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Follicle Cryopreservation

Site: Oregon National Primate Research Center

Principal Investigator: Mary B. Zelinski, PhD

Co-Investigator: Richard R. Yeoman, PhD


While fertility preservation for males has been available for several decades using sperm cryopreservation, attempts to similarly preserve mature female oocytes and ovarian tissue have been more challenging.  Tissue damage during the freezing and thawing process has the potential to irreversibly disrupt the 3-dimensional structure of the follicle necessary for the development of a fully viable mature oocyte. The goal of this research is to provide the direction necessary by testing existing technologies that represent the state-of-the-art in cryopreservation of ovarian tissue samples in nonhuman primates.  In addition, novel methods that will improve tissue viability after thaw as well as minimize operator time and effort will be developed.

Experimental endpoints include successful in vitro maturation of follicles, fertilization of resulting mature oocytes, embryo development and establishment of pregnancy.  After rigorous testing in nonhuman primates, the findings will be rapidly applied to the cryopreservation of human ovarian tissue in order to expand fertility preservation options for women at the time of diagnosis of cancer and other serious diseases.

The objectives of this project are to:

  • Compare two methods of ovarian tissue cryopreservation (slow freeze vs. vitrification) in the nonhuman primate, and assess follicle and oocyte viability in all stages of follicle maturity
  • Assess the fertilization and embryonic potential of primate oocytes derived from cryopreserved ovarian tissue and cumulus-oocyte complexes
  • Adapt new cryo-technology under development around the globe to the nonhuman primate model

Key Experiments

  • Compare vitrification to slow freeze methods in rhesus monkey ovarian tissue by assessing a) histology of tissue preservation; b) in follicle maturation (IFM), i.e., isolation of individual secondary follicles to monitor survival, growth and functional characteristics using the encapsulated 3-dimensional (3D) culture system recently established in the nonhuman primate; and c) isolation of the matured oocyte and assessment of fertilization, early embryonic development, and ultimately production of live offspring.
  • Compare vitrification to slow freeze methods in cumulus–oocyte complexes (COCs) derived from small antral follicles in rhesus monkey ovarian tissue and assessing oocyte maturation in vitro, fertilization, early embryonic development and ultimately production of live offspring.
  • Develop novel cryopreservation methods based on cutting-edge technologies derived from the cryobiology field (i.e., novel cryoprotectants) in rhesus monkey ovarian tissue and COCs
  • Test methods for transporting ovarian tissues (currently used for tissue transplantation) rapidly between sites for optimal preservation of morphology and function

Consortium Support and Impact
The successful execution of this project and eventual application of preservation technology will require close collaboration with other members of the Oncofertility Consortium. Specifically, the project requires close cooperation with the team working to improve methods to mature primate follicles in vitro and the Biomaterials Core. In addition, a primary goal of the National Physicians Cooperative is to ultimately translate the results of these studies to the preservation of human ovarian tissue and follicles. While the human application of methods to cryopreserve and recover ovarian tissue will fill a significant need in fertility preservation in women, it is also expected to lead to new social, legal, and ethical concerns that will impact how patients and physicians discuss infertility (see Social Science and Oncofertility), particularly with regard to pediatric cancer patients.  Education of patients and physicians and training of oncofertility specialists also be necessary to ensure that cutting-edge procedures associated with follicle removal and storage become part of routine cancer care worldwide


Ting AY, Yeoman RR, Lawson MS, Zelinski MB. In vitro development of secondary follicles from cryopreserved rhesus macaque ovarian tissue after slow-rate freeze or vitrification. Hum Reprod. 2011 Jun 24.

Xu J, Lawson MS, Yeoman RR, Pau KY, Barrett SL, Zelinski MB, Stouffer RL. Secondary follicle growth and oocyte maturation during encapsulated three-dimensional culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin. Hum Reprod. 2011 Feb 28.

Jin, S, Lei, L, Shea, LD, Zelinski, MB, Stouffer, RL, and Woodruff, TK. Markers of growth and development in primate primordial follicles are preserved after slow cryopreservationFertility and Sterility, 2010.

Peluffo MC, Barrett SL, Stouffer RL, Hennebold JD, Zelinski MB. Cumulus-Oocyte Complexes from Small Antral Follicles During the Early Follicular Phase of Menstrual Cycles in Rhesus Monkeys Yield Oocytes That Reinitiate Meiosis and Fertilize In vitro. Biol Reprod 83:525-532, 2010.  PMID  20519694.

Smitz J, Dolmans MM, Donnez J, Fortune JE, Hovatta O, Jewgenow K, Picton HM, Plancha C, Shea LD, Stouffer RL, Telfer EE, Woodruff TK, and Zelinski MB. Current achievements and future research directions in ovarian tissue culture, in vitro follico development and transplantation: implications for fertility preservation. Human Reproduction Update, 2010.

Smitz J, Dolmans MM, Donnez J, Fortune JE, Hovatta O, Jewgenow K, Picton HM, Plancha C, Shea LD, Stouffer RL, Telfer EE, Woodruff TK, and Zelinski MB. Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation (Chinese excerpt). Human Reproduction Update, Vol.16, No.4 pp. 395–414, 2010.

Xu J, Bernuci MP, Lawson MS, Yeoman RR, Fisher TE, Zelinski MB, Stouffer RL. Survival, growth, and maturation of secondary follicles from prepubertal, young and older adult, rhesus monkeys during encapsulated three-dimensional (3D) culture: effects of gonadotropins and insulin. Reproduction, Aug. 2010. PMID  20729335

Xu M, West-Farrell ER, Stouffer RL, Shea LD, Woodruff TK, and Zelinski MB. Encapsulated Three-Dimensional Culture Supports Development of Nonhuman Primate Secondary Follicles. Biology of Reproduction. (3):587-94 Sep.8, 2009

This research was supported by the Oncofertility Consortium®, funded by the National Institutes of Health through the NIH Roadmap for Medical Research, Grant UL1DE19587 and RL1HD058293.

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