While cancer therapies are continually being improved and an increasing proportion of all patients reach full remission with their fertility intact (deVita et al, 1993; ...
Human Reproduction vol 11 no 8 pp 1668-1673, 1996
Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients
J.M.Shaw1-4, J.Bowles2, P.Koopman2, E.C.Wood3 and A.O.Trounson1 'Laboratories for Human and Animal Reproductive Biology. Institute for Reproduction and Development, Monash University, Clayton, Victoria 3168, Centre for Molecular and Cellular Biology, The University of Queensland, Brisbane, Queensland 4072 and 3 Monash IVF, 89 Bridge Rd, Richmond, Victoria, Australia ^ 0 whom correspondence should be addressed
Key words: cancer/cryopreservation/mouse/ovary/transplantation
Introduction There are some instances in which girls and young women require medical treatments which can result in sterility, including some cancer treatments and ovariectomy. While cancer therapies are continually being improved and an increasing proportion of all patients reach full remission with their fertility intact (deVita et al, 1993; Collichio and Pandya 1994; Dow 1995; Madsen et al., 1995) the incidence of amenorrhoea may reach 62% and with combined-modality therapy 42% of treated women reach menopause by age 31 years (Meister and Meadows, 1993). There is currently no way of predicting whether or not a patient will remain fertile after chemo- or radiotherapy.. As a precaution, the patients who need these 1668
Transplantation of fresh and frozen tissues carries a risk of disease transmission. Viruses including human immunodeficiency virus (HTV) (Patijn et al, 1993; Petersen et al, 1993; Nicholson and Johnson, 1994), hepatitis (Wreghitt et al, 1994; Pereira et al, 1995) and cancers (Weissmann et al, 1995) can be transmitted by grafts, and cancers have been known to return to patients in remission after the replacement of autologous cryopreserved bone marrow (van Rhee et al, 1994; Beaujean et al, 1995; Roy et al, 1995). It is therefore possible that blood-borne cancers such as leukaemia, systemic cancers such as lymphoma and metastasizing cancers, could be transmitted by grafts of ovarian tissue. To estabb'sh whether lymphoma could be transmitted with fresh or frozen ovarian tissue grafts, we performed a series of experiments on AKR strain mice. Most individuals of this strain spontaneously develop lymphomas after 9 months of © European Society for Human Reproduction and Embryology
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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 frozenthawed 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
types of treatments but wish to have their own children in the future may want to have their oocytes, embryos or ovarian tissue collected and stored for their own future use. Ovarian tissue freezing has several potential advantages over oocyte and embryo freezing. Mature oocytes can only be collected for a limited time in each menstrual cycle and are rarely available in large numbers. Although mature oocytes can be successfully cryopreserved for the mouse (Carroll et al., 1993; Bos-Mikich et al, 1995), oocyte freezing has met with little success in most other species including man (Parks and Ruffing, 1992; Trounson and Bongso, 1996). The cryopreservation of embryos is relatively efficient (Trounson and Bongso 1996) but is only an option for patients from whom mature eggs can be collected and fertilized. Thus they must have a partner or accept fertilization by donor spermatozoa. Ovarian tissue can be collected by laparoscopy at any stage of the menstrual cycle. The ovarian cortex of young women contains several hundred thousand primordial follicles (Knobil and Neill, 1994). Therefore even small pieces (1 mm3) of cortex may contain several hundred primary follicles. It has not yet been demonstrated that cryopreserved ovarian tissues can restore fertility to women, but live young have been obtained from fresh and frozen ovarian tissue grafts in mice (Parrott, 1960; Carroll and Gosden, 1993) and sheep (Gosden et al, 1994). Cryopreserved primate ovarian tissue (marmoset monkey) grafted into nude mice also develops apparently normal antral follicles (Candy et al, 1995). Autotransplantation of ovarian tissue has not been reported for the human, but it is likely that ovarian tissue which is cryopreserved while large numbers of healthy primordial follicles are present may restore fertility. This would be of value to patients who are at risk of entering premature menopause following medical treatments (e.g. ovariectomy or cancer therapy).
Cryopreservation and grafting of ovaries with cancer age (Rosner et al, 1993). To verify that the lymphoma was of donor origin and was not induced by the procedure or developed spontaneously, cells from males with lymphoma were injected into normal females (Drobyski et al, 1993). The male cells contained Y specific sequences which could be detected by polymerase chain reaction (PCR). Once the injected females developed lymphoma their ovaries were grafted into young healthy female recipients which would not be expected to develop cancer for at least 4 months. The tissues of the donor and recipient females were assayed by PCR to demonstrate whether cells containing the Y-specific sequence were present. Materials and methods
Ovarian grafting Donor females were killed by cervical dislocation and their ovaries removed by dissection The ovaries were placed in phosphate buffered saline (PBS) and each cut into pieces around 1 mm3 in size with a no 22 scalpel blade (Swann-Morton, Sheffield, UK). The pieces were grafted either immediately or after cryopreservation, into female recipients of the same strain. The grafting procedure was based on that of Jones and Krohn (1960) and is described in detail in Harari et al. (1996) and Shaw et al (1996). Briefly, the recipient's reproductive tract was exteriorized and the bursa opened to expose the ovary. Both of the recipient's own ovanes were removed. A small piece (1/8-1/4) of fresh or frozen donor ovary was then inserted into the bursa. The bursal membrane was closed with three stitches (9—0 novafil suture, Davis and Geek, Cyanamid Aust Pty Ltd, Clayton, Australia), before returning the reproductive tract to the abdominal cavity. The skin incision was closed with clips (9 mm Auto clips Becton Dickinson, Kjioxfield, Australia) The recipients were monitored daily for signs of ill health In one experiment, ovarian tissue was grafted under the kidney capsule. The kidney was exteriorized through a 1 cm incision through the flank of the anaesthetized recipient The membrane surrounding the kidney was then perforated to allow a small (
