cancer patients who choose to have their ovaries frozen at Northwestern.
The 3D culture system has provided new ideas for fertility preservation options. It also serves as a model for understanding fundamental biological principles, follicular development across species, age-related decline in follicle quality, and the molecular basis of follicular diseases. “I think that’s ultimately the true power of what we’ve been able to do,” says Shea. His research team is working on the next generation of biomaterials, using a new formulation of the hydrogel. Combined with imaging techniques and biochemical analysis, the synthetic environment of the 3D culture system will allow researchers to track molecular and cellular events during follicle development.
This work is supported in part by the National Institute of Biomedical Imaging and Bioengineering and the NIH Roadmap for Medical Research.
Xu M, Banc A, Woodruff TK, Shea LD. Secondary follicle growth and oocyte maturation by culture in alginate hydrogel following cryopreservation of the ovary or individual follicles. Biotechnol Bioeng. 2009 June 1;103(2):378-86.
West-Farrell ER, Xu M, Gomberg MA, Chow YH, Woodruff TK, Shea LD. The mouse follicle microenvironment regulates antrum formation and steroid production: alterations in gene expression profiles. Biol Reprod. 2009 March;80(3):432-9. Epub 2008 Nov 12.