1 Department of OB/GYN, Eulji University School of Medicine, Seoul and 2 Department of Animal Sciences, Chungbuk National University, Chong Ju, Korea
3 To whom correspondence should be addressed at: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, CedarsSinai Medical Center, Los Angeles, CA 90048, USA. Email: medssk{at}attglobal.net
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Abstract |
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Key words: cryopreservation/microtubule/oocyte/ovarian tissue/transplantation
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Introduction |
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Embryo freezing can be offered prior to cancer treatment, but it is only an option for patients who have a partner or are willing to accept fertilization by donor sperm. Cryopreservation of oocytes does not require a partner, but it has its own limitations (Kim et al., 2001a). Obviously, both embryo and oocyte freezing cannot be offered to pre-pubertal girls. These two strategies can delay cancer treatment, which is not acceptable to many cancer patients. An emerging technology, ovarian tissue cryopreservation, has several potential advantages. In this procedure, hundreds of immature oocytes are cryopreserved without the necessity of ovarian stimulation and delay in initiating cancer treatment. It offers the potential for restoration of natural fertility with less ethical dilemma.
However, it is impossible to achieve fertilization without obtaining mature oocytes. The practical strategy to grow and mature oocytes in stored ovarian tissue currently appears to be autotransplantation of frozenthawed ovarian tissue. Restoration of endocrine functions after autotransplantation of fresh or frozenthawed ovarian tissue in humans has been demonstrated (Oktay and Karlikaya, 2000; Callejo et al., 2001
; Oktay et al., 2001
; Radford et al., 2001
; Kim et al., 2004b
; Tryde Schmidt et al., 2004
). Nevertheless, it is still difficult to obtain healthy, mature oocytes from ovarian grafts for fertilization (Oktay et al., 2004
). To date, there is only one report of live birth after transplantation of ovarian tissue in humans (Donnez et al., 2004
).
Although ethical and safety issues associated with growing human follicles to maturity in animal hosts are of concern, xenotransplantation is an alternative strategy to develop immature oocytes in stored ovarian tissue. With this strategy, transmission and relapse of cancer in patients can be completely eliminated. Previous studies demonstrated that follicles were matured to the antral stage after xenografting human ovarian tissue to severe combined immunodeficient (SCID) mice (Weissman et al., 1999; Gook et al., 2001
, 2003
; Kim et al., 2002
). However, it is unknown whether human oocytes developed in animal hosts are normal. The aim of the present study was to investigate the integrity of the human oocytes retrieved from antral follicles that were grown and matured in hosts after transplantation of cryopreserved ovarian tissue.
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Materials and methods |
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Freezing and thawing
Prepared thin ovarian cortical sections (5x5x 1 mm) were transferred into 1 ml cryogenic vials (Nunc; Intermed, Kamstrup, Denmark) containing 1.5 mol/l dimethylsulphoxide (DMSO) (Sigma, St Louis, MO, USA) with 1% human serum albumin and 0.1 mol/l sucrose. The vials were gently shaken for 30 min at 4°C to promote equilibration, cooled in a programmable freezer as per our ovarian freezing programme (cooled at 2°C/min to 7°C, seeding manually at 7°C, 0.3°C/min to 40°C, 10°C/min to 120°C) and plunged into liquid nitrogen (196°C) for storage.
For transplantation, cryopreserved ovarian tissue was thawed by rapid thawing method (100°C/min) in a warm water bath (30°C), and washed stepwise for 3 min each in rehydration media to minimize osmotic damage (1.0 mol/l DMSO +0.1 mol/l sucrose; 0.5 mol/l DMSO +0.1 mol/l sucrose; 0.1 mol/l sucrose).
Experimental animals
Thirty-five (25 female, 10 male) homozygous SCID mice at 68 weeks of age were obtained from the Jackson Laboratory, Bar Harbor, Maine, USA. These animals were housed in air-filtered positive pressure isolators with free access to sterilized water and food.
Xenotransplantation
Thawed human ovarian tissue was incubated in minimum essential medium (Sigma) containing 500 IU/ml penicillin G and 382 IU/ml streptomycin for 30 min at 37°C in the incubator (5% CO2 in air). The animals were anaesthetized with tribromoethanol (0.01 ml/g of body weight). First, gonadectomy was performed through a dorsomedian incision in female mice and through a ventromedian incision in male mice. After confirming the complete removal of gonads bilaterally, human ovarian cortical tissue (5x5 mm) was placed into the subcutaneous space (two grafts per animal) and anchored with 50 nylon sutures to the muscle layer.
Ovarian stimulation and monitoring
After transplantation, the animals were observed for 20 weeks in a sterile condition. Five animals (four females and one male) died during this observational period. The remaining 30 animals were stimulated with 5 IU of pregnant mare's serum gonadotrophin (PMSG, Intervet, UK) every second day for 2 weeks starting at 20 weeks after transplantation. At the end of the stimulation cycle, 10 IU of hCG was injected i.p. Approximately 36 h after the hCG administration, the animals were euthanized with CO2 gas and ovarian grafts were recovered. Blood samples were collected for radioimmunoassay of estradial concentrations at the time the animals were decapitated.
Oocyte retrieval and in vitro maturation
Recovered grafts were grossly inspected. The visible antral follicles (>2 mm) were counted and dissected. Oocytes were retrieved from dissected antral follicles (26 mm in diameter) under the dissecting microscope. The majority of the recovered oocytes appeared to be immature, thus these oocytes with cumulus oophorus were cultured in 200 µl microdrops of in vitro maturation (IVM) medium for 3648 h at 37°C in the incubator (5% CO2 in air). Our IVM medium was composed of 20% human follicular fluid, 10 IU/ml recombinant FSH (rFSH), 20 IU/ml hCG, 20 ng/ml estadiol (E2) in a TCM 199 medium (Sigma).
Immunocytochemical assessment of microtubules and DNA
Before fixation, cumulus cells were removed using a combination of 0.1 mg/ml hyaluronidase and manual pipetting of the oocyte. Denuded oocytes were permeabilized in a modified buffer M (Simerly and Schatten, 1993) for 20 min at 37°C, fixed in methanol at 20°C for 10 min and stored in phosphate-buffered saline (PBS) containing 0.02% sodium azide and 0.1% bovine serum albumin (BSA) for 25 days at 4°C. Microtubule localization was performed using
-tubulin monoclonal antibody (Sigma). Fixed oocytes were incubated for 90 min at 37°C with antibody diluted 1:300 in PBS. After several washes with PBS containing 0.5% Triton X-100 and 0.5% BSA, oocytes were incubated in a block solution (Simerly and Schatten, 1993
) at 37°C for 1 h. The blocking was followed by incubation in fluorescein isothiocyanate-labelled goat anti-mouse antibody (Sigma) for 1 h. DNA was observed by exposure to 10 µg/ml propidium iodide (Sigma) for 1 h. Stained oocytes were then mounted under a coverslip with antifade mounting medium (Universal Mount: Fisher Scientific Co., Huntsville, AL, USA) to retard photobleaching. Slides were examined using a laser-scanning confocal microscope (BIO-RAD MRC 1024, Richmond, CA, USA). All images were recorded and archived on an erasable magnetic optical diskette and downloaded to a dye sublimation printer (Sony, Tokyo, Japan) using Adobe Photoshop Software (Adobe, Mountain View, CA, USA).
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Results |
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Discussion |
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Most growing follicles cannot survive cryoinjury, whereas the survival rate of primordial follicles in ovarian tissue after freezing and thawing is >70% (Newton et al., 1996). As a consequence, cryopreservation of ovarian tissue is basically storing immature follicles at low temperature. The main challenge is how to develop these stored follicles to the mature stage for fertilization. Theoretically, there are three strategies, which include autotransplantation (either orthotopically or heterotopically), xenotransplantation, and in vitro culture (Kim et al., 2001a
).
The most desirable strategy is to develop immature oocytes entirely in an in vitro culture system, because embryos could then be transferred free of disease to the patient after cancer treatment. However, it is not technically feasible yet, because the culture techniques and media presently available are inadequate to sustain the long periods of follicular development required in humans (Kim et al., 2004a).
At present, autotransplantation of stored ovarian tissue seems to be the only clinically practical strategy, and is already being practised on a tentative clinical basis under institutional review board guidelines. Nevertheless, one serious and imminent concern with autotransplantation of ovarian tissue in cancer patients is that stored tissue can harbour malignant cells and subsequently transmit microscopic metastatic disease. Shaw et al. (1996) reported that healthy AKR mice which were grafted with fresh or cryobanked ovarian tissue from donor mice with lymphoma, died of lymphoma within 23 weeks of grafting.
Clinically, however, ovarian metastasis is rare in many cancers such as Wilm's tumour and Hodgkin's disease. The risk of transferring cancer cells depends on the disease type, activity, stage, and the mass of malignant cells transferred. Although Kim et al. (2001b) reported that ovarian tissue harvested from lymphoma patients may be safe for autotransplantation, it is not known whether ovarian tissue transplantation is safe for women with other cancers.
Xenotransplantation of ovarian tissue from cancer patients could eliminate the risk of cancer transmission, but it has been used purely for experimental purposes and will not be an object for clinical applications, unless the safety and ethical issues can be resolved.
Nevertheless, the potential value of xenotransplantation of ovarian tissue as a strategy to preserve endangered animal species is enormous, not to mention its value as an investigative tool for follicular development and ovarian physiology.
The high survival rate of primordial follicles in frozenthawed ovarian tissue is reassuring (Newton et al., 1996; Gook et al., 1999
), However, these findings were based on morphological evaluation by light microscopy, which does not indicate the functional viability and developmental competence of these follicles. In other words, we would like to know if follicles in ovarian tissue after transplantation can not only survive, but also grow to the mature stage. Xenotransplantation has been a powerful experimental tool to study the viability and developmental potential of the follicles. It has been already demonstrated that transplantation of ovarian tissue from cat, sheep, African elephant, monkey and human to immunodeficient mice can support follicular development up to the antral stage (Gosden et al., 1994a
; Gunasena et al., 1998
; Candy et al., 1995
; Weissman et al., 1999
; Gook et al., 2001
). Furthermore, we demonstrated evidence of ovulation and corpus luteum formation after hCG administration in human ovarian tissue xenografted to immunodeficient mice (Kim et al., 2002
).
To date, we have very limited knowledge on xenotransplantation of human ovarian tissue. Although we have accumulated abundant information about the clinical use of gonadotrophins in infertility patients, there is a paucity of information about their optimal use in mice after xenotransplantation of human ovarian tissue. In fact, we do not know the most effective gonadotrophin preparation to stimulate follicles in ovarian tissue xenografted into SCID mice as well as the optimal dosage and duration of gonadotrophin stimulation.
Although follicular growth and maturation can be successfully stimulated with FSH alone, we chose to use a gonadotrophin preparation consisting of both LH and FSH because the hormonal milieu of the animal host may differ from that of the human. If endogenous LH levels are inadequate, the process of normal steroidogenesis, luteinization and ovulation can be affected (Strauss and Steinkampf, 1995). Our previous study showed that follicle stimulation with PMSG resulted in follicular maturation, ovulation and corpus luteum formation in xenografted human ovarian tissue (Kim et al., 2002
). In the present study the E2 levels of female mice with antral follicles in the xenografts were in the range of 75517 pg/ml. Although PMSG worked well, it should be investigated if the other human gonadotrophins such as hMG or rFSH could be more effective and improve the quality of oocytes.
Revascularization of the graft is crucial for the survival of follicles, as ischaemia is the main cause of follicular loss after ovarian tissue transplantation. The optimal site for transplantation should support the speedy establishment of revascularization to the graft. The subcapsular space of the kidney has been a site of choice for xenotransplantation because of the profuse blood supply in the area. Another potential site explored for xenotransplantation is the subcutaneous space. Although the blood supply in the subcutaneous space is not as good as in the subcapsular space of the kidney, the subcutaneous site has many advantages including the simplicity of the transplant, ample space for follicular development, convenience of monitoring and easy accessibility to follicles. For the same reasons, the subcutaneous space has been favoured as a site for heterotopic autotransplantation of human ovarian tissue.
The follicular growth pattern in the ovarian graft may not correlate with the normal folliculogenesis occurring in the in situ ovary. No human follicles developed >6 mm in diameter in animal hosts after xenotransplantation, nevertheless, mature oocytes could be observed (Gook et al., 2003). Oktay et al. (2004)
also noticed that oocyte maturity in ovarian tissue transplanted to a heterotopic site seemed to be attained at 1011 mm diameter, contrasting with 1617 mm in orthotopic ovaries. Gougeon (1986)
classified the antral follicle >2 mm in diameter as a class 5 follicle, which is considered to be a full grown follicle. Thus, we can hypothesize that the retrieved oocytes from the xenografts have a potential to complete meiotic maturation with IVM, because the oocytes for this study were collected from antral follicles >2 mm in diameter.
Contrary to our expectation, the maturation rate of the oocytes in this study was very low (only two out of 11 oocytes). This may be due to the inadequacy of our IVM system. However, the oocyte maturation rate in our clinical IVM study using the same culture media has been >60%, which is comparable to that reported in the literature (Picton, 2002; Le Du et al., 2005
). Alternatively, this poor maturation can be explained by intrinsic differences in oocytes grown in the xenograft. Anomalies in nuclear and cytoplasmic maturation would compromise both the meiotic and developmental competencies of in vitro matured human oocytes (Combelles et al., 2002
). Further studies with a large sample size will be required to clarify this issue.
Gook et al. (2003) observed all stages of nuclear maturation of the human oocytes including the MII stage in the antral follicles grown in the xenografts. Of note, these MII oocytes were only detected in follicles >2.7 mm in diameter (Gook et al., 2003
). Although this study provided the morphological evidence of nuclear maturation within the oocytes, there was no information about cytoplasmic maturation. It is therefore still unknown whether oocytes developed in animal hosts are truly competent for normal fertilization and embryo development.
The best way to test the functional competency of the oocytes, in theory, would be the observation of embryo development after fertilization of the retrieved oocytes in vitro. However, it may not be acceptable to fertilize human oocytes retrieved from ovarian tissue xenografted into an animal host because of ethical and safety issues. Alternatively, the quality and competency of the oocyte can be assessed by examining microtubule organization and chromosome configuration in the mature oocyte, since normal fertilization and embryo development could be determined by the organization of the chromosomes and microtubules during the process of nuclear and cytoplasmic maturation of the oocyte. Meiotic competence and expression can be altered by multiple factors such as germinal vesicle chromatin organization (Wickramasinghe et al., 1991). Furthermore, disruption of microtubule organization might underlie failures in chromosome segregation or organelle allocation during later development (Van Blerkom et al., 1995
).
The present study, for the first time, demonstrated the patterns of microtubule organization and chromosome configuration in human oocytes retrieved from the antral follicles developed in animal hosts. Our results showed abnormal nuclear and cytoplasmic maturation in the human oocytes developed in animal hosts and matured in vitro. Although our current study did not intend to identify the cause of these abnormalities, we can speculate that it may be due to: (i) freezethaw injury to the follicles; (ii) lack of optimal ovarian stimulation protocols; (iii) suboptimal conditions of animal hosts for the growth of human follicles; (iv) inadequate in vitro culture systems, as IVM of human oocytes still remains an experimental approach.
It is intriguing that a higher recovery rate of antral follicles (>2 mm in diameter) and oocytes was found from the ovarian tissue transplanted to male mice compared to female mice. Previously, Weissman et al. (1999) observed that male mice, with high concentration of androgen, were better hosts for the development of antral follicles than were female mice. They hypothesized that endogenous androgen production as substrate for estrogen can support antral follicle growth as there is a relative deficiency of stroma in the graft. This hypothesis may not be applied directly to our study, because we performed bilateral gonadectomy in both male and female mice before transplantation. However, high androgen levels in the male mice at the time of transplantation could influence the survival of the follicles. It may require further investigation if the androgen milieu of male mice did indeed support graft survival and favour the development of antral follicles after transplantation.
As this is a pilot study with limited data, our results should be scrutinized by a larger scale study in the future. One of the areas to focus on is to clarify the effect of IVM on the integrity of oocytes by comparing IVM and non-IVM oocytes, as any suboptimal IVM condition may be the reason for disorganized chromatin and microtubule patterns. Nevertheless, the findings of our pilot study can be summarized as follows: (i) retrieval of human oocytes from the xenografts can be achieved; (ii) retrieved oocytes, if not meiotically mature, can be further matured to the MII stage by IVM; (iii) development of human oocytes in animal hosts may increase the chance of abnormal nuclear and cytoplasmic maturation. In light of this finding, clinical application of ovarian tissue transplantation should be pursued with caution, because there is no reason to believe that the development of oocytes after autotransplantation of cryopreserved human ovarian tissue should be normal.
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Acknowledgements |
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References |
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Submitted on February 20, 2005; resubmitted on April 13, 2005; accepted on April 19, 2005.
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