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a Division of Reproductive Sciences, Department of Obstetrics and Gynecology and
b Department of Pathobiology and Laboratory Medicine, The Toronto Hospital, University of Toronto, Toronto, Ontario, Canada M5G 2C4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A promising field with respect to preservation of gonadal function and fertility is ovarian cortex transplantation. The ability of ovarian allografts to restore fertility has been demonstrated in several species including the mouse [3–5], hamster [6], rabbit [7], and sheep [8]. Recently, several mice strains carrying mutations that render them immunodeficient have been extensively used to study ovarian tissue xenografting and the biology of the ovarian follicle. Fresh or cryopreserved ovarian tissue from the marmoset monkey [9], the sheep [8], the cat [8], and the human [10] xenografted under the kidney capsule of immunodeficient mice has been shown to develop apparently normal antral follicles. Encouraged by these promising results, we conducted a series of experiments with the aim of establishing a new mouse xenograft model that would be both practical and effective for the maturation and collection of human oocytes. We chose to use the s.c. space for transplantation since, if vascularization and implantation ensued, there would be adequate room for follicle development to occur to the preovulatory stage. In contrast, the kidney capsule or ovarian bursa, while well vascularized, are unlikely to be able to support complete human preovulatory follicle development. The objectives of our study were 1) to evaluate the s.c. space in the non-obese diabetic-severe combined immune deficiency (NOD-SCID) mouse as a new site for human ovarian cortex transplantation and 2) to determine the optimal transplant conditions for maintenance of follicle health and responsiveness to exogenous gonadotropin stimulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male and female NOD-SCID mice (6–8 wk of age) were obtained from the breeding colony of the Samuel Lunenfeld Research Institute at the Mount Sinai Hospital, Toronto. The animals were housed in a high-efficiency particulate air-filtered, positive-pressure room. Cages were filter-topped, and animals had free access to sterilized food and water under 12L:12D conditions. A solution of 200 mg sulfamethoxazole and trimethoprim (40 mg/5 ml; Novo-Trimel; Novapharm, Toronto, ON, Canada) was added to the drinking water for 7 days every other week (15 ml in 1 L of water). Upon arriving from the breeding suite, mice were allowed to acclimate for 1 wk. All procedures, tests, and injections were performed under a laminar flow hood in the positive-pressure room. Approval for the study was obtained from the Samuel Lunenfeld Research Institute Animal Research Committee.
Validation of the Subcutaneous Transplantation Site
Female mice were randomly assigned to one of the following experimental groups: sham operated (group 1; n = 5); ovariectomized (group 2; n = 7); ovariectomized with s.c. ovarian allografting (group 3; n = 10). Mice were anesthetized by an i.p. injection of 2,2,2-tribromoethyl alcohol (0.4 mg/g BW) in 2-methyl-2-butanol. During surgery, mice were kept on a warming plate (~37°C) covered a with sterile towel. Ovaries were externalized through small dorsolateral incisions. Sham-operated animals received no further treatment; the ovaries were replaced to the abdominal cavity, and body wall incisions were sutured. Both ovaries of animals in groups 2 and 3 were removed, with care taken to remove all ovarian tissue. Group 2 (ovariectomized) animals received no further surgery, and the incisions were closed as described above. In group 3 (ovariectomized) animals, an s.c. pocket was developed above the flank, using blunt dissection through the original left skin incision, and one ovary was inserted into the space within seconds from removal. After surgery, the mice received an i.m. injection of 8 µg gentamicin (Novopharm, Toronto, ON, Canada) and 8 µg tobramycin (Nebcin; Eli Lilly Canada Inc., Toronto, ON, Canada) and then were allowed to recover.
Vaginal smears were taken daily, starting on Day 9 postsurgery, from all mice in groups 1–3. Using sterile pipettes and sterile normal saline, the vagina of each mouse was flushed gently and the cells were smeared onto a slide. The smears were left to air dry and then stained with an aqueous solution of methylene blue. The stage of the estrous cycle was determined from the cell types observed in the smear [11].
Human Tissue Collection and Transplantation
Human ovarian tissue was collected from patients undergoing gynecological surgery for various benign conditions following informed consent and approval from the Institutional Review Board. The patients (n = 13) ranged in age from 24 to 38 yr and had normal cycles prior to surgery. The stage of the menstrual cycle was not available. Immediately after removal, cortical biopsies were collected PBS containing 8 IU/L of highly purified human urinary FSH (Fertinorm; Serono Canada, Oakville, ON, Canada). A fresh piece of tissue from each donor was kept for histological examination in order to confirm the presence of follicles.
Xenografting of ovarian cortex was performed within 2 h after removal from the patient to minimize ischemic damage. Animals were prepared as described above. In female mice, the ovaries were left intact. Pieces of ovarian cortex were minced into small pieces of 1–2 mm3. Through a small dorso-median transverse incision, slices of ovarian tissue were placed bilaterally into the s.c. space above the flank. Three pieces of tissue were inserted on each side without suturing. The wound margin incision was fixed with a single clip, an antibiotic was given (as described above), and the animals were allowed to recover.
Twelve weeks after surgery, ovarian stimulation, consisting of daily i.p. injections of human menopausal gonadotropin (Pergonal, Serono Canada) at a dose of 4 IU/day were commenced. Ovarian stimulation was carried out for 14 days. Follicle survival and development after stimulation were compared in the recovered ovarian cortex for the following variables:
A. Male versus female mice as recipients In order to evaluate which gender and associated hormonal milieu is more favorable for the human ovarian xenograft model, follicle survival and development were compared between male (n = 30) and female (n = 20) mice as hosts.
B. Cold versus warm transport medium In order to determine which transport conditions are more conductive for follicle survival and health, we compared the results of transplantation following tissue transport on ice (n = 7) and at 37°C (n = 32).
C. Intact versus pituitary down-regulated mice To evaluate the effects of endogenous mouse pituitary and ovarian function on human follicle survival and response to stimulation, we compared the results of ovarian tissue xenografting in intact mice (n = 36, 16 males and 20 females) versus mice that underwent pituitary down-regulation (n = 9, 4 males and 5 females). The GnRH agonist leuprolide acetate, (0.25 mg i.m.; Depot Lupron; Abbott Laboratories, Montreal, PQ, Canada) was administered 2 wk prior to stimulation.
Histology
Upon the completion of ovarian stimulation, the mice were killed using carbon dioxide gas and the grafts were dissected out from the s.c. space. The recovered grafts were placed in Bouin's fixative overnight; they were then transferred into 70% ethanol and stored at 4°C until processing. Fixed tissues were embedded in paraffin blocks, sectioned, and stained with hematoxylin and eosin. The sections were examined for the presence of follicles, and their developmental stage was determined. Follicles were classified as follows: primordial follicles with one layer of flattened granulosa cells surrounding the oocyte, primary follicles having up to two layers of cuboid granulosa cells, preantral follicles having three or more layers of granulosa cells but no antrum, and antral follicles with an antral cavity.
Proliferating Cell Nuclear Antigen
Ovarian tissue sections originating from seven patients were randomly selected and evaluated for immunolocalization of proliferating cell nuclear antigen (PCNA) in 14 mice (one male and one female for each source of ovarian tissue). For this purpose, after overnight incubation in 4% paraformaldehyde, tissue was washed, dehydrated, and stored in 70% ethanol at 4°C until embedding. Sections were deparaffinized with xylene, quickly rehydrated in ethanol, and heat-treated in citrate buffer to enhance antigen retrieval. Sections were preincubated with 10% nonimmune porcine serum in PBS with 0.1% Triton X (PBS-T) for 10 min at room temperature. The primary antibody, monoclonal mouse anti-human PCNA (Dako, Carpinteria, CA) was added (1:50 dilution in PBS-T supplemented with 1% normal porcine serum), and the sections were incubated overnight at 4°C in a humidified chamber. The secondary antibody, biotinylated porcine anti-mouse antibody (Multilink; Dako) was added, in 1:100 dilution in PBS-T, for 1 h at room temperature. The final distribution of antibody was determined by streptavidin-Texas Red conjugate (Calbiochem, San Diego, CA) diluted 1:150 in PBS-T for 30 min at 4°C. After washing, sections were counterstained with the nuclear fluorochrome 4,6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co., St. Louis, MO) to confirm subcellular localization of PCNA staining. Mouse testis samples were used as positive controls. Positive staining for PCNA was observed only in mitotically dividing spermatogonia. Ovarian cortex xenografts treated according to the above protocol, but with the primary or secondary antibody omitted, served as negative controls, and no PCNA staining was observed. All specimens were examined using a Zeiss (Carl Zeiss, Thornwood, NY) fluorescent microscope with appropriate filters.
Data Analysis
The statistical package Sigmastat (Jandel Corporation, San Raphael, CA) was used for data analysis. Nominal data were expressed as mean ± SEM and compared using one-way ANOVA. Follicle survival and development in the various study groups were compared using chi-square analysis or Fisher's exact test. A p value of < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animals recovered from surgeries and survived the postoperative period with the exception of three that developed infections and died. Smears from all sham-operated animals (group 1) demonstrated cyclic changes in vaginal cytology characteristic of estrous cycles by 9 days after surgery. Cornified epithelial cells were seen at intervals of 5.8 ± 1.8 days. Four of seven ovariectomized mice (group 2) did not demonstrate any cornified epithelial cells in smears throughout the monitoring period, whereas three animals demonstrated sporadic incidence of epithelial cells at irregular intervals, most probably the result of mechanical stimulation of the vaginal mucosa. Vaginal smears in mice that had received s.c. ovarian allografts (group 3) showed presence of cornified epithelial cells as early as 9 days after transplantation. Within 14 days from surgery, all animals showed estrogen effects. Vaginal cornification occurred at intervals of 7.1 ± 1.9 days, which was not statistically different from the value for group 1 (p = 0.24).
Follicle Survival and Development in Human Xenografts
On autopsy, human xenografts were distinguishable from the surrounding host s.c. tissue and could be recovered in 50 of 54 (92.6%) mice. In mice in which ovarian tissue could not be recovered, small fibrotic bands of white tissue were noted, suggesting that the graft had undergone reabsorption. Overall, ovarian follicles were identified in 37 of 50 (74%) of the recovered xenografts. Twelve weeks after transplantation, human gonadotropin stimulation resulted in the development of preantral or larger follicles (Fig. 1, top) in 19 of 37 (51%) of the grafts, including several graafian follicles up to 6 mm in diameter that could be palpated and visualized through the mouse skin (Fig. 1, middle and lower panels).
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Male versus Female Mice as Hosts
Overall, no significant difference was noted in the presence of primary or primordial follicles in male versus female mice as hosts (20 of 30 [66.6%] vs. 17 of 20 [85%]; p = 0.26). However, after ovarian stimulation, significantly more preantral or larger follicles were present in male as compared to female mice as hosts (13 of 17 [76.5%] vs. 6 of 20 [30%], respectively; p < 0.05).
Cold versus Warm Transport Medium
Follicular survival and development were significantly increased when warm tissue transport medium was used as compared to cold transport (26 of 32 [81.3%] vs. 2 of 7 [28.6%], respectively; p < 0.05). Significantly more follicles were present in female mice after warm transport (12 of 16 or 75%) as compared to cold transport (1 of 5 or 20%; p < 0.05). In males, however, the same trend was noted (14 of 16 or 87.5% in warm transport medium as compared to 1 of 2 or 50%; not significant, probably due to the small numbers involved).
Pituitary Down-Regulated versus Intact Mice
Similar degrees of follicle survival and development were noted in intact compared to GnRH-a down-regulated mice (overall, 24 of 36 [75%] in intact vs. 7 of 9 [77.8%] in GnRH-a treated mice; not significant). Comparing male versus female mice as hosts, a similar rate of follicular survival and development was noted in intact (12 of 16, 75%) versus GnRH-a down-regulated males (3 of 4, 75%; not significant) and in intact females (12 of 20, 80%) versus down-regulated females (4 of 5, 80%; not significant).
PCNA
The overall health of stimulated follicles obtained was confirmed by the status of PCNA expression. Nuclei of granulosa cells and oocytes in primordial follicles did not contain detectable levels of PCNA immunoreactivity (not shown). Both in primary follicles and in more advanced stages of follicle development, PCNA immunoreactivity was observed in both granulosa cells and the oocyte (Fig. 2), indicating active cellular proliferation in growing follicles.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experience with s.c. allografts of ovarian tissue in humans is limited. In a single study that has not been repeated, s.c. transplantation of cryopreserved human ovarian tissue transplanted s.c. was able to restore menstrual cycles in women. [16]. While the option of allografting frozen-thawed ovarian cortex to cancer survivors seems attractive, it may carry a risk of transmission of microscopic metastatic disease, which could theoretically result in relapse. Such relapses have been described following autologous frozen-thawed bone marrow transplantation [17]. Young patients with bloodborne cancers such as leukemia, systemic cancers such as lymphoma, and metastatic disease may all be at risk. In a mouse model [18], when small slices of ovarian tissue from donor mice with lymphoma were grafted into healthy mice, 7 of 7 recipients of fresh tissue and 6 of 7 recipients of frozen tissue developed lymphoma. Confirmation of the safety of ovarian tissue for autologous transplantation based solely on absence of malignant cells by light microscopy [2] may not be sufficient; and in the absence of reliable technology to detect the presence of remnant cancer cells in frozen-thawed ovarian tissue, transplantation may be too hazardous. Transplantation of fresh or frozen-thawed ovarian cortex into an animal host, with subsequent maturation and collection of oocytes in the animal may therefore offer considerable advantages to cancer survivors. First, the possibility of cancer transmission and relapse is eliminated, since cancer cells could not penetrate the zona pellucida—thus allowing transfer of cancer-free embryos. Second, patients would be spared the risks and complications inherent with surgical ovarian tissue transplantation, and the clinical stimulation and retrieval of oocytes.
Immunodeficient rodents are promising research models for ovarian cortex xenografting. Nude (athymic, therefore lacking T lymphocytes) [9, 19] and severe combined immune deficiency (SCID) (lacking both B and T cell immunity) [10, 20] mouse strains are the most commonly used for this purpose. However, the SCID mutation does not appear to be entirely stable in blocking lymphocyte development, and with maturation, 2–20% of SCID mice develop a few clones of functional B and T cells, thus producing detectable levels of IgG [21]. Incomplete immunosuppression of SCID mice may become a serious problem when these animals are used for long-term xenografting of ovarian cortex, since partial or complete rejection of foreign tissue could ensue. In addition, nude and SCID mice have functional macrophages and high levels of natural killer (NK) cell and complement activity, which contribute to a nonspecific immune response [21]. In order to obviate potential rejection problems, we have selected a new mouse strain, which includes the non-obese diabetic (NOD) mutation, in which T lymphocyte-mediated pancreatic B cell destruction occurs. Shultz et al. [22] recently developed a NOD strain congenic for the SCID mutation and homozygous for this recessive mutation (NOD-Scid/Scid). NOD-SCID mice are not only deficient in functional B and T lymphocytes and diabetes-resistant, but are also markedly deficient in NK, macrophages, and complement activity. Since the NOD-SCID strain appears to be stable over time, we have selected this strain of mice for our experimental model.
Using NOD-SCID mice, we have demonstrated that s.c. xenogeneic ovarian tissue transplantation is feasible. The viability of xenografted human ovarian tissue in NOD-SCID mice was confirmed by the presence of primordial and primary follicles, which developed into preantral and antral follicles following stimulation with human gonadotropins. Staining of granulosa cells with PCNA provided firm evidence that the follicles were proliferating, since PCNA is not expressed in atretic follicles or in the pregranulosa cells of primordial follicles [23]. It has been observed previously that most developing follicles, and up to 50% of primordial follicles, undergo atresia during transplantation, probably due to ischemia [24, 25]. The differential survival of primordial follicles versus larger developing follicles in grafts is probably related to the lower metabolic rate of the small follicles [20]. Hence, the majority of follicles that develop in xenografts are primordial follicles that have entered the growth phase after grafting.
In an elegant study using SCID mice made deficient in pituitary gonadotropins, Oktay et al. [10] have recently shown that follicles do not progress beyond the stage of two granulosa cell layers without gonadotropin support. Hence, in order to evaluate the effect of gonadotropin administration, it is necessary to differentiate primordial and primary follicles, which have not entered the stage of gonadotropin responsiveness, from preantral or antral follicles that require gonadotropin support. Unlike Oktay et al. [10], who used FSH only for follicle stimulation, we have used a gonadotropin preparation consisting of both LH and FSH. Since only a small piece of ovarian cortex was used for transplantation, we hypothesized that there may be a relative deficiency of stroma and of endogenous androgen production as substrate for estrogen. This hypothesis receives some support from our finding in the present study that male mice, with high serum concentrations of androgen, were better hosts for the development of antral follicles than were female mice. Consequently, a combination of LH and FSH may be more effective in stimulating follicle development. In addition, it is not known what gonadotropin dose is required for stimulation of ovarian follicles in s.c. xenografts, where nonphysiological vascularization may affect hormone availability. Thus, we empirically used a relatively high daily dose of 4 IU of human menopausal gonadotropin. A dose-finding study for the optimal dose and gonadotropin preparation to be used in the xenograft model is planned.
Since it takes about 3 mo for human primordial follicles to enter the gonadotropin-dependent growth phase [26], a waiting period of 12 wk was used before stimulation commenced. After 2 wk of stimulation, some of the antral follicles that had developed were as large as 6 mm in diameter and could be easily seen and palpated through the mouse skin. Thus, the s.c. space not only provides ample room for growth and development of follicles, but also allows for simple and convenient monitoring and direct access for needle aspiration. With the kidney capsule and ovarian bursa for xenografting, follicle monitoring may be extremely difficult and the retrieval of oocytes requires laparotomy. Subcutaneous xenografting, therefore, has the potential for repetitive stimulation and egg-retrieval cycles. The rate-limiting factor for repetitive stimulation would be the life expectancy of the NOD-SCID mouse, which is around 1 yr. This relatively short life span may preclude the use of mice as temporary quarters of ovarian cortex if allografting the tissue back to the patient is to be considered. Since confirmation of a disease-free state may require a longer period of time, freezing and thawing of tissue for allografting in cancer survivors may be more appropriate, once all safety issues have been resolved.
Another major advantage of the s.c. site is related to the simplicity of the actual transplantation procedure. Compared to transplantation to the kidney capsule and ovarian bursa, s.c. transplantation is a quick procedure that requires only minimal training and operator experience and is associated with very low mouse morbidity and mortality rates, especially in immunosuppressed animals.
An important factor for the successful transplantation of ovarian cortex in immunodeficient mice is the quick establishment of rich blood supply. Revascularization of the graft is crucial for the survival of ovarian follicles after xenografting. Therefore, the profuse blood supply in the subcapsular region of the kidney and in the ovarian bursa has been the main reason to choose these sites for transplantation. However, the present study suggests that the choice of site may not be critical, since the tissue placed in the s.c. space seems to achieve a vascular supply very quickly. The factors responsible for stimulation of angiogenesis after transplantation are largely unknown. Dissen et al. [27], using ovarian cortex allografting adjacent to the jugular vein, showed that revascularization of the graft was initiated within 48 h of transplantation. This vascularization process was accompanied by a striking increase in gene expression of selected angiogenic factors, most prominently that of vascular endothelial growth factor. These authors also suggested that gonadotropin secretion following transplantation plays a role in the vascular response.
Although revascularization occurs as early as 2 days after grafting [27], a significant degree of cellular damage may occur during that period. Some of this damage may be caused by reactive oxygen species generated during ischemia-reperfusion. It has been recently demonstrated that the detrimental effects of ischemia-reperfusion injury on ovarian cortex allografts may be significantly reduced with use of antioxidants, such as vitamin E [28]. Our experience with vitamin E administration (unpublished results) failed to suggest any beneficial effects.
Pituitary gonadotropins also play a role in ischemia-reperfusion injury. Of the two pituitary gonadotropins, FSH is the more important in this context. FSH stimulates granulosa cell mitosis and inhibits granulosa cell apoptosis [29–31]. Treatment of rat antral follicles with increasing doses of FSH caused a dose-dependent suppression of DNA fragmentation, reaching a maximal suppression of 60% [31]. Thus, withdrawal of trophic and mitogenic factors such as pituitary gonadotropins and ovarian steroids can lead to the apoptotic demise of cells. For this reason, we have decided to include FSH routinely in our transport medium. Further research is necessary to determine the role of FSH in the ovarian xenograft model.
A factor found to have major significance in terms of follicle survival and development was the temperature of the transport medium. Unlike other organ transplant models, ovarian tissue transport at 37°C was found to be significantly superior to transport on ice. It is possible that oocytes in primordial and primary follicles have sensitivity to chilling effects similar to the known chilling sensitivity of mature oocytes and follicles [32]. Further confirmation of this aspect of the xenograft procedure seems mandatory before firm conclusions can be made. Although ovariectomy as suggested by earlier work improves the success of ovarian cortex transplantation [12] in rodents and is therefore currently being practiced in similar experimental models [18, 20], we failed to observe any beneficial effects of "medical gonadectomy" by GnRH-a in male or female recipients.
In summary, we have established a new model for human ovarian cortex xenografting in immunodeficient mice. Subcutaneous transplantation is a quick and simple procedure that maintains follicle viability. Primordial and primary follicles in ovarian xenografts retain the ability to become responsive to gonadotropins and develop into advanced follicles following stimulation. Warm tissue transport and the use of male mice as recipients seem to yield the best results. Direct monitoring of follicle development can be conducted through inspection and palpation of the mouse skin. Thus, s.c. transplantation may provide a safe and suitable environment for follicle growth and oocyte maturation that would obviate many of the problems and concerns related to ovarian banking for cancer patients [33–36]. Several challenging questions have yet to be answered. 1) Can oocytes derived from xenografted ovarian cortex mature and become meiotically competent so that normal fertilization and embryo development would occur? 2) How can tissue and follicle survival during the transplantation procedure be improved? 3) Which transplant site in the mouse is most conducive to follicle and oocyte health? 4) Which is the optimal stimulation protocol for use with xenografted ovarian cortex? 5) Can the same results be achieved with frozen-thawed ovarian tissue? Research on these and many other aspects of ovarian tissue xenografting is currently under way in several laboratories including our own.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: R.F. Casper, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, The Toronto Hospital 6-246 Eaton Wing North, 200 Elizabeth Street, Toronto, ON, Canada M5G 2C4. FAX: 416 340 4022; r.casper{at}utoronto.ca
Accepted: January 22, 1999.
Received: November 18, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 H. Kaneko, K. Kikuchi, J. Noguchi, M. Hosoe, and T. Akita Maturation and Fertilization of Porcine Oocytes from Primordial Follicles by a Combination of Xenografting and In Vitro Culture Biol Reprod, November 1, 2003; 69(5): 1488 - 1493. [Abstract] [Full Text] [PDF]
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S.S. Kim Ovarian tissue banking for cancer patients: To do or not to do? Hum. Reprod., September 1, 2003; 18(9): 1759 - 1761. [Abstract] [Full Text] [PDF]
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D. A. Gook, D.H. Edgar, J. Borg, J. Archer, P.J. Lutjen, and J.C. McBain Oocyte maturation, follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting Hum. Reprod., September 1, 2003; 18(9): 1772 - 1781. [Abstract] [Full Text] [PDF]
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T. Israely, H. Dafni, D. Granot, N. Nevo, A. Tsafriri, and M. Neeman Vascular Remodeling and Angiogenesis in Ectopic Ovarian Transplants: A Crucial Role of Pericytes and Vascular Smooth Muscle Cells in Maintenance of Ovarian Grafts Biol Reprod, June 1, 2003; 68(6): 2055 - 2064. [Abstract] [Full Text] [PDF]
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 M. Snow, S.-L. Cox, G. Jenkin, A. Trounson, and J. Shaw Generation of Live Young from Xenografted Mouse Ovaries Science, September 27, 2002; 297(5590): 2227 - 2227. [Full Text] [PDF]
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J. Liu, J. Van der Elst, R. Van den Broecke, and M. Dhont Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries Hum. Reprod., March 1, 2002; 17(3): 605 - 611. [Abstract] [Full Text] [PDF]
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D. A. Gook, B.A. McCully, D.H. Edgar, and J.C. McBain Development of antral follicles in human cryopreserved ovarian tissue following xenografting Hum. Reprod., March 1, 2001; 16(3): 417 - 422. [Abstract] [Full Text] [PDF]
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J. Liu, J. Van der Elst, R. Van den Broecke, and M. Dhont Live Offspring by In Vitro Fertilization of Oocytes from Cryopreserved Primordial Mouse Follicles after Sequential In Vivo Transplantation and In Vitro Maturation Biol Reprod, January 1, 2001; 64(1): 171 - 178. [Abstract] [Full Text]
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J. Qu, P. A. Godin, M. Nisolle, and J. Donnez Distribution and epidermal growth factor receptor expression of primordial follicles in human ovarian tissue before and after cryopreservation Hum. Reprod., February 1, 2000; 15(2): 302 - 310. [Abstract] [Full Text] [PDF]
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 D. C. Linch, R. G. Gosden, T. Tulandi, S.-L. Tan, and S. L. Hancock Hodgkin's Lymphoma: Choice of Therapy and Late Complications Hematology, January 1, 2000; 2000(1): 205 - 221. [Abstract] [Full Text] [PDF]
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 M. R. Soules Commentary: Posthumous Harvesting of Gametes A Physician's Perspective J. Law Med. Ethics, December 1, 1999; 27(4): 362 - 365. [PDF]
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