1 Department of Obstetrics and Gynecology, McGill University, Royal Victoria Hospital, Montreal, QC H3A 1A1, 2 Laboratory of Experimental Surgery, Centre Hospitalier de lUniversité de Montréal, Notre-Dame Hospital, Montréal, QC H2L 4M1, Canada and 3 Department of Obstetrics and Gynecology, EulJi Medical School, Seoul, Korea
4 Current address: The Jones Institute for Reproductive Medicine, 601 Colley Avenue, Norfolk, VA, USA
5 Current address: Department of Urology, Huashan Hospital, Fudan University, Shanghai, 200040, P. R. China
6 To whom correspondence should be addressed at: The Jones Institute for Reproductive Medicine, 601 Colley Ave, Norfolk, VA 23507, USA. e-mail: yinh{at}evms.edu
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Abstract |
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Key words: cryopreservation/fertility/follicles/ovary/testis
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Introduction |
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Vascular transplants could, in theory, reinstate natural fertility after cryopreservation of ovaries and testes, but the diminutive calibre of blood vessels in small laboratory animals is demanding for microsurgery (Lee, 1974; Denjean et al., 1982
; Cornier et al., 1985
). We now report transplantation of the intact rat ovary and its proximal reproductive tract using vascular surgery. We have also investigated the possibility of allotransplantation, which might represent a radical alternative to existing oocyte donation technology.
Following initial success with rat ovarian isotransplants, which was summarized in correspondence to Nature (Wang et al., 2002), we extended our study to investigate storage of ovaries at low temperatures above freezing and tolerance of allotransplantation with moderate immunosuppression. We also tested testicular transplants either using fresh or cryopreserved organs. The rationale for the male was the same as for females. Oligo- or azoospermia can arise from many causes, including cancer treatment (Damani et al., 2002
; Thomson et al., 2002
). Assisted reproduction involving frozen semen or microsurgical sperm aspiration for ICSI with cryopreservation if appropriate (Podsiadly et al., 1996
; Patrizio, 2000
) are widely practised, but cannot be used for pre-pubertal boys (Bahadur et al., 2000
). Testicular banking offers a possibility of fertility conservation if the organ can be successfully transplanted, and this option too has now been tested in the rat model.
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Materials and methods |
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Ovary transplantation
In addition to intact controls and oophorectomized controls to compare the effects of hypo-estrogenism on hormone levels, there were five groups of ovarian transplant. Animals received fresh organs which had been held for <1 h at 4°C, or were maintained ischaemic under the same conditions for 24 h before transplantation, or received cryopreserved organs. Further groups received allotransplanted ovaries of BrownNorway rats. They were either gavaged daily with cyclosporin A in olive oil (4 mg/kg; Novartis, Canada) for 30 or 60 days, or were given only the oil vehicle.
Testis transplantation
Three groups of testicular transplants were compared, namely, intact controls, fresh isotransplants and cryopreserved isotransplants.
Organ cryopreservation
Seven ovarian isotransplants and five testicular transplants were cryopreserved using an equilibrium cooling protocol before surgery. The organs were perfused slowly with ice-cold M2 medium (Sigma, USA) containing 0.1 mol/l fructose and a gradient of dimethylsulphoxide (DMSO) rising from 0 to 1.5 mol/l at a rate of 0.35 ml/min for 30 min. The female organs were cooled in cryovials (Nalge Company, USA) and the testes, which were larger than ovaries, were prepared in cryobags (Planer Products Ltd, UK). The chamber of a controlled rate freezer (CryoLogic, Australia) was modified to hold cryovials or cryobags by changing core sizes, and the freezer was programmed to cool from 0 to 7°C at 2°C/min. The organs were held for 5 min before inducing ice nucleation. Subsequently, they were cooled at 0.3°C/min to 40°C, and then at 10°C/min to 85°C before plunging into liquid nitrogen for overnight storage. Thawing was carried out rapidly (>100°C/min) by swirling the organs in a water bath at 40°C. The cryoprotectant was washed out by reversing the concentration gradient by perfusion. The organs were held in Wisconsin solution at 4°C until transplanted (<1 h).
Monitoring transplant function in vivo
Vaginal smears were prepared daily for monitoring ovarian activity for 60 days after surgery. Four rats with fresh and four with cryopreserved isotransplants, whose cycles had been restored after 30 days, were tested for fertility with Lewis males for another 30 days. Two additional animals with fresh transplants were kept for an additional 50 days for testing ovulatory capacity by priming with i.p. injections of 50 IU equine CG and 50 IU hCG 48 h later so that ovulation could be verified the following day.
At autopsy, the ovaries and reproductive tracts were removed and examined for signs of ovulation and/or pregnancy. The Fallopian tubes and uteri were perfused with Trypan blue dye (0.025%, v:v; Sigma) to check if the tubes were patent. The completeness of oophorectomy on the contralateral side was confirmed at this time. Likewise the male gonad and tract were examined and organs of both sexes were weighed and then fixed in Bouins fluid overnight. The tissues were prepared as 6 µm serial sections stained with haematoxylin and eosin. The slides of ovaries were coded and the numbers of primordial and growing follicles were counted blindly in every tenth ovarian section and testes were studied morphologically.
Hormone assays
Rats were euthanized with carbon dioxide at a median time of 67.8 days (female) or 42 days (male) after transplantation. During terminal anaesthesia, blood was withdrawn by cardiac puncture and stored as serum at 80°C. The concentrations of FSH and estradiol-17 in female rats and testosterone in male rats were measured using radioimmunoassay (sensitivity of 0.1 ng FSH per tube, Amersham Pharmacia Biotech, USA) and chemiluminescence (sensitivity of 37 pmol/l estradiol-17
; Bayer, USA) and ADVIA CentaurTM System (sensitivity of 0.35 nmol/l testosterone; Bayer, USA).
Statistics
The data were analysed by analysis of variance and Students t-test, P < 0.05 being considered significant.
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Results |
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All fresh ovarian transplants were still present at autopsy and were showing signs of normal function up to 60 days post-operation. Vaginal smears indicated that estrous cycles had not been interrupted apart from brief irregularity after surgery. Ovarian and uterine weights were normal, nor were serum FSH or estradiol-17
significantly different to intact controls (Table I). These findings were consistent with the ovarian morphology, which was indistinguishable from controls [Figure 1(i), (ii)]. In addition, the numbers of primordial and growing follicles were not significantly different between controls and fresh transplants (Figure 2). These results indicated that the whole organ had survived with its intact vascular supply, because a non-vascularized implant would have lost follicles. Two additional animals injected with gonadotrophins after
120 days after transplantation responded by forming corpora lutea, indicating competence for ovulation.
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The morphology of the Fallopian tube was not affected by 24 h of cold ischaemia [Figure 3(i)], and uterine tissues on both sizes of the utero-uterine anastomosis were histologically indistinguishable and normal [Figure 3(ii), (iii)]. Only one animal was pregnant at autopsy, and it was in the cryopreserved group. It was not clear why other animals with transplants failed either to mate or to become pregnant because their uteri were patent according to the Trypan blue dye test. Fallopian tubes were sometimes shrunken, suggesting that there may have been some necrosis, and tubo-ovarian and other adhesions might have physically obstructed gamete transport and/or caused ovulatory dysfunction. The animal which became pregnant had the second largest number of follicles in its group, and its ovary contained 13 hyperaemic corpora lutea, which is typical of a young rat with a single ovary. The fetuses and their placentas were normal in appearance and estimated to be 16 days post-conception. One had implanted on each side of the uterine anastomosis, identified by a remnant of suture material.
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Discussion |
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Most experience to date has been based upon cryopreservation of gonadal tissue slices. Provided the tissue is thin enough, immersion in cryoprotectants may be adequate for avoiding serious damage from intracellular ice crystals during cooling or rewarming. Successful transplantation of ovarian tissue from a number of species has now been recorded, including laboratory rodents (Carroll and Gosden, 1993; Harp et al., 1994
; Candy et al., 2000
), farm animals (Gosden et al., 1994
; Baird et al., 1999
) and monkeys (Schnorr et al., 2002
). Moreover, a few case reports indicate there are prospects for clinical application (Oktay and Karlikaya 2000
; Radford et al., 2001
). The attraction of this strategy is that both the endocrinology and natural fertility of the individuals would be restored by transplantation. The disadvantage is that implantation of tissue without vascular reanastomosis leads to ischaemic damage to the tissue and depletion of germ cells and their supporting somatic cells. Fortunately for the ovary, primordial follicles appear to be relatively resistant to ischaemia, although losses always occur (Nugent et al., 1998
). Likewise, there is damage to the seminiferous tubules when slices of testicular tissue are transplanted (Schlatt et al., 2002
; Shinohara et al., 2002
). Whilst orthotopic transplantation provides a possibility of restoring natural fertility, heterotopic transplants require assisted reproductive technology to realize the fertile potential of the tissue. A more radical procedure is to recover spermatogonial stem cells from the testis for cryopreservation. Experiments in rodents have demonstrated that these cells restore fertility to the sterilized testis after transfer to the seminiferous tubules (Avarbock et al., 1996
). It is questionable if germ cell transfer or tissue implantation can achieve sufficiently high efficiency for restoring a full lifespan of fertility.
Gonadal transplants have been tested throughout the 20th century, and some extraordinary claims, which were often not verifiable, were made about their efficiency in humans (Nugent et al., 1997). Attempts were also made to cryopreserve whole organs, such as the kidney, but they were either unsuccessful or unrepeatable (Guttman et al., 1976
). One of the most serious problems was vascular injury, probably resulting from osmotic damage or chemical toxicity from cryoprotective agents. Our study shows that, even after storage at liquid nitrogen temperature, a whole rat ovary can be transplanted successfully and restore follicular activity and secretion. Moreover, there is a possibility of pregnancy after these procedures, indicating potential new strategies for fertility conservation in animals and humans. However, ovarian allotransplants were vigorously rejected in this species, which appears to rule out ovarian donation except when the donor and host have a close tissue match. Unlike the ovaries, testicular isotransplants did not survive if the organs had been cryopreserved, although fresh organs were more successful.
In the rat model, vascular surgery is technically demanding, but the small size of the organs is favourable for cryopreservation. The vascular architecture of the reproductive system in this species requires the ovary to be transplanted with its adjacent Fallopian tube and a segment of the uterus. In larger species and in humans, an en-bloc transplant would not be required, for which anastomosis of the ovarian pedicle would be technically straightforward. Indeed, this procedure has already been used in monkeys (Scott et al., 1981) and sheep (Jeremias et al., 2002
). In clinical practice, we envisage that one of the pair of ovaries would be left in situ so that an intact pedicle is available for exchanging the sterilized organ with the frozen and thawed ones once the patient is ready for autotransplantation. In the present study the equilibrium cooling method was used with a standard cryoprotectant (DMSO) because this has already proved successful with ovarian tissue (Baird et al., 1999
). The cryoprotectant was introduced and washed out by slow perfusion, and a sugar was added as a further precaution to reduce osmotic damage. This model enabled function of the transplants to be monitored non-invasively using vaginal smears to indicate estrogen secretion. Confirmatory endocrine data were obtained by serum hormone measurement and by gross and microscopical examination of the gonads. There was concordance between gonadal morphology and size and the circulating levels of gonadotrophins and sex steroids, which were correspondingly elevated and decreased, respectively, in hypogonadal animals. However, the ultimate test of function in successful transplants is fertility, and this was demonstrated by pregnancy in one rat which, surprisingly, was in the cryopreserved ovary group. Clearly it is desirable to confirm and extend this study, although the labour and surgical skill required will likely delay progress towards optimizing protocols, and avoiding thrombi and necrosis. Evidently, a period of about 1 h ischaemia was not detrimental to function because the fresh transplants did not suffer significant follicle losses. However, 24 h of ischaemia had detrimental effects, although the mean number of follicles was higher than in the cryopreserved group. This result implies that there may be significant impairment in potential fertility if, for example, organs are shipped on ice between centres without cryopreserving. Should these techniques ever be applied for the conservation of fertility in animals or patients, cryopreservation protocols and alternative strategies need to be refined and tested. Vitrification, which can prevent ice formation in tissue, should be considered although ultra-rapid freezing of large organs of primates and farm animals is problematic and equilibration with high concentrations of cryoprotective agents will be lengthy (Al Aghbari and Menino, 2002
). These problems will also have to be addressed for conventional cryopreservation methods.
The results of testicular transplantation were much less successful than with the ovary, probably because of the long spermatic vessels and vulnerability of this organ to ischaemia. With fresh transplants, three out of seven organs maintained spermatogenesis and secreted testosterone. Nevertheless, some damage was observed, and tubules were narrower, implying that Sertoli cells and/or germ cells were less abundant and the daily production of sperm was reduced. None of the cryopreserved testes were functional after transplantation, and testosterone levels were in the castrate range. These disappointing results should not discourage attempts using modified protocols because testicular banking could benefit some patients, especially pre-pubertal children, seeking fertility conservation.
Lastly, studies were carried out to investigate the survival of gonadal allotransplants. The rationale for these studies was that organ donation could theoretically be used for treating patients with premature gonadal failure or gonadal agenesis, and might therefore serve as an alternative to conventional oocyte donation. Combinations of inbred rat strains were chosen on the basis of extensive studies of other organ transplants, and the dose of cyclosporin was selected because it permitted rat cardiac allotransplants to survive when the Lewis and BrownNorway combination was used (Harrison and Madwed, 1999). However, these doses were unsuccessful in preventing rejection of allotransplants of ovaries. Estrous cycles were never reinitiated, follicles disappeared completely and serum FSH and estradiol levels were in the castrate range. It therefore appears that ovarian allotransplantation is not an option, unless there is a closer match of MHC haplotypes or more aggressive immunosuppression is used.
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Acknowledgements |
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References |
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Avarbock, M.R., Brinster, C.J. and Brinster, R.L. (1996) Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat. Med., 2, 693696.[ISI][Medline]
Aziz, N.M. (2002) Cancer survivorship research: challenge and opportunity. J. Nutr., 132, (Suppl. 11), 3494S3503S.
Bahadur, G., Chatterjee, R. and Ralph, D. (2000) Testicular tissue cryopreservation in boys. Ethical and legal issues: case report. Hum. Reprod., 15, 14161420.
Baird, D.T., Webb, R., Campbell, B.K. Harkness, L.M. and Gosden, R.G. (1999) Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at 196 C. Endocrinology, 140, 462471.
Candy, C.J., Wood, M.J. and Whittingham, D.G. (2000) Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries. Hum. Reprod., 15, 13001304.
Carroll, J. and Gosden, R.G. (1993) Transplantation of frozenthawed mouse primordial follicles. Hum. Reprod., 8, 11631167.[Abstract]
Cornier, E., Sibella, P. and Chatelet, F. (1985) Histological study and functional results of tubal and ovarian transplants in the rat (isografts and allografts treated with cyclosporin A). J. Gynecol. Obstet. Biol. Reprod. (Paris), 14, 567573.[Medline]
Damani, M.N., Master, V., Meng, M.V. Burgess, C., Turek, P., Oates, R.D. and Masters, V. (2002) Postchemotherapy ejaculatory azoospermia: fatherhood with sperm from testis tissue with intracytoplasmic sperm injection. J. Clin. Oncol., 15, 930936.[CrossRef]
Denjean, R., Boeckx, W., Gordts, S. and Brosens, I. (1982) Ovarian transplantation by selective microvascular anastomoses in the rabbit. Br. J. Obstet. Gynaecol., 89, 652656.[ISI][Medline]
Fabbri, R., Porcu, E., Marsella, T. Rocchetta, G., Venturoli, S. and Flamigni, C.2001) Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum. Reprod., 16, 411416.
Faddy, M.J., Gosden, R.G., Gougeon, A. Richardson, S.J. and Nelson, J.F. (1992) Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum. Reprod., 7, 13421346.[Abstract]
Gosden, R.G., Baird, D.T., Wade, J.C. and Webb, R. (1994) Restoration of fertility to oophorectomized sheep by ovarian autografts stored at 196 degrees C. Hum. Reprod., 9, 597603.[Abstract]
Grundy, R., Gosden, R.G., Hewitt, M. Leiper, A., Spondeas, H.A. and Walker, D. (2001) Fertility preservation for children treated for cancer (1): scientific advances and research dilemmas. Arch. Dis. Child., 84, 355359.
Guttman, F.M., Robitaille, P., Lizin, J. and Blanchard, H. (1976) Functional parameters of frozen-thawed autotransplanted canine kidneys. Surg. Forum., 27, 305306.[ISI][Medline]
Harp, R., Leibach, J., Black, J., Keldahl, C. and Karow, A. (1994) Cryopreservation of murine ovarian tissue. Cryobiology, 31, 336343.[CrossRef][ISI][Medline]
Harrison, P.C. and Madwed, J.B. (1999) Anti-LFA-1 alpha reduces the dose of cyclosporin A needed to produce immunosuppression in heterotopic cardiac transplanted rats. J. Heart Lung Transplant, 18, 279284.[CrossRef][ISI][Medline]
Jeremias, E., Bedaiwy, M.A., Gurunluoglu, R. Biscotti, C.V., Siemionow, M. and Falcone, T. (2002) Heterotopic autotransplantation of the ovary with microvascular anastomosis: a novel surgical technique. Fertil. Steril., 77, 12781282.[CrossRef][ISI][Medline]
Kim, S.S., Battaglia, D.E. and Soules, M.R. (2001) The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil. Steril., 75, 10491056.[CrossRef][ISI][Medline]
Lee, S. (1974) Experience with organ transplantation in the rat. IX. Implantation vs transplantation of the ovary. J. Res. Ins. Sci. Korea, 6, 112.
Meirow, D. (2000) Reproduction post-chemotherapy in young cancer patients. Mol. Cell Endocrinol., 169, 123131.[CrossRef][ISI][Medline]
Morita, Y., Perez, G.I., Paris, F., Miranda, S.R., Ehleiter, D., Haimovitz-Friedman, A. Fuks, Z., Xie, Z., Reed, J.C., Schuchman, E.H. et al. (2000) Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy. Nat. Med., 6, 11091114.[CrossRef][ISI][Medline]
Nugent, D., Meirow, D., Brook, P.F., Aubard, Y. and Gosden, R.G. (1997) Transplantation in reproductive medicine: previous experience, present knowledge and future prospects. Hum. Reprod. Update, 3, 267280.
Nugent, D., Newton, H., Gallivan, L. and Gosden, R.G. (1998) Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts. J. Reprod. Fertil., 114, 341346.[Abstract]
Oktay, K. and Karlikaya, G. (2000) Ovarian function after transplantation of frozen, banked autologous ovarian tissue. N. Engl. J. Med., 342, 1919.
Oktay, K., Economos, K., Kan, M., Rucinski, J., Veeck, L. and Rosenwaks, Z. (2001) Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm. J. Am. Med. Assoc., 286, 14901493.
Patrizio, P. (2000) Cryopreservation of epididymal sperm. Mol. Cell Endocrinol., 169, 1114.[CrossRef][ISI][Medline]
Perez, G.I., Robles, R., Knudson, C.M., Flaws, J., Korsmeyer, S.J. and Tilly, L. (1999) Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency. Nature Genet., 21, 200203.[CrossRef][ISI][Medline]
Picton, H.M., Kim, S.S. and Gosden, R.G. (2000) Cryopreservation of gonadal tissue and cells. Br. Med. Bull., 56, 603615.[Abstract]
Podsiadly, B.T., Woolcott, R.J., Stanger, J.D. and Stevenson, K. (1996) Pregnancy resulting from intracytoplasmic injection of cryopreserved spermatozoa recovered from testicular biopsy. Hum. Reprod., 11, 13061308.[Abstract]
Poirot, C., Vacher-Lavenu, M.C., Helardot, P., Guibert, J., Brugieres, L. and Jouannet, P. (2002) Human ovarian tissue cryopreservation: indications and feasibility. Hum. Reprod., 17, 14471452.
Radford, J.A., Lieberman, B.A., Brison, D.R., Smith, A.R., Critchlow, J.D., Russell, S.A., Watson, A.J., Clayton, J.A., Harris, M., Gosden, R.G. and Shalet, S.M. (2001) Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkins lymphoma. Lancet, 357, 11721175.[CrossRef][ISI][Medline]
Schlatt, S., Kim, S.S. and Gosden, R. (2002) Spermatogenesis and steroidogenesis in mouse, hamster and monkey testicular tissue after cryopreservation and heterotopic grafting to castrated hosts. Reproduction, 124, 339346.
Schnorr, J., Oehninger, S., Toner, J. Hsiu, J., Lazendorf, S., Williams, R. and Hodgen, G. (2002) Functional studies of subcutaneous ovarian transplants in non-human primates: steroidogenesis, endometrial development, ovulation, menstrual patterns and gamete morphology. Hum. Reprod., 17, 612619.
Scott, J.R., Keye, W.R., Poulson, A.M. and Reynolds, W.A. (1981) Microsurgical ovarian transplantation in the primate. Fertil. Steril., 36, 512515.[ISI][Medline]
Shinohara, T., Inoue, K., Ogonuki, N. Kamatsu-Shinohara, M., Miki, H., Nakata, K., Kurome, M., Nagashima, H., Toyokuni, S. Honjo, T. et al. (2002) Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination. Hum. Reprod., 17, 30393045.
Thomson, A.B., Campbell, A.J., Irvine, D.C. Anderson, R.A., Kelnar, C.J. and Wallace, W.H. (2002) Semen quality and spermatozoal DNA integrity in survivors of childhood cancer: a casecontrol study. Lancet, 3, 361367.
Tilly, J.L. and Kolesnick, R.N. (1999) Sphingolipid signaling in gonadal development and function. Chem. Phys. Lipids, 102, 149155.[CrossRef][ISI][Medline]
Trounson, A. and Mohr, L. (1983) Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature, 305, 707709.[ISI][Medline]
Wang, X., Chen, H., Yin, H. Kim, S.S., Lin Tan, S. and Gosden, R.G.(2002) Fertility after intact ovary transplantation. Nature, 415, 385.
Witherington, R., Black, J.B. and Karow, A.M. Jr (1977) Semen cryopreservation: an update. J. Urol., 118, 510512.[ISI][Medline]
Submitted on August 28, 2002; resubmitted on January 24, 2003; accepted on February 13, 2003.