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Developmentally competent oocytes can be collected from xenografted ovarian tissues; however, optimal xenograft conditions need to be established for this technique to be of use in assisted reproduction. In the present study, common wombat ovarian tissue was xenografted under the kidney capsule of nude mice to clarify the role of recipient gonadal status and donor tissue age on graft establishment, follicle development and oocyte recovery. Eighty-nine per cent of all grafts were recovered; of these, 78% contained growing follicles. In female graft recipients, follicle development to the antral stage occurred earlier in ovariectomised recipients compared with intact graft recipients. Similarly, follicle development occurred earlier in recipients of pouch young ovarian tissue grafts when compared with subadult xenografts. Follicle development proceeded to the antral stage in subadult grafts placed under the kidney capsule of male recipient mice, albeit at a slower rate than subadult grafts placed in female recipients. Oocytes were collected from grafts placed in female and male recipients, but no mature oocytes were observed at the time of collection, nor could these oocytes be matured in vitro. The present study demonstrated that common wombat pouch young tissue xenografted to female recipient mice, and subadult ovarian tissue xenografted to male recipient mice, can develop to the antral stage and can therefore facilitate oocyte collection. However, mature oocytes were not obtained using the current protocol.

Primordial and growing follicles are abundant within the ovaries of healthy young female mammals. While our understanding of follicular dynamics is based mainly on studies of normal ovaries in intact animals, techniques such as ovarian grafting and in vitro culture, in particular when used in combination with cryopreservation, have provided both significant additional insights and a source of mature oocytes. Primordial follicles are small, quiescent, and most commonly located within the collagen-rich outer portion of the ovary. Providing that appropriate collection, handling, freezing, and thawing methods are used, they can tolerate cryopreservation very well irrespective of whether they are frozen within a whole ovary (small species), as ovarian pieces, or as individual, isolated, follicles. Animal studies show that grafts of such fresh or frozen materials can, providing that they contain viable follicles, form mature fertilizable oocytes, produce hormones, and support pregnancies to term. Grafts can be returned to the original donor (autograft), but grafting between histocompatible individuals of the same species and between species (xenografts) is also possible. Cryopreservation and grafting are therefore useful both as practical and as experimental tools. Clinically, ovarian tissue has started to be collected and frozen for patients who are at risk of ovarian failure. Very recent case reports show that such frozen ovarian tissue autografts can support the return of menses and antral follicle formation in patients, although as yet no pregnancies have been established.

Ovaries from a marsupial, the tammar wallaby (Macropus eugenii), were grafted into a eutherian recipient at known stages of development to ascertain whether normal development would occur. Xenografted ovaries from pouch young < 20 days old, before the onset of meiosis, retained few germ cells and developed tubule-like structures reminiscent of seminiferous cords. Ovaries from 50-day-old pouch young, which contain primordial follicles, developed into antral follicles and corpora lutea within the eutherian host, and produced hormones that stimulated the reproductive tract of the host. The timing of onset of antrum formation and the progress of follicle development were advanced relative to the timing of events in ovaries in situ. Frozen-thawed ovaries from 50-day-old donors developed into preantral follicles, but at a reduced rate and number. This finding shows that gonads of a marsupial species can develop as xenografts in a eutherian, forming large antral follicles. Accelerated follicular development in xenografts provides a potentially valuable model for studying the factors that control follicle development. Assisted reproduction of endangered marsupials may also be feasible using follicles from pouch young grown as xenografts in a eutherian host.

The Northern Hairy-nosed Wombat (Lasiorhinus krefftii) is a highly endangered marsupial species and every possible option for sustaining the species needs to be explored. One important approach may be the development of assisted reproductive technologies in the non-endangered Common Wombat (Vombatus ursinus) and Southern Hairy-nosed Wombat (Lasiorhinus latifrons) for application in breeding the Northern Hairy-nosed Wombat. In this study, it was examined whether cryopreserved Wombat ovarian tissue would develop following xenografting to immunologically deficient rats. Ovarian tissue was collected from Common Wombats (n = 3) and cryopreserved as small cortical pieces. After thawing the cortical pieces were grafted underneath the kidney capsule of Nude rats (n = 16). The grafts were recovered at 2, 4, and 10 weeks after transplantation and their gross and histological appearance investigated. Two weeks after grafting (n = 2), the tissue was revascularized and healthy primordial follicles were present. At week 4 (n = 2), some follicular development was present. At week 10, six rats received human chorionic gonadotrophin (hCG) to trigger follicle and oocyte maturation while another six rats were not given any treatment. The administration of hCG did not induce preovulatory follicles and oocyte maturation although type 5 follicles were present in ovarian tissue collected 10 weeks posttransplantation in both treated and untreated groups. This study demonstrates for the first time that Wombat ovarian tissue can survive and function when grafted into immunocompromized rats and that Wombat ovarian follicles can be recruited to growth and development in an ovarian xenograft. This model system has the potential to produce mature oocytes from endangered species for use in assisted reproductive technologies such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and mature oocytes from non-endangered species for nuclear transfer which may be necessary for the preservation of critically endangered species.

Xenografting of ovarian tissue salvaged from valuable dogs to an immunologically incompetent recipient is a possible mechanism to allow the ovarian tissue to be used for recovery of fertilizable oocytes. In this study, follicular development was assessed after xenotransplantation of fresh canine ovarian tissue into immunodeficient mice. Fresh prepubertal canine ovarian tissue was grafted beneath the kidney capsule of 7-week-old ovariectomized severe combined immunodeficient mice. At intervals after grafting, the recipient mice were killed, necropsied and the xenografts were recovered. The number and stage of development of follicles were assessed quantitatively by histological examination of serial sections of the xenografts. By day 56 after grafting, recruitment of primordial follicles had occurred but follicular development to the antral stage was not observed. The recipients showed persistent vaginal cornification and uterine dilation, which indicates that the grafts were producing hormones. However, these changes are not consistent with the oestrous cycle of either bitches or mice, indicating that inappropriate communication may occur between the recipient hypothalamic-pituitary glands and the axis of the xenograft gonad.

Ovarian grafting provides a strategy for clinical infertility treatment and is starting to be used in conjunction with ovarian tissue storage for patients at risk of early ovarian failure. As patients are starting to return for their frozen stored tissue we need to ascertain how to maximise follicle survival when this tissue is grafted back to the patient. For research purposes ovarian tissue is commonly grafted to the kidney capsule as the rich capillary bed at this site favours rapid graft revascularization. This is however not an ideal site for natural conceptions or for the harvest of mature oocytes for in vitro fertilization. While oocytes would be relatively easy to recover from grafts on the abdominal wall or subcutaneous tissue graft revascularization at these sites is slower and evidence indicates that fewer follicles survive. As gonadotropins can upregulate angiogenic growth factors in the ovary this study was designed to test whether the administration of exogenous gonadotropins would increase the number of surviving follicles in grafts placed at less vascularised sites. We showed that exogenous gonadotrophins, given to either the donor or the recipient, could increase the number of developing follicles but the magnitude of this effect was influenced by the timing of the injections relative to the time of grafting.

Ovarian tissue storage has several potentially very valuable clinical applications, including the management of young female patients that are at risk of premature menopause. Ovarian tissue collection, used alone or in combination with oocyte and embryo cryopreservation, may help these patients safeguard their own future fertility. All available evidence from animal studies indicates that grafting of frozen ovarian tissue should be feasible in the human. This study on the mouse shows that frozen thawed ovarian tissue grafts can restore long term fertility to previously ovariectomised recipients. This, and other available evidence, indicates that ovarian tissue collection and storage, used alone or in combination with oocyte or embryo collection, may help safeguard the fertility of patients at risk of premature menopause.

Embryo cryopreservation is a widely used and relatively well-established procedure. By contrast, ovarian tissue and unfertilized oocytes are only rarely cryopreserved, even though for germ line storage these often would be preferable to embryo cryopreservation. There are many reasons for this discrepancy. Unfertilized mature (MII) stage oocytes are more difficult to cryopreserve than cleavage stage embryos of the same species. Many factors contribute to this including the oocyte's surface to volume ratio, single membrane, temperature-sensitive metaphase spindle and zona, and its susceptibility to parthenogenetic activation and chill-injury. A completely different set of problems applies to primordial follicles. Oocytes in primordial follicles are very small and tolerate cryopreservation by slow cooling very well. The problem lies in the difficulty in producing mature oocytes from these primordial follicles. Better and/or more convenient cryopreservation procedures for both oocytes and ovarian tissue are being developed. This paper describes some of the advances in this area and outlines the relative merits and limitations of several currently available egg and ovarian tissue cryopreservation procedures.

Cryopreservation of ovarian tissue is a potential alternative or addition to cryopreservation of embryos or oocytes for sustaining fertility in women at risk of premature ovarian failure. Grafts of cryopreserved ovarian tissue have resulted in live-born mice and sheep. While human applications are in their infancy, frozen human ovarian tissue grafted to the kidney capsules of immunodeficient mice has produced normally developing ovarian follicles.

Girls and young women who require ovariectomy or cancer therapy may consider having their own eggs, embryos or ovarian tissue stored (cryopreserved) for their own future use. Ovarian tissue is simple to collect and contains large numbers of germ cells. Transplantation of fresh and frozen-thawed ovarian tissue in healthy sheep and mice has resulted in normal live young. Similar techniques may be effective in the human but it is unclear whether ovarian tissue cryopreservation and grafting is suitable for ovaries from individuals with cancer or infections. If cancer cells were present in an ovary at the time of collection and survived cryopreservation and grafting they could establish cancer in the recipient. We therefore performed ovarian cryopreservation and transplantation trials using a mouse lymphoma model. This established that the lymphoma was transmitted by grafts of both fresh and frozen ovarian tissue. The normal healthy recipient mice died 9-43 days after receiving a small piece (1 mm3) of ovarian tissue from a donor with lymphoma. We conclude that ovarian tissue which is collected, cryopreserved and grafted while it contains cancer cells has the potential to spread the cancer to the graft recipient.