Department of Obstetrics and Gynecology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, A81, Cleveland, Ohio, USA
1 To whom correspondence should be addressed. e-mail: falcont{at}ccf.org
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Abstract |
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Key words: cancer/fertility preservation/ischaemic injury/ovarian freezing/ovarian tissue banking
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Introduction |
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Using present techniques, ischaemic injury to the transplanted tissue results in the loss of virtually the entire growing follicle population and a significant number of primordial follicles (Newton et al., 1996). This could limit its long-term viability. Alternatively the primordial follicles in the ovarian tissue can be matured in vitro or in an immune-deficient animal host. The former cannot be accomplished with present technology and the latter has significant ethical and biological issues, such as transmission of virus or viral particles. The focus of this contribution will be about the potential of cryopreserving an intact ovary followed by its transplantation. Although the published work is encouraging, many technical and laboratory nuances need to be resolved.
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Which cryoprotective agent to be used? |
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Similar results were obtained by Candy et al. (1997). They suggested that to achieve optimal cryoprotection, it is essential that freezing protocols allow uniform penetration of CPA throughout the ovarian tissue. Consequently, the CPA permeation rate is an important limiting factor in developing better cryopreservation protocols for ovarian tissues (Candy et al., 1997
). Besides the importance of the diffusion rate, Newton et al. (1998
) also studied the importance of the diffusion temperature. They concluded that at a higher temperature (37°C) all CPA penetrate the ovarian tissue at a faster rate. Recent evidence shows that using such a protocol will result in the development of a metaphase II oocyte from xenografting cryopreservedthawed human ovarian cortical strips in immunodeficient mice (Gook et al., 2003
).
Tissue penetration of cryoprotectant in bigger pieces of tissue or whole organ systems is even more complicated. The transplant literature, such as with kidneys, have several protocols that strive to protect the tissue prior to transplantation. These protocols try to perfuse the whole organ with a CPA. The potential for cryopreserving the entire ovary has the same methodological limitations. The cryoprotectant can be perfused through the ovarian vessels using a special pump to achieve the appropriate tissue penetration.
The fact that the ovary has different heterogeneous cellular components adds to the technical challenge of intact ovary freezing as different cell types have different properties which may alter their requirements for cryoprotectants. Conse quently, the differential effect of a single CPA on the epithelial, germ and sex cordstromal cell components of the ovary may affect their post-thaw survival and function. However, unlike the disappointing results of earlier trials of kidney cryopreservation, using this approach, pregnancies in rats (Wang et al., 2002) and partial restoration of hormonal function in sheep were recently reported (Bedaiwy et al., 2003
).
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Vascularized ovarian grafts: is it possible? |
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In our trials to establish an animal model for studying different aspects of vascularized ovarian grafts, we proposed anastomosis of the ovarian vessels to the deep inferior epigastric vessels in a sheep model. The inferior epigastric vessels are branches of the external iliac artery that lie superior to the inguinal ligament and posterior to the rectus muscle. They are easily accessible without entering the peritoneal cavity. Furthermore there is an extensive experience in the plastic surgery literature in the use of these vessels for breast reconstructive surgery. Normal hormonal functions, higher primordial follicular counts as well as normal ovarian histological architecture were observed in animals with patent vascular anastomosis compared to those with occluded anastomosis. This experiment proved the feasibility of using the deep inferior epigastric vessels as a potential recipient site for vascularized fresh ovarian grafts (Jeremias et al., 2002). It provides an easy access to the microvascular anastomosis technique as well as future oocyte harvesting.
Similarly, in another experiment we tested the feasibility of transplanting intact frozenthawed ovary with microvascular anastomosis of the ovarian vascular pedicle to the deep inferior epigastric vessels using the same technique. No significant differences were observed between the mean values of apoptosis and follicular viability in cryopreservedthawed ovarian cortical strips placed along the course of the deep inferior epigastric vessels without vascular anastomosis and cryopreservedthawed intact ovaries, demonstrating that cryosurvival of whole ovary is as good as cortical strips, at least in the sheep. After autotransplantation of the whole organ, post-operative FSH and serum estradiol levels were similar to pre-operative values within 1 week in animals with patent vascular anastomosis at necropsy. From this study we concluded that transplantation of intact frozenthawed ovary is technically feasible. Using this approach, immediate restoration of vascular supply and ovarian hormonal function is possible (Bedaiwy et al., 2003). These provisional results should also be documented in long-term studies in humans. Although there is some question of systemic thrombotic risks after vascular transplantation, this has not been the experience in the reconstructive plastic surgery literature, where extremely complex autografting with a vascular anastomosis is used routinely in plastic surgery, with few life-threatening complications. Moreover, pregnancy in transplanted mouse uterus after long-term cold ischaemic preservation was reported (Racho El-Akouri et al., 2003
).
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Autografting of human ovarian tissue: current status and limitations |
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The potential for whole organ transplant |
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Another limitation for intact organ transplantation is the need for the removal and storage of an intact ovary or an ovarian segment with its vasculature. Several technology companies are working on developing cryo-chambers that will hold the size of a human ovary. Cryopreservation protocols are currently almost exclusively limited to fragmented non-vascular cortex pieces. However, in recent years improvements in freezing protocols for vessel grafts, and reports of the cryopreservation of composite tissue and animal organs, point to the possibility of organ cryopreservation. Our preliminary data support this observation (Bedaiwy et al., 2003).
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Conclusions |
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References |
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Submitted on December 2, 2003; accepted on March 23, 2004.