BACKGROUND Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research.

In vitro ovarian follicle cultures may provide fertility-preserving options to women facing premature infertility due to cancer therapies. An encapsulated three-dimensional (3-D) culture system utilizing biomaterials to maintain cell-cell communication and support follicle development to produce a mature oocyte has been developed for the mouse. We tested whether this encapsulated 3-D system would also support development of nonhuman primate preantral follicles, for which in vitro growth has not been reported. Three questions were investigated: Does the cycle stage at which the follicles are isolated affect follicle development? Does the rigidity of the hydrogel influence follicle survival and growth? Do follicles require luteinizing hormone (LH), in addition to follicle-stimulating hormone (FSH), for steroidogenesis? Secondary follicles were isolated from adult rhesus monkeys, encapsulated within alginate hydrogels, and cultured individually for

This encapsulated 3D culture model permits further studies on the endocrine and local factors that influence primate follicle growth and oocyte maturation, with relevance to enhancing fertility preservation options in women.

A three-dimensional culture system supports the development of primate preantral follicles to the antral stage with appreciable steroid production. This study assessed i) whether in vitro developmental competence of follicles is age dependent, ii) the role of gonadotropins and insulin in supporting folliculogenesis, and iii) anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) production by growing follicles. Ovaries were obtained from prepubertal, young, and older adult rhesus macaques. Secondary follicles were encapsulated into alginate beads and cultured individually for 40 days in media containing 0.05 or 5 μg/ml insulin, with or without recombinant human (rh) FSH (500 mIU/ml). No follicles survived in the culture without rhFSH. In the presence of rhFSH, survival was lower for follicles from older animals, whereas growth, i.e. follicle diameter, was less by day 40 for follicles from prepubertal animals. The surviving follicles were categorized as no-grow (NG;≤250 μm), slow-grow (SG; 250–500 μm), and fast-grow (FG;≥500 μm) according to their diameters. SG follicles cultured with 5 μg/ml insulin produced more ovarian steroids than those cultured with 0.05 μg/ml insulin by week 5. SG and FG follicles produced more AMH and VEGF than the NG, and levels peaked at weeks 2 and 5 respectively. After 100 ng/ml rh chorionic gonadotropin treatment for 34 h, more healthy oocytes were retrieved from young adults whose follicles were cultured with 5 μg/ml insulin. This culture system offers an opportunity to characterize the endocrine and paracrine function of primate follicles that influence follicle growth and oocyte maturation.

The removal and cryo-storage of ovarian cortical biopsies is now offered as a fertility preservation option for young women. The only available option to restore fertility using this tissue is by transplantation which may not be a viable option for all patients. The full potential of this tissue to restore fertility could be achieved by the development of in vitro systems that support oocyte development from the most immature stages to maturation. The techniques of in vitro growth (IVG) combined with in vitro maturation (IVM) are being developed in human but comparing different systems has been difficult because of the scarcity of tissue and non-human primates are being used as model systems. There are many challenges to developing a complete culture system that will support human oocyte development and this review outlines the approaches being taken by several groups to support each of the stages of oocyte development using tissue from women and non-human primate models.

This work was supported by The Oncofertility Consortium (NIH U54 RR024347-HD058294, PL1-EB008542), NIH U54-HD18185 (Eunice Kennedy Shriver Specialized Cooperative Centers Program in Reproduction and Infertility Research), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), Oregon National Primate Research Center 8P51OD011092. There are no conflicts of interest.

Radiation and high-dose chemotherapy may render women with cancer prematurely sterile, a side-effect that would be avoided if ovarian tissue that had been removed before treatment could be made to function afterwards. Live offspring have been produced from transplanted ovarian tissue in mice and sheep but not in monkeys or humans, although sex steroid hormones are still secreted. Here we describe the successful transplantation of fresh ovarian tissue to a different site in a monkey, which has led to the birth of a healthy female after oocyte production, fertilization and transfer to a surrogate mother. The ectopically grafted tissue functions without surgical connection to major blood vessels and sets the stage for the transplantation of cryopreserved ovarian tissue in humans.

Twenty-three rhesus monkeys were subjected to 9 days of ovarian hyperstimulation with sequential exposure to human follicle-stimulating hormone (hFSH) and then human luteinizing hormone (hLH) + hFSH. Six animals (26%) did not exhibit sustained, elevated levels of circulating estradiol, primarily due to the occurrence of a premature surge of endogenous LH (n = 4). Seventeen animals (74%) responded with supraphysiologic levels of circulating estradiol (peak value: means = 4480 pg/ml) and received human chorionic gonadotropin (hCG) on Day 10. Oocytes were collected 26 h later by aspiration of large antral follicles. Oocyte quantity (means = 18/animal) and quality (63% mature) were evaluated by in vitro fertilization (IVF), embryonic development, and embryo transfer to foster mothers. Modified conditions for the successful fertilization of oocytes used a Tyrode's augmented (TALP) medium supplemented with 0.3% bovine serum albumin (BSA). Oocytes were inseminated at the metaphase II stage with ejaculated, washed sperm (50 100 x 10(3)/ml) preexposed at ambient temperature to caffeine and dibutyryl cyclic adenosine 3'5'-monophosphate. Successful fertilization ranged from 26% to 75%. In one experiment, 5 of 11 embryos produced by IVF developed in vitro to hatched blastocysts. Embryo freezing employed a propanediol-based protocol and was applied to early cleavage-stage embryos with 100% (5 of 5) post-thaw survival. Two frozen-thawed embryos were transferred transtubally on 3 occasions into rhesus monkeys during the early luteal phase of spontaneous menstrual cycles. One pregnancy resulted, which proceeded normally to the unassisted delivery of a male offspring 170 days after the LH surge. We conclude that this sequential regimen of human gonadotropins provides a cohort of oocytes from rhesus monkeys that will complete meiotic maturation and fertilize in vitro, with embryonic development proceeding in vitro and in vivo. The production of putative antibodies to human gonadotropins, assessed by the presence of Protein A-precipitated hCG binding components in sera, limits the repeated use of monkeys in the hyperstimulation protocol. Nevertheless, this model system should facilitate further studies on oocyte maturation, fertilization, and early embryogenesis in primates.