Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantation

S. Samuel Kim1,3, Hee Gyu Kang1, Nam Hyung Kim2, Hoi Chang Lee1 and Hyang Heun Lee1

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, Cedars–Sinai Medical Center, Los Angeles, CA 90048, USA. Email: medssk{at}attglobal.net


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Previous studies showed that immature oocytes stored in ovarian tissue could develop to the mature stage after transplantation. However, the quality and competency of the oocytes developed in xenografted ovarian tissue have never been investigated. As a pilot study to investigate this uncharted issue, we evaluated microtubule organization and chromatin configuration of human oocytes harvested from xenografted frozen–thawed ovarian tissue. METHODS: Frozen–thawed human ovarian tissue was transplanted into severe combined immunodeficient mice. All animals were stimulated with gonadotrophin from 20 weeks after transplantation. Grafts were recovered 36 h after hCG administration. The oocytes were retrieved from the antral follicles (>2 mm diameter), cultured in vitro, stained for microtubule and chromatin localization. RESULTS: Five oocytes from 21 female mice and seven oocytes from nine male mice were retrieved. Immunocytochemical examinations of these oocytes after in vitro maturation revealed only two developed to the metaphase II stage. Most oocytes were between prophase and metaphase with abnormal microtubule organization and chromatin configuration. CONCLUSIONS: Immature oocytes in stored human ovarian tissue can grow to maturity in host animals after xenotransplantation. Retrieval of oocytes from the xenograft can be carried out and is reproducible. However, many oocytes, grown in host animals and further matured in vitro, showed aberrant microtubule organization and chromatin patterns.

Key words: cryopreservation/microtubule/oocyte/ovarian tissue/transplantation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Intensive cancer treatment can cause premature gonadal failure and sterility. Indeed, most of the patients undergoing haematopoietic cell transplant will lose fertility due to high dose alkylating agents and/or ionizing radiation (Sanders et al., 1988Go). A mathematical model estimated that a 90% reduction of the germ cell population before the age of 14 years could result in permanent ovarian failure by 27 years of age in women (Faddy and Gosden, 1995Go). As sterility can impact on the quality of life for many young cancer survivors, there has been growing awareness of the importance of fertility conservation for young cancer patients. Although some fertility conservation options are currently available, there is no robust safeguard for female cancer patients.

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., 2001aGo). 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 frozen–thawed ovarian tissue. Restoration of endocrine functions after autotransplantation of fresh or frozen–thawed ovarian tissue in humans has been demonstrated (Oktay and Karlikaya, 2000Go; Callejo et al., 2001Go; Oktay et al., 2001Go; Radford et al., 2001Go; Kim et al., 2004bGo; Tryde Schmidt et al., 2004Go). Nevertheless, it is still difficult to obtain healthy, mature oocytes from ovarian grafts for fertilization (Oktay et al., 2004Go). To date, there is only one report of live birth after transplantation of ovarian tissue in humans (Donnez et al., 2004Go).

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., 1999Go; Gook et al., 2001Go, 2003Go; Kim et al., 2002Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Collection and preparation of human ovarian tissue
The study was approved by the Institutional Review Board of the Eulji University Medical Center. Ovarian tissue was collected from the patients (20–35 years of age) who underwent an elective repeat Caesarean section after obtaining informed written consent. During a Caesarean section a small piece of the ovary (1x1 cm) was obtained by wedge resection and transported to the laboratory in Leibovitz L-15 medium (Sigma, St Louis, MO, USA) at room temperature in half an hour. Ovarian cortex was prepared as 1–2 mm thick slices in Leibovitz L-15 medium at room temperature and cut into 5x5 mm sections.

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 6–8 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 5–0 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 (2–6 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 36–48 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, 1993Go) 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 2–5 days at 4°C. Microtubule localization was performed using {alpha}-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, 1993Go) 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Recovered grafts and follicles
The graft recovery rate was 93% in female mice (39 grafts from 21 female mice) and 100% in male mice (18 grafts from nine male mice). The size of the recovered grafts was reduced to ~50% of the original in both female and male mice. Of 39 recovered grafts from female mice, a total of 13 antral follicles was isolated. Of 18 recovered grafts from male mice, a total of 13 antral follicles was isolated (Table I). Good vascular supply to the antral follicles was evident (Figure 1). Antral follicles <2 mm in diameter were not counted for this study. All of the antral follicles recovered were 2–5 mm in diameter with the exception of two 6 mm antral follicles from male mice (Figure 1). The estradiol levels of female mice with antral follicles were in the range of 75–517 pg/ml.


View this table:
[in this window]
[in a new window]
 
Table I. Recovery of grafts, antral follicles and oocytes developed in male versus female severe combined immunodeficient mice after xenotransplantation

 


View larger version (51K):
[in this window]
[in a new window]
 
Figure 1. Antral follicles isolated from xenografts. (A) Well established vascular supply to the follicle can be easily seen (arrowhead: antral follicle, arrow: blood vessels). (B) Growing antral follicles in the human ovarian tissue grafted to a male severe combined immunodeficient mouse. (C) Isolated fully grown antral follicles. It appears to be one single preovulatory follicle, but in fact, it consists of two follicles positioned back to back.

 
Recovered oocytes
Of 13 antral follicles dissected from the grafts in female mice, five oocyte–cumulus complexes (OCC) were recovered, but none appeared to be mature. Of 13 antral follicles dissected from the grafts in male mice, six OCC were recovered. One additional oocyte with no cumulus oophorus (naked oocyte) was discarded. One of the six OCC showed expanding cumulus, although the precise maturation status of the oocyte was uncertain (Figure 2). The other five OCC were considered immature as the cumulus appeared to be dark and compact (Figure 2). Therefore, all of the recovered OCC were cultured in IVM medium for 36–48 h. Following culture, OCC were denuded for the assessment of oocyte quality and stage. Two from the male mouse group were matured to metaphase II (MII) oocytes (Figure 2), but none from the female mouse group. Two oocytes were arrested at the germinal vesicle (GV) stage. The rest of oocytes were between the GV and MII stage. The diameter of the oocytes was between 85 and 110 µm.



View larger version (60K):
[in this window]
[in a new window]
 
Figure 2. Images of retrieved oocytes viewed by inverted microscopy. Scale bar=50 µm. (A) An immature oocyte–cumulus complex (OCC) with dark and compact cumulus oophorus. (B) An OCC with expanding cumulus. (C) A metaphase II (MII) oocyte with a polar body after in vitro maturation. Please note the cytoplasmic granularity and thickened zona pellucida.

 
Microtubule organization and chromatin patterns
Immunocytochemical examinations of retrieved oocytes after 36–48 h of in vitro culture revealed the various stages and patterns of microtubule organization and chromatin configuration. (i) Two oocytes matured to the MII stage showed a metaphase plate and organized spindles. The meiotic spindle of the first MII oocyte appeared to be slightly clumpy, and not all chromatin was affixed to metaphase plate (Figure 3A). The spindle and chromatin organization in the second MII oocyte appeared to be relatively normal (Figure 3B). (ii) One oocyte appeared to be at the MI stage with the condensed chromatin (Figure 3C). (iii) Six oocytes appeared to be in the stages between early GV breakdown and MII. All six oocytes, however, showed disorganized chromatin and microtubule patterns (Figure 3D, E, F). (iv) The patterns of microtubule organization and chromatin configuration in two GV oocytes were compatible with the prophase I stage. Nevertheless, noticeable microtubule organizing centres adjacent to the nucleus in the ooplasm of these GV oocytes (Figure 3G, F) might be indicative of stress from the in vitro culture condition and/or the suboptimal in vivo environment of hosts for the growth of human follicles.



View larger version (69K):
[in this window]
[in a new window]
 
Figure 3. Immunocytochemical localization of microtubules and chromatins in human oocytes retrieved from xenografts. Chromatin is represented in red (propodium iodide) and microtubules in green (fluorescein isothiocyanate). Original magnification x600. (A) The meiotic spindle of the metaphase II oocyte is slightly clumpy, and one chromosome is dislocated from the metaphase plate (arrow). (B) A metaphase II oocyte with relatively normal-looking meiotic spindle and chromatin, although chromatin staining is faint. (C) An oocyte at the metaphase I stage with condensed chromatin. (D and E) An oocyte at late prophase with nuclear fragmentation. (F) An oocyte at prometaphase with severe nuclear fragmentation. (G and H) Germinal vesicle oocytes with noticeable microtubule organizing centres (arrows).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The potential of ovarian tissue cryobanking as a strategy to preserve fertility for women faced with premature ovarian failure has been entertained since 1994, when restoration of fertility after autotransplantation of frozen–thawed ovarian tissue was first demonstrated in the ewe (Gosden et al., 1994bGo). Ten years later, the first live baby was born after orthotopic transplantation of frozen–thawed ovarian tissue in a woman with Hodgkin's lymphoma (Donnez et al., 2004Go). Despite this promising report, there is still lingering scepticism and concerns about the ovarian tissue cryopreservation and transplantation to preserve fertility in cancer patients.

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., 1996Go). 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., 2001aGo).

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., 2004aGo).

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)Go reported that healthy AKR mice which were grafted with fresh or cryobanked ovarian tissue from donor mice with lymphoma, died of lymphoma within 2–3 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)Go 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 frozen–thawed ovarian tissue is reassuring (Newton et al., 1996Go; Gook et al., 1999Go), 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., 1994aGo; Gunasena et al., 1998Go; Candy et al., 1995Go; Weissman et al., 1999Go; Gook et al., 2001Go). Furthermore, we demonstrated evidence of ovulation and corpus luteum formation after hCG administration in human ovarian tissue xenografted to immunodeficient mice (Kim et al., 2002Go).

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, 1995Go). 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., 2002Go). In the present study the E2 levels of female mice with antral follicles in the xenografts were in the range of 75–517 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., 2003Go). Oktay et al. (2004)Go also noticed that oocyte maturity in ovarian tissue transplanted to a heterotopic site seemed to be attained at 10–11 mm diameter, contrasting with 16–17 mm in orthotopic ovaries. Gougeon (1986)Go 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, 2002Go; Le Du et al., 2005Go). 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., 2002Go). Further studies with a large sample size will be required to clarify this issue.

Gook et al. (2003)Go 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., 2003Go). 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., 1991Go). Furthermore, disruption of microtubule organization might underlie failures in chromosome segregation or organelle allocation during later development (Van Blerkom et al., 1995Go).

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) freeze–thaw 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)Go 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Dr David Battaglia and Dr Hang Yin for their expert opinions on the immunofluorescent microtubule and chromatin images of the oocytes. This study was supported by Korea Research Foundation (KRF-2002-042-E00053).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Callejo J, Salvador C, Miralles A, Vilaseca S, Lailla JM and Balasch J (2001) Long-term ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab 86, 4489–4494.[Abstract/Free Full Text]

Candy CJ, Wood MJ and Whittingham DG (1995) Follicular development in cryopreserved marmoset ovarian tissue after transplantion. Hum Reprod 10, 2334–2338.[Abstract]

Combelles CMH, Cekleniak NA, Racowsky C and Albertini DF (2002) Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes. Hum Reprod 17, 1006–1016.[Abstract/Free Full Text]

Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B and van LA (2004) Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 364, 1405–1410.[CrossRef][ISI][Medline]

Faddy MJ and Gosden RG (1995) A mathematical model of follicle dynamics in the human ovary. Hum Reprod 10, 770–775.[Abstract]

Gook DA, Edgar DH and Stern C (1999) Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1,2-propanediol. Hum Reprod 14, 2061–2068.[Abstract/Free Full Text]

Gook DA, McCully BA, Edgar DH and McBain JC (2001) Development of antral follicles in human cryopreserved ovarian tissue following xenografting. Hum Reprod 16, 417–422.[Abstract/Free Full Text]

Gook DA, Edgar DH, Borg J, Archer J, Lutjen PJ and McBain JC (2003) Oocyte maturation, follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting. Hum Reprod 18, 1772–1781.[Abstract/Free Full Text]

Gosden RG, Boulton MI, Grant K and Webb R (1994a) Follicular development from ovarian xenografts in SCID mice. J Reprod Fertil 101, 619–623.[ISI][Medline]

Gosden RG, Baird DT, Wade JC and Webb R (1994b) Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -196 degrees C. Hum Reprod 9, 597–603.[Abstract]

Gougeon A (1986) Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1, 81–87.[Abstract]

Gunasena KT, Lakey JR, Villines PM, Bush M, Raath C, Critser ES, McGann LE and Critser JK (1998) Antral follicles develop in xenografted cryopreserved African elephant (Loxodonta africana) ovarian tissue. Anim Reprod Sci 53, 265–275.[CrossRef][ISI][Medline]

Kim SS, Battaglia DE and Soules MR (2001a) The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril 75, 1049–1056.[CrossRef][ISI][Medline]

Kim SS, Radford J, Harris M, Varley J, Rutherford AJ, Lieberman B, Shalet S and Gosden R (2001b) Ovarian tissue harvested from lymphoma patients to preserve fertility may be safe for autotransplantation. Hum Reprod 16, 2056–2060.[Abstract/Free Full Text]

Kim SS, Soules MR and Battaglia DE (2002) Follicular development, ovulation, and corpus luteum formation in cryopreserved human ovarian tissue after xenotransplantation. Fertil Steril 78, 77–82.[CrossRef][ISI][Medline]

Kim SS, Yin H and Gosden RG (2004a) Cryobanking of ovarian and testicular tissue for children and young adults. In Tulandi T and Gosden RG (eds) Preservation of Fertility. Taylor & Francis, London, pp. 157–175.

Kim SS, Hwang IT and Lee HC (2004b) Heterotopic autotransplantation of cryobanked human ovarian tissue as a strategy to restore ovarian function. Fertil Steril 82, 930–932.[CrossRef][ISI][Medline]

Le Du A, Kadoch IJ, Bourcigaux N, Doumerc S, Bourrier M-C, Chevalier N et al. (2005) In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome: the French experience. Hum Reprod 20, 420–424.[Abstract/Free Full Text]

Newton H, Aubard Y, Rutherford A, Sharma V and Gosden R (1996) Low temperature storage and grafting of human ovarian tissue. Hum Reprod 11, 1487–1491.[Abstract/Free Full Text]

Oktay K and Karlikaya G (2000) Ovarian function after transplantation of frozen, banked autologous ovarian tissue. New Engl J Med 342, 1919.[Free Full Text]

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, 1490–1493.[Abstract/Free Full Text]

Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, Opsahl M and Rosenwaks Z (2004) Embryo development after heterotopic transplantation of cryopreserved ovarian tissue. Lancet 363, 837–840.[CrossRef][ISI][Medline]

Picton HM (2002) Oocyte maturation in vitro. Curr Opin Obstet Gynecol 14, 295–302.[CrossRef][ISI][Medline]

Radford JA, Lieberman BA, Brison DR, Smith AR, Critchlow JD, Russell SA, Watson AJ, Clayton JA, Harris M, Gosden RG et al. (2001) Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin's lymphoma. Lancet 357, 1172–1175.[CrossRef][ISI][Medline]

Sanders JE, Buckner CD, Amos D, Levy W, Appelbaum FR, Doney K, Storb R, Sullivan KM, Witherspoon RP and Thomas ED (1988) Ovarian function following marrow transplantation for aplastic anemia or leukemia. J Clin Oncol 6, 813–818.[Abstract/Free Full Text]

Shaw JM, Bowles J, Koopman P, Wood EC and Trounson AO (1996) Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients. Hum Reprod 11, 1668–1673.[Abstract]

Simerly C (1993) Schatten G Techniques for localization of specific molecules in oocytes and embryos. Meth Enzymol 225, 516–552.[ISI][Medline]

Strauss JF and Steinkampf MP (1995) Pituitary-ovarian interactions during follicular maturation and ovulation. Am J Obstet Gynecol 172, 726–735.[CrossRef][ISI][Medline]

Tryde Schmidt KL, Yding AC, Starup J, Loft A, Byskov AG and Nyboe AA (2004) Orthotopic autotransplantation of cryopreserved ovarian tissue to a woman cured of cancer—follicular growth, steroid production and oocyte retrieval. Reprod Biomed Online 8, 448–453.[ISI][Medline]

Van Blerkom J, Davis P, Merriam J and Sinclair J (1995) Nuclear and cytoplasmic dynamics of sperm penetration, pronuclear formation and microtubule organization during fertilization and early preimplantation development in the human. Hum Reprod Update 1, 429–461.[Abstract]

Weissman A, Gotlieb L, Colgan T, Jurisicova A, Greenblatt EM and Casper RF (1999) Preliminary experience with subcutaneous human ovarian cortex transplantation in the NOD-SCID mouse. Biol Reprod 60, 1462–1467.[Abstract/Free Full Text]

Wickramasinghe D, Ebert KM and Albertini DF (1991) Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes. Dev Biol 143, 162–172.[CrossRef][ISI][Medline]

Submitted on February 20, 2005; resubmitted on April 13, 2005; accepted on April 19, 2005.





This Article
Abstract
Full Text (PDF )
All Versions of this Article:
20/9/2502    most recent
dei099v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Samuel Kim, S.
Articles by Lee, H. H.
PubMed
PubMed Citation
Articles by Samuel Kim, S.
Articles by Lee, H. H.