1 Laboratory of Reproductive Biology, Section 5712, 2 The Fertility Clinic, Section 4071, University Hospital of Copenhagen, Rigshospitalet, DK-2100 Copenhagen and 3 The Fertility Clinic, University Hospital of Aarhus, Skejby Sygehus, DK-8200 Aarhus, Denmark
4 To whom correspondence should be addressed at: Laboratory of Reproductive Biology, Section 5712, The Juliane Marie Center for Children, Women and Reproduction, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. e-mail: yding{at}rh.dk
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Abstract |
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Key words: cryopreservation/ovary/survival of follicles/transportation/xenotransplantation
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Introduction |
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The number of viable primordial follicles in the ovarian tissue following reimplantation and revascularization is decisive for the clinical benefit of this technique, both in terms of development of pre-ovulatory follicles and for the longevity of the transplant. The ovaries gradually loose their follicles over time and are exhausted of follicles at the time of menopause. The total number of follicles in an ovary at any given age as obtained from the literature may be used to estimate the number of follicles in a transplant but huge variations exist and only actual reimplantation studies will provide information for deciding on an age limit and the necessary number of follicles in a transplant. In this regard the study by Radford et al. (2001) is interesting: here the ovary of a 36 year old woman having received two previous hefty gonadotoxic treatments still contained enough functional follicles to regain endocrine function following a period of cryopreservation. The cryopreservation procedure itself also reduces the number of surviving follicles. In sheep, only a small percentage of primordial follicles seems to be lost during the actual freezing process, whereas the period following implantation and revascularization accounts for 6070% of the total follicle loss (Baird et al., 1999
). Collectively, only limited data are available to allow prediction of the outcome of replacing ovarian tissue and more solid guidelines await actual clinical studies.
Until now, cryopreservation of ovarian tissue has been a specialist task, which in many countries is centralized to only a limited number of centres. This practice may deprive some patients of the option of having ovarian tissue cryopreserved. The initiation of treatment may not be postponed and may not allow the time required for referral to a centre where ovarian cryopreservation is performed. In addition, seriously ill patients may not want or cannot easily be moved from one centre to another.
Since the vast majority of follicles seem to be lost during the reimplantation period (Baird et al., 1999), the present study was undertaken to evaluate the possibility of removing ovarian tissue at the local hospital, performing a crude preparation of the ovarian cortex on site and transporting the cortical tissue chilled on ice to another centre, where the actual cryopreservation took place. The survival of primordial follicles in the thawed ovarian cortex was evaluated after transplantation to ovariectomized immunodeficient mice for a period of 4 weeks.
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Materials and methods |
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Case 1
A 26 year old nulliparous woman was diagnosed with Hodgkins disease in the beginning of 2000. She received nine series of ABVD (adriamycine, bleomycine, vinblastin, decarbazine), during which period she maintained regular menstrual cycles. In June 2001 she had a relapse, and consequently high-dose chemotherapy was planned with ABVD/COOP (cyclophosphamide, oncovine, procarbazine, prednisolone)x5 followed by ABVDx3. Her right ovary was removed laparoscopically for cryopreservation before the onset of this treatment. A total of 24 cortical pieces were obtained.
Case 2
A 38 year old nulliparous woman was diagnosed with breast cancer in December 2001. Her right ovary was removed laparoscopically for cryopreservation before the initiation of chemotherapy. She subsequently received seven series CEF comprising 6.1 g cyclophosphamide, 616 mg epirubicin and 6.1 g 5-fluoruracil. A total of 21 cortical pieces were obtained.
Case 3
An 11 year old girl was diagnosed with Ewings sarcoma of the superior ramus of the pubic bone in March 2002. High-dose chemotherapy in the EURO-Ewing 99 protocol and local irradiation was planned following laparoscopic removal of her left ovary. A total of 15 cortical pieces were obtained from the tissue.
Case 4
A 13 year old girl was diagnosed with a synovial sarcoma of the right knee in June 2001 and high-dose chemotherapy in the EURO-Ewing 99 protocol was planned. Prior to this her right ovary was removed laparoscopically for cryopreservation. The girl had entered menarche 6 months earlier and had menstruated four times. A total of 31 cortical pieces were obtained from the ovary.
As the four cases included two girls, 1113 years old, and two adults, two control patients were included, a 12 year old girl and an adult.
Control 1
A 12 year old girl was diagnosed with a germ cell tumour of the brain in 1999, for which she was initially operated and received local radiation therapy as well as chemotherapy. She received a total of 960 mg Etoposide and 5.1 g Endoxane. Because of relapse bone marrow, transplantation was planned following high-dose chemotherapy (alkylating agents). She had her right ovary removed laparoscopically in February 2002. A total of 12 cortical pieces were obtained.
Control 2
A 22 year old woman was diagnosed with non-Hodgkins lymphoma in August 2001. Chemotherapy with high doses of alkylating agents was planned, and she had her left ovary removed laparoscopically for cryopreservation prior to this. Subsequently she received a total of 8.1 g cyclophosphamide, 540 mg adriamycine and 12 mg vincristine. A total of 36 cortical pieces were obtained.
Transportation and cryopreservation procedure
Immediately after recovery of the ovary the cortex was partly isolated. The ovary was cut into two equal halves and the cortex of each half trimmed into an approximate thickness of 35 mm using an isotonic saline solution to wash away blood contamination. The fragments were placed in a pre-cooled standard IVF medium (MediCult A/S, Denmark) and kept on ice for the transport to Copenhagen, which lasted 34 h and included an air flight. At the airport the plastic container containing the ovarian tissue was placed in a pocket, while the icebox passed the security check in order to avoid any unnecessary irradiation of the ovarian tissue. Arriving at the laboratory, where cryopreservation took place, the cortex was immediately trimmed further to 12 mm of thickness and cut into 5x5 mm fragments that were rinsed several times with an isotonic saline solution. This procedure was used for all patients, i.e. those having the ovarian cortex transported and also for the two control patients that had the ovary removed on site. From each patient, one of these fragments was removed for histology prior to cryopreservation. All other fragments were transferred to 30 ml of 0.1 mol/l sucrose and 1.5 mol/l ethylene glycol in phosphate-buffered saline, and equilibrated for 30 min at 1°C on a tilting table. The fragments of cortex were stored in 1.8 ml cryovials (Nunc A/S, Denmark), each containing 1 ml of cryoprotectant, and cryopreserved using a programmable Planer freezer (Planner K10; Planner Ltd, UK). The following programme was used: 2°C/min to 9°C, 5 min of soaking, then manual seeding for ice crystal nucleation induction, 0.3°C/min to 40°C, 10°C/min to 140°C, at which temperature the samples were plunged into liquid nitrogen at 196°C (Newton, 1996). For quality control of the cryopreservation procedure, one sample from each patient was thawed rapidly in a 37°C water bath. From each patient, one piece was thawed for histology and one for transplantation. From the fresh tissue a total of 26, 22, 20, 22, 28, 50 sections were counted from case 1, 2, 3, 4, control 1 and control 2 respectively; from the frozenthawed tissue the numbers of counted sections were 26, 12, 22, 46, 18, 16 respectively, and from the transplanted tissue the numbers were 46, 44, 40, 179, 22, 22 respectively.
The ovaries of the two patients serving as controls were prepared in the same manner as mentioned above, but preparation took place immediately after removal of the ovaries.
Immunodeficient mice and transplantation of frozenthawed ovarian tissue
The immunodeficient mice (strain: Bom NMRI-nu) were kept in our regular animal house with free access to water and food. The immunodeficient mice were at an age of 78 weeks (M&B A/S, Denmark) and were ovariectomized under general anaesthesia. One to 2 weeks after ovariectomy, frozenthawed ovarian cortical pieces isolated from the patient and controls were grafted under the skin. Under general anaesthesia, two small pockets were created on each side of the back of the mouse using a pair of scissors. Before being transferred to the mouse, the frozenthawed cortical piece was divided into four small equal-sized pieces. This allowed for the replacement of a total of four small cortical pieces, one in each pocket, per mouse. Only one fragment of cortex was thawed from each patient in order to allow one transplantation experiment to be performed. Without further manipulation the tissue was left in the mouse for a period of 4 weeks, after which time the mouse was killed and the human tissue recovered. Each tissue fragment was fixed in Bouins solution and prepared for histology. The entire tissue fragment was cut into sections of 30 µm in thickness, mounted on glass slides and stained with haematoxylin Mayer and Schiffs reagent. All sections containing ovarian tissue were reviewed from each individual patient and every second section was used to evaluate the number of follicles present.
Fishers exact test was used to compare the developmental stages of follicles before and after transplantation (i.e. primordial follicles versus more advanced stages of follicular development).
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Results |
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Discussion |
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One previous study found a significantly enhanced survival of human ovarian follicles following transport in warm (37°C) medium as compared to cold (on ice) (Weissman et al., 1999). However, that study differs from the present study in several ways; xenografting of ovarian cortex was performed without cryopreservation within 2 h after removal from the patient. In addition, the transport medium was supplemented with FSH in order to maintain follicular health and responsiveness. Our study included cryopreservation of the ovarian cortex and we did not find it appropriate to stimulate follicular growth prior to freezing, where primordial follicles are believed to survive well because they are in a state of low metabolic activity. However, the present study evaluated only transport of ovarian tissue chilled on ice and did not evaluate the effectiveness of transport in warm medium and does not exclude the possibility that follicles actually may survive better if transported close to body temperature. Further studies need to evaluate the effect of temperature during transportation and establish the present method as a full-scale clinical application, but collectively the present dataalthough limitedindicate that transport of human ovarian cortex chilled on ice provide surviving follicles following transplantation and may be viewed as an option for women who may otherwise be deprived the chance of ovarian cryopreservation.
The present study also demonstrated that in five out of six patients a significantly larger proportion of follicles in the pieces of ovarian cortex transplanted to the ovariectomized mice were found in the primary and secondary follicles. This may express early signs of follicular growth, although it cannot be excluded that the follicles were present in the initial frozenthawed tissue. Further, it may suggest that the follicles survived cryopreservation and were able to respond to stimulation with murine gonadotrophins. Thereby our data support and extend those previously published (Van den Broecke et al., 2001) showing that the immunodeficient mouse is a suitable model to evaluate the survival of cryopreserved human follicles.
The number of follicles in the transplanted tissue after a period of 4 weeks comprises 1040% of that in the fresh tissue. This shows that a number of follicles survive the entire procedure, but does not provide information on the fraction of primordial follicles that survive the different steps of manipulation from laparoscopy to storage in liquid nitrogen. However, the follicular density of human ovarian cortex varies a lot; actually a recent study from our laboratory has shown that the follicular density in cortical fragments prepared for cryopreservation representing entire ovaries varies more than two orders of magnitude (Schmidt et al., 2003
). Therefore, comparison of the follicular density or number of follicles between different pieces of cortex only provides little if any information.
Ovarian tissue from the two control patients was prepared for cryopreservation immediately after removal of the tissue. When frozenthawed cortical tissue from these patients was replaced to immunodeficient mice in the same way as for the case patients, the picture of follicular survival seemed to be the same, suggesting that at least this limited period prior to cryopreservation may be without major importance to follicular survival. In sheep it has been estimated that 7% of follicles are lost due to the cryopreservation procedure, whereas 6070% are lost during the revascularization process in connection with transplantation (Baird et al., 1999
). It may therefore be speculated that the period prior to cryopreservation may be of less importance to the overall result.
The most commonly used cryoprotectants in connection with freezing human ovarian tissue is propanediol or dimethylsulphoxide. The original study by Newton et al. (1996) actually showed best survival of follicles frozen with ethylene glycol as cryoprotectant. However, the different cryoprotectants of that study also included fetal calf serum and, in order to avoid that, we performed studies on whole mouse ovaries, in which follicle survival was evaluated testing the combinations of cryoprotectants as used by Newton et al. (1996
) and those reported here. We found significantly better follicle survival with the combination of cryoprotectants as reported here (unpublished data) and the present study confirms ethylene glycol as an alternative to propanediol and dimethylsulphoxide.
In conclusion, the present study shows that at least a fraction of primordial follicles from ovaries of each of four patients survive a period of 4 h on ice prior to cryopreservation. These results show that ovarian cryopreservation may be developed into a service that patients attending a local hospital without the facilities to perform ovarian cryopreservation may benefit from. However, this procedure needs further evaluation in both animal and in clinical studies, where the measure of success is in return of ovarian function and pregnancies.
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Acknowledgements |
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References |
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Submitted on October 11, 2002; resubmitted on May 12, 2003; accepted on September 8, 2003.