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Gonadal tissue cryopreservation and transplantation
RBMOnline - Vol 4. Suppl. 1. 64–67 Reproductive BioMedicine Online; www.rbmonline.com/Article/165 on web 14 July 2001
Gonadal tissue cryopreservation and transplantation RG Gosden Department of Obstetrics and Gynecology, McGill University, Women’s Pavilion (F3.38), Royal Victoria Hospital, 687 Pine Avenue West, Montreal, PQ H3A 1A1, Canada Correspondence: Tel. (514) 842 1231 ex 1231; Fax: (514) 843 1662; e-mail: [email protected]
Abstract Transplantation of ovarian and testicular tissue has been practised for over a century, mainly for experimental purposes. It is now being considered as a potential strategy for preserving fertility in young patients, including children, undergoing sterilizing treatment for cancer and other diseases. Ovarian tissue biopsies can be stored at liquid nitrogen temperatures indefinitely so that, after thawing, they can be returned as either ortho- or heterotopic grafts to the original patient. A different approach is needed for preserving male germ cells to restore fertile potential. Experimental studies have shown that spermatogonial stem cells injected into the rete testis/seminiferous tubules can re-initiate spermatogenesis after sterilizing treatment with alkylating agents; alternatively, in prepubertal cases, testicular biopsies that have been cryopreserved can be grafted subcutaneously to generate enough spermatozoa for intracytoplasmic sperm injection (ICSI). These strategies have been demonstrated in animal models and are now undergoing clinical testing. Keywords: cryopreservation, germ cells, ovary, testis, transplantation
64
Objectives
Historical sketch
In the past two decades, new protocols for managing haematological and solid tumours have led to major improvements in the chances of long-term survival. This has been particularly striking in young people, including children, and it has been estimated that in the West at least one in 1000 adults alive today is a survivor of a childhood malignancy (Birch et al., 1988). Unfortunately, this success with aggressive chemotherapy treatment, especially when alkylating agents are included, often comes at the price of hypogonadism and infertility (Apperley and Reddy, 1995). In some cases, the effect is transient and spermatogenesis and menses resume soon after treatment, but in others, it is permanent and irreversible or there is a risk of premature menopause, particularly in those aged >30 years (Blumenfeld and Haim, 1997). In females, the probability of sterilization varies with age and the numbers of primordial follicles in the ovary, but the age factor is negligible or absent in males. Menopausal symptoms in women are treatable with oestrogen replacement therapy, but fertility requires egg donation unless patients have frozen-banked embryos or oocytes. Besides, neither of these latter strategies can guarantee a child, and there may not be time for an assisted reproduction procedure before cancer treatment. Adult male patients should be offered sperm banking automatically, although specimens are more often of poor quality and prepubertal boys do not have any option. Fertility conservation is an important issue for young people who have not started or completed their family size, and where the current technology is unable to meet their needs in full. Consequently, attempts are underway to preserve gonadal tissue at low temperatures for subsequent re-implantation, because this strategy could be more universally applicable.
Over two centuries ago, Scottish surgeon-anatomist, John Hunter, carried out pioneering transplants using cockerel gonads and speculated about cryopreserving various organs and even the whole body. In 1895, a New York surgeon, Robert Morris, used donated ovarian tissue from young women to try to overcome climacteric symptoms in prematurely menopausal patients, at least one of whom later became pregnant. Some 30 years later, Serge Voronoff attracted publicity to testicular grafts, with which he hoped to reverse supposed hypogonadism in ageing men (Gosden and Aubard, 1996). Alexis Carrell, who had earlier won a Nobel Prize for pioneering organ grafts, was doubtful about allografts, although it was not until after the Second World War that Peter Medawar provided an experimental foundation for Carrell’s assumption. Nevertheless, ovarian and testicular transplants have occupied a central place in experimental endocrinology for many years, albeit mainly as autografts or isografts to avoid the problem of rejection. Ovarian implants without vascular anastomoses restored natural fertility, and, at a heterotopic site such as under the renal capsule or subcutaneously, oestrogen production and cyclicity resumed (Felicio et al., 1983; Harp et al., 1994; Gunasena et al., 1997a). The remarkably high efficiency of the simple technique can be attributed to rapid revascularization of grafts and relative tolerance of primordial follicles to ischaemia (Dissen et al., 1994). Soon after the breakthrough discovery of the cryoprotective properties of glycerol in the late 1940s, attempts were made to bank gonadal tissues from rodents. The tissues were cooled to approximately –80°C using methods that are crude by the standards of today’s technology, but successes were
Gonadal tissue cryopreservation and transplantation - RG Gosden
nevertheless reported. After freezing, thawing and transplanting to adult hosts, there was extensive tissue destruction, but enough follicles survived to restore oestrous cycles and spermatogenesis was initiated from testes of prepubertal rats (Deanesly, 1954). The crowning achievement was restoration of fertility to oophorectomized mice by frozen–thawed isografts (Parrott, 1960) but, since there were no obvious applications for gonadal tissue banking, research progress paused until the 1990s.
Gonadal transplantation Avascular implants Although implantation of tissue (e.g., cornea, heart valves and bone) without re-anastomosis of blood vessels is well established, it is rare with highly vascular organs. The ovary is well adapted for this technique, however, especially for small laboratory animals and, being cortically distributed, the primordial follicles are among the first cells to benefit from angiogenesis. Moreover, the metabolic requirements of these follicles are likely to be low because they are non-growing, which may help them to resist hypoxia. Up to 50% or more survive until the organ is revascularized, whereas growing follicles usually die (Dissen et al., 1994; Gosden et al., 1994b). Production of reactive oxygen species as a result of ischaemiareperfusion injury probably accounts for some of the losses, because treatment with antioxidants improves follicle survival and inhibits formation of lipid peroxides (Nugent et al., 1998). Studies have seldom been carried out with testicular implants since Deanesly’s pioneering work nearly 50 years ago, but the essential validity of her findings have been confirmed recently (Gosden et al., 2000). Since farm animal and primate ovaries are bulkier, more fibrous and have a more dispersed follicle population, there was doubt whether implants could be as effective in other animals as in rodents. Nevertheless, there is now good evidence that follicles in fresh and frozen–thawed tissue from a range of larger species can survive grafting, although few studies have been quantitative. Cortical slices can restore fertility in sheep by autografting (Gosden et al., 1994a; Aubard et al., 1999) and, in cats, monkeys, humans and even elephants, xenografted follicles have survived in immunodeficient mice (Gosden et al., 1994b; Newton et al., 1996; Gunasena et al., 1997b). A preliminary report of an ovarian transplant in a patient has also been described (Oktay and Karlikaya, 2000), and more data from clinical studies are expected in the near future.
Follicle and germ cell transfer In theory, cryopreservation is expected to be more effective with isolated primordial follicles, rather than leaving them in situ, where thermal and chemical equilibration with cryoprotective agents is slower. Methods have therefore been developed for enzymatically disaggregating the ovary to harvest follicles for banking and retransplantation. A two-step technique involving partial disaggregation in a medium containing collagenase followed by teasing with fine needles can extract small follicles from even fibrous organs, such as human ovaries (Oktay et al., 1997). The number of follicles harvested per hour by a single operator is low (<100), although the recovery rate and viability are both >50%. In theory, it
should be possible to inject the follicles into the ovarian parenchyma to restore fertility to postmenopausal ovaries, although this has only been attempted in small animals. In mice, the sterilized ovary is too small to retain a suspension of follicles, but if they are incorporated into a vehicle, such as clotted plasma or collagen gel, the implant can be attached to a resected host organ. The transferred follicles restore ovulatory function, even after cryopreservation (Gosden, 1990; Carroll and Gosden, 1993). However attractive in theory, this approach is impracticable for all but experimental purposes, because the process of harvesting large numbers of primordial follicles from human ovaries is laborious, and follicle quality deteriorates progressively. Moreover, since follicles can be preserved more successfully and conveniently in tissue slices, which are easier to transplant, no net advantage is gained. For experimental purposes, however, a combination of grafting and growth in vitro might find applications in the future (Liu et al., 2000). In males there is an exciting possibility that spermatogonial stem cells could be used to restore fertility after banking, by analogy with routine clinical practices with bone marrow. In a series of pioneering experiments, Ralph Brinster’s group has shown that when a suspension containing gonocytes from immature mouse testes is injected into seminiferous tubules the testis can be repopulated and spermatogenesis is restored; in some cases fertility returns after sterilization with busulphan (Brinster and Avarbock, 1994; Brinster and Zimmerman, 1994; Ogawa et al., 2000). What is more, the germ cells could be frozen-banked in advance (Avarbock et al., 1996). The cells are apparently able to transmigrate between the Sertoli cells to their normal location in the basal compartment of the tubule. Ongoing research in other species, including humans, aims to develop this strategy for practical purposes (Schlatt et al., 1999, Brook et al., 2001), including applications for oncology patients. In that case, spermatogonia from testicular biopsies could be banked for transfer when the patients are in full remission. This method would serve as a back-up for routine semen cryopreservation and provides the attractive incentive of initiating spermatogenesis in tissue obtained before prepubertal boys undergo chemotherapy. What is more, if the efficiency of recolonization could attain the same levels as in rodents (Nagano et al., 1999), the technique might restore natural fertility in men.
Vascular grafts Transplantation of a whole organ by vascular re-anastomosis is more attractive in theory than either tissue/cell transfer, but more technically demanding from a surgical and, especially, a cryobiological standpoint. Removing a whole organ from exposure to systemic chemotherapy, plus storage at low temperatures with subsequent autotransplantation stands the best chance of restoring long-term fertility. Where a patient requires a single acute treatment with whole body/abdominal radiation and when oophoropexy is not ideal (Tulandi and AlTook, 1998), the ovaries could be removed, perfused with preserving medium (e.g. University of Wisconsin medium) and chilled for up to 48 h. Judging from clinical experience with renal allotransplantation, this approach might succeed with ovaries and testes, but it will have few applications in practice and has not been tested yet.
65
Gonadal tissue cryopreservation and transplantation - RG Gosden
Where chemotherapy is used, the organs would have to be removed for longer (preferably until the patient is cured), and this requires cryopreservation to liquid nitrogen temperatures. Storage of whole organs has been problematic, largely because of damage to blood vessels, and more research is needed to improve protocols. Animal models are already available for microsurgical reanastomosis of vessels for ovarian and testicular transplantation in rats and rabbits, and these could serve for testing freeze-thaw protocols (Lee et al., 1971; Winston and McClure Browne, 1974).
Cryopreservation Cryopreservation of gametes and embryos has occupied a central place in reproductive technology for many years, but comparatively little attention has been given to tissue banking until recently. Ensuring the survival of gonadal tissue is more challenging, because tissue is more bulky and heterogeneous and difficult to protect from the risks of osmotic swelling, intracellular ice and solution effects. Chilling above the freezing point is an additional hazard for highly sensitive oocytes at metaphase II stage, but it is not known if immature germ cells in ovarian tissue share this disadvantage. It has been surprisingly easy, however, to obtain good survival rates for primordial follicles in thin ovarian cortical slices using conventional equilibrium cooling methods (slow freezing–rapid thawing) (Newton et al., 1996; Hovatta et al., 1996; Baird et al., 1999; Candy et al., 2000). A number of cryoprotective agents (CPA) have proved to be effective (dimethylsulfoxide, 1,2-propanediol and ethylene glycol), glycerol being least successful because it is less permeating and, unless the CPA enters the cell, lysis is almost inevitable. Equilibrium time in CPA is obviously longer than for gametes and embryos, but histological and ultrastructural studies indicate that this need not be very lengthy (<1 h) (Newton et al., 1998; Gook et al., 1999). Monosaccharide or protein is often added to the CPA, but it is not clear whether the benefit applies to tissues undergoing cryopreservation. Finally, there are safety concerns where patients have systemic disease or gynaecological cancer, unless they are in remission, and animal studies have highlighted the risk of reintroducing disease with an ovarian graft (Shaw et al., 1996). More research is needed to determine which diseases, such as Hodgkin’s disease, do not metastasise to the ovary (Meirow et al., 1998). In circumstances where grafting is contraindicated, hope must rest on the development of new methods for culturing follicles in vitro if cryopreserved gonadal tissue is to realize its potential without danger (Newton et al., 1999).
References
66
Apperley JF, Reddy N 1995 Mechanisms and management of treatment-related gonadal failure in chemoradiotherapy. Blood Reviews 9, 93–116. Aubard Y, Piver P, Cognié Y et al. 1999 Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Human Reproduction 14, 2149–2154. Avarbock MR, Brinster CJ, Brinster RL 1996 Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nature Medicine 2, 693–696. Baird DT, Webb R, Campbell BK et al. 1999 Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at –196°C. Endocrinology 140, 462–471. Birch JM, Marsden HB, Morris Jones PH et al. 1988 Improvements
in survival from childhood cancer: results of a population based survey over 30 years. British Medical Journal 286, 1372–1376. Blumenfeld Z, Haim N 1997 Prevention of gonadal damage during cytotoxic therapy. Annals of Medicine 29, 199–206. Brinster RL, Avarbock MR 1994 Germline transmission of donor haplotype following spermatogonial transfer. Proceedings of the National Academy of Sciences of the USA 91, 11303–11307. Brinster RL, Zimmerman JW 1994 Spermatogenesis following male germ cell transplantation. Proceedings of the National Academy of Sciences of the USA 91, 11298–11302. Brook PF, Radford JA, Shalet SM et al. 2001 Isolation of germ cells from human testicular tissue for low temperature storage and autotransplantation. Fertility and Sterility 75, 269–274. Candy CJ, Wood MJ, Whittingham DG 2000 Restoration of normal reproductive lifespan after grafting of cryopreserved mouse ovaries. Human Reproduction 15, 1300–1304. Carroll J, Gosden RG 1993 Transplantation of frozen-thawed mouse primordial follicles. Human Reproduction 8, 1163–1167. Deanesly R 1954 Spermatogenesis and endocrine activity in grafts of frozen and thawed rat testis. Journal of Endocrinology 11, 201–206. Dissen GA, Lara HE, Fahrenbach WH et al. 1994 Immature rat ovaries become revascularized rapidly after autotransplantation and show a gonadotropin-dependent increase in angiogenic factor gene expression. Endocrinology 134, 1146–1154. Felicio LS, Nelson JF, Gosden RG, Finch CE 1983 Restoration of ovulatory cycles by young ovarian grafts in aging mice: potentiation by long-term ovariectomy decreases with age. Proceedings of the National Academy of Sciences of the USA 80, 6076–6080. Gook DA, Edgar DH, Stern C 1999 Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1,2-propanediol. Human Reproduction 14, 2061–2068. Gosden RG 1990 Restitution of fertility in sterilized mice by transferring primordial ovarian follicles. Human Reproduction 5, 499–504. Gosden RG, Aubard Y 1996 Ovarian and Testicular Tissue Transplantation, Medical Intelligence Unit. Landes, Austin, Texas. Gosden RG, Baird DT, Wade JC, Webb R 1994a Restoration of fertility to oophorectomized sheep by ovarian autografts stored at –196°C. Human Reproduction 9, 597–603. Gosden RG, Boulton M, Grant K, Webb R 1994b Follicular development from ovarian xenografts in SCID mice. Journal of Reproduction and Fertility 101, 619–623. Gosden RG, Matthews SJ, Kim SS, Schlatt S 2000 Potential fertility in mice after isografting cryopreserved testicular tissue. Biology of Reproduction 62 (suppl. 1), 190. Gunasena KT, Villines PM, Critser ES, Critser JK 1997a Live births after autologous transplant of cryopreserved mouse ovaries. Human Reproduction 12, 101–106. Gunasena KT, Laky JRT, Villines PM et al. 1997b Allogenic and xenogenic transplantation of cryopreserved ovarian tissue to athymic mice. Biology of Reproduction 57, 226–231. Harp R, Leibach J, Black J et al. 1994 Cryopreservation of murine ovarian tissue. Cryobiology 31, 336–343. Hovatta O, Silye R, Kransz T et al. 1996 Cryopreservation of human ovarian tissue using dimethysulphoxide and propanediol-sucrose as a cryoprotectant. Human Reproduction 11, 1268–1272. Lee S, Tung KS, Orloff MJ 1971 Testicular transplantation in the rat. Transplantation Proceedings 3, 586–590. Liu J, van der Elst J, van den Broeke R et al. 2000 Maturation of mouse primordial follicles by combination of grafting and invitro culture. Human Reproduction 62, 1218–1223. Meirow D, Yehuda DB, Prus D et al. 1998 Ovarian tissue banking in patients with Hodgkin’s disease: is it safe? Fertility and Sterility 69, 996–1000. Nagano M, Avarbock MR, Brinster RL 1999 Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biology of Reproduction 60, 1429–1436.
Gonadal tissue cryopreservation and transplantation - RG Gosden
Newton H, Aubard Y, Rutherford A et al. 1996 Low temperature storage and grafting of human ovarian tissue. Human Reproduction 11, 1487–1491. Newton H, Fisher J, Arnold JRP et al. 1998 Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Human Reproduction 13, 376–380. Newton H, Picton H, Gosden RG 1999 In vitro growth of oocytegranulosa cell complexes isolated from cryopreserved ovine tissue. Journal of Reproduction and Fertility 115, 141–150. Nugent D, Newton H, Gallivan L, Gosden RG 1998 Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts. Journal of Reproduction and Fertility 114, 341–346. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL 2000 Transplantation of male germ line stem cells restores fertility in infertile mice. Nature Medicine 6, 29–34. Oktay K, Nugent D, Newton H et al. 1997 Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertility and Sterility 67, 481–486.
Oktay K, Karlikaya G 2000 Ovarian function after transplantation of frozen, banked autologous ovarian tissue. New England Journal of Medicine 342, 1919. Parrott DVM 1960 The fertility of mice with orthotopic ovarian grafts derived from frozen tissue. Journal of Reproduction and Fertility 1, 230–241. Schlatt S, Rosiepen G, Weinbauer GF et al. 1999 Germ cell transfer into rat, bovine, monkey and human testes. Human Reproduction 14, 144–150. Shaw JM, Bowles J, Koopman P et al. 1996 Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients. Human Reproduction 11, 1668–1673. Tulandi T, Al-Took S 1998 Laparoscopic ovarian suspension before irradiation. Fertility and Sterility 70, 381–383. Winston RML, McClure Browne JC 1974 Pregnancy following autograft transplantation of fallopian tube and ovary in the rabbit. Lancet ii, 494–495.
67
Gonadal tissue cryopreservation and transplantation RG Gosden Department of Obstetrics and Gynecology, McGill University, Women’s Pavilion (F3.38), Royal Victoria Hospital, 687 Pine Avenue West, Montreal, PQ H3A 1A1, Canada Correspondence: Tel. (514) 842 1231 ex 1231; Fax: (514) 843 1662; e-mail: [email protected]
Abstract Transplantation of ovarian and testicular tissue has been practised for over a century, mainly for experimental purposes. It is now being considered as a potential strategy for preserving fertility in young patients, including children, undergoing sterilizing treatment for cancer and other diseases. Ovarian tissue biopsies can be stored at liquid nitrogen temperatures indefinitely so that, after thawing, they can be returned as either ortho- or heterotopic grafts to the original patient. A different approach is needed for preserving male germ cells to restore fertile potential. Experimental studies have shown that spermatogonial stem cells injected into the rete testis/seminiferous tubules can re-initiate spermatogenesis after sterilizing treatment with alkylating agents; alternatively, in prepubertal cases, testicular biopsies that have been cryopreserved can be grafted subcutaneously to generate enough spermatozoa for intracytoplasmic sperm injection (ICSI). These strategies have been demonstrated in animal models and are now undergoing clinical testing. Keywords: cryopreservation, germ cells, ovary, testis, transplantation
64
Objectives
Historical sketch
In the past two decades, new protocols for managing haematological and solid tumours have led to major improvements in the chances of long-term survival. This has been particularly striking in young people, including children, and it has been estimated that in the West at least one in 1000 adults alive today is a survivor of a childhood malignancy (Birch et al., 1988). Unfortunately, this success with aggressive chemotherapy treatment, especially when alkylating agents are included, often comes at the price of hypogonadism and infertility (Apperley and Reddy, 1995). In some cases, the effect is transient and spermatogenesis and menses resume soon after treatment, but in others, it is permanent and irreversible or there is a risk of premature menopause, particularly in those aged >30 years (Blumenfeld and Haim, 1997). In females, the probability of sterilization varies with age and the numbers of primordial follicles in the ovary, but the age factor is negligible or absent in males. Menopausal symptoms in women are treatable with oestrogen replacement therapy, but fertility requires egg donation unless patients have frozen-banked embryos or oocytes. Besides, neither of these latter strategies can guarantee a child, and there may not be time for an assisted reproduction procedure before cancer treatment. Adult male patients should be offered sperm banking automatically, although specimens are more often of poor quality and prepubertal boys do not have any option. Fertility conservation is an important issue for young people who have not started or completed their family size, and where the current technology is unable to meet their needs in full. Consequently, attempts are underway to preserve gonadal tissue at low temperatures for subsequent re-implantation, because this strategy could be more universally applicable.
Over two centuries ago, Scottish surgeon-anatomist, John Hunter, carried out pioneering transplants using cockerel gonads and speculated about cryopreserving various organs and even the whole body. In 1895, a New York surgeon, Robert Morris, used donated ovarian tissue from young women to try to overcome climacteric symptoms in prematurely menopausal patients, at least one of whom later became pregnant. Some 30 years later, Serge Voronoff attracted publicity to testicular grafts, with which he hoped to reverse supposed hypogonadism in ageing men (Gosden and Aubard, 1996). Alexis Carrell, who had earlier won a Nobel Prize for pioneering organ grafts, was doubtful about allografts, although it was not until after the Second World War that Peter Medawar provided an experimental foundation for Carrell’s assumption. Nevertheless, ovarian and testicular transplants have occupied a central place in experimental endocrinology for many years, albeit mainly as autografts or isografts to avoid the problem of rejection. Ovarian implants without vascular anastomoses restored natural fertility, and, at a heterotopic site such as under the renal capsule or subcutaneously, oestrogen production and cyclicity resumed (Felicio et al., 1983; Harp et al., 1994; Gunasena et al., 1997a). The remarkably high efficiency of the simple technique can be attributed to rapid revascularization of grafts and relative tolerance of primordial follicles to ischaemia (Dissen et al., 1994). Soon after the breakthrough discovery of the cryoprotective properties of glycerol in the late 1940s, attempts were made to bank gonadal tissues from rodents. The tissues were cooled to approximately –80°C using methods that are crude by the standards of today’s technology, but successes were
Gonadal tissue cryopreservation and transplantation - RG Gosden
nevertheless reported. After freezing, thawing and transplanting to adult hosts, there was extensive tissue destruction, but enough follicles survived to restore oestrous cycles and spermatogenesis was initiated from testes of prepubertal rats (Deanesly, 1954). The crowning achievement was restoration of fertility to oophorectomized mice by frozen–thawed isografts (Parrott, 1960) but, since there were no obvious applications for gonadal tissue banking, research progress paused until the 1990s.
Gonadal transplantation Avascular implants Although implantation of tissue (e.g., cornea, heart valves and bone) without re-anastomosis of blood vessels is well established, it is rare with highly vascular organs. The ovary is well adapted for this technique, however, especially for small laboratory animals and, being cortically distributed, the primordial follicles are among the first cells to benefit from angiogenesis. Moreover, the metabolic requirements of these follicles are likely to be low because they are non-growing, which may help them to resist hypoxia. Up to 50% or more survive until the organ is revascularized, whereas growing follicles usually die (Dissen et al., 1994; Gosden et al., 1994b). Production of reactive oxygen species as a result of ischaemiareperfusion injury probably accounts for some of the losses, because treatment with antioxidants improves follicle survival and inhibits formation of lipid peroxides (Nugent et al., 1998). Studies have seldom been carried out with testicular implants since Deanesly’s pioneering work nearly 50 years ago, but the essential validity of her findings have been confirmed recently (Gosden et al., 2000). Since farm animal and primate ovaries are bulkier, more fibrous and have a more dispersed follicle population, there was doubt whether implants could be as effective in other animals as in rodents. Nevertheless, there is now good evidence that follicles in fresh and frozen–thawed tissue from a range of larger species can survive grafting, although few studies have been quantitative. Cortical slices can restore fertility in sheep by autografting (Gosden et al., 1994a; Aubard et al., 1999) and, in cats, monkeys, humans and even elephants, xenografted follicles have survived in immunodeficient mice (Gosden et al., 1994b; Newton et al., 1996; Gunasena et al., 1997b). A preliminary report of an ovarian transplant in a patient has also been described (Oktay and Karlikaya, 2000), and more data from clinical studies are expected in the near future.
Follicle and germ cell transfer In theory, cryopreservation is expected to be more effective with isolated primordial follicles, rather than leaving them in situ, where thermal and chemical equilibration with cryoprotective agents is slower. Methods have therefore been developed for enzymatically disaggregating the ovary to harvest follicles for banking and retransplantation. A two-step technique involving partial disaggregation in a medium containing collagenase followed by teasing with fine needles can extract small follicles from even fibrous organs, such as human ovaries (Oktay et al., 1997). The number of follicles harvested per hour by a single operator is low (<100), although the recovery rate and viability are both >50%. In theory, it
should be possible to inject the follicles into the ovarian parenchyma to restore fertility to postmenopausal ovaries, although this has only been attempted in small animals. In mice, the sterilized ovary is too small to retain a suspension of follicles, but if they are incorporated into a vehicle, such as clotted plasma or collagen gel, the implant can be attached to a resected host organ. The transferred follicles restore ovulatory function, even after cryopreservation (Gosden, 1990; Carroll and Gosden, 1993). However attractive in theory, this approach is impracticable for all but experimental purposes, because the process of harvesting large numbers of primordial follicles from human ovaries is laborious, and follicle quality deteriorates progressively. Moreover, since follicles can be preserved more successfully and conveniently in tissue slices, which are easier to transplant, no net advantage is gained. For experimental purposes, however, a combination of grafting and growth in vitro might find applications in the future (Liu et al., 2000). In males there is an exciting possibility that spermatogonial stem cells could be used to restore fertility after banking, by analogy with routine clinical practices with bone marrow. In a series of pioneering experiments, Ralph Brinster’s group has shown that when a suspension containing gonocytes from immature mouse testes is injected into seminiferous tubules the testis can be repopulated and spermatogenesis is restored; in some cases fertility returns after sterilization with busulphan (Brinster and Avarbock, 1994; Brinster and Zimmerman, 1994; Ogawa et al., 2000). What is more, the germ cells could be frozen-banked in advance (Avarbock et al., 1996). The cells are apparently able to transmigrate between the Sertoli cells to their normal location in the basal compartment of the tubule. Ongoing research in other species, including humans, aims to develop this strategy for practical purposes (Schlatt et al., 1999, Brook et al., 2001), including applications for oncology patients. In that case, spermatogonia from testicular biopsies could be banked for transfer when the patients are in full remission. This method would serve as a back-up for routine semen cryopreservation and provides the attractive incentive of initiating spermatogenesis in tissue obtained before prepubertal boys undergo chemotherapy. What is more, if the efficiency of recolonization could attain the same levels as in rodents (Nagano et al., 1999), the technique might restore natural fertility in men.
Vascular grafts Transplantation of a whole organ by vascular re-anastomosis is more attractive in theory than either tissue/cell transfer, but more technically demanding from a surgical and, especially, a cryobiological standpoint. Removing a whole organ from exposure to systemic chemotherapy, plus storage at low temperatures with subsequent autotransplantation stands the best chance of restoring long-term fertility. Where a patient requires a single acute treatment with whole body/abdominal radiation and when oophoropexy is not ideal (Tulandi and AlTook, 1998), the ovaries could be removed, perfused with preserving medium (e.g. University of Wisconsin medium) and chilled for up to 48 h. Judging from clinical experience with renal allotransplantation, this approach might succeed with ovaries and testes, but it will have few applications in practice and has not been tested yet.
65
Gonadal tissue cryopreservation and transplantation - RG Gosden
Where chemotherapy is used, the organs would have to be removed for longer (preferably until the patient is cured), and this requires cryopreservation to liquid nitrogen temperatures. Storage of whole organs has been problematic, largely because of damage to blood vessels, and more research is needed to improve protocols. Animal models are already available for microsurgical reanastomosis of vessels for ovarian and testicular transplantation in rats and rabbits, and these could serve for testing freeze-thaw protocols (Lee et al., 1971; Winston and McClure Browne, 1974).
Cryopreservation Cryopreservation of gametes and embryos has occupied a central place in reproductive technology for many years, but comparatively little attention has been given to tissue banking until recently. Ensuring the survival of gonadal tissue is more challenging, because tissue is more bulky and heterogeneous and difficult to protect from the risks of osmotic swelling, intracellular ice and solution effects. Chilling above the freezing point is an additional hazard for highly sensitive oocytes at metaphase II stage, but it is not known if immature germ cells in ovarian tissue share this disadvantage. It has been surprisingly easy, however, to obtain good survival rates for primordial follicles in thin ovarian cortical slices using conventional equilibrium cooling methods (slow freezing–rapid thawing) (Newton et al., 1996; Hovatta et al., 1996; Baird et al., 1999; Candy et al., 2000). A number of cryoprotective agents (CPA) have proved to be effective (dimethylsulfoxide, 1,2-propanediol and ethylene glycol), glycerol being least successful because it is less permeating and, unless the CPA enters the cell, lysis is almost inevitable. Equilibrium time in CPA is obviously longer than for gametes and embryos, but histological and ultrastructural studies indicate that this need not be very lengthy (<1 h) (Newton et al., 1998; Gook et al., 1999). Monosaccharide or protein is often added to the CPA, but it is not clear whether the benefit applies to tissues undergoing cryopreservation. Finally, there are safety concerns where patients have systemic disease or gynaecological cancer, unless they are in remission, and animal studies have highlighted the risk of reintroducing disease with an ovarian graft (Shaw et al., 1996). More research is needed to determine which diseases, such as Hodgkin’s disease, do not metastasise to the ovary (Meirow et al., 1998). In circumstances where grafting is contraindicated, hope must rest on the development of new methods for culturing follicles in vitro if cryopreserved gonadal tissue is to realize its potential without danger (Newton et al., 1999).
References
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Apperley JF, Reddy N 1995 Mechanisms and management of treatment-related gonadal failure in chemoradiotherapy. Blood Reviews 9, 93–116. Aubard Y, Piver P, Cognié Y et al. 1999 Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Human Reproduction 14, 2149–2154. Avarbock MR, Brinster CJ, Brinster RL 1996 Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nature Medicine 2, 693–696. Baird DT, Webb R, Campbell BK et al. 1999 Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at –196°C. Endocrinology 140, 462–471. Birch JM, Marsden HB, Morris Jones PH et al. 1988 Improvements
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