Survival of primordial follicles following prolonged transportation of ovarian tissue prior to cryopreservation

K.L.T. Schmidt1,2, E. Ernst3, A.G. Byskov1, A. Nyboe Andersen2 and C. Yding Andersen1,4

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Cryopreservation of ovarian tissue for fertility preservation is becoming increasingly common. Treatment of diseases that may deprive the ovaries of follicles is often performed at local hospitals that are without the necessary facilities and expertise to cryopreserve ovarian tissue. The aim of the present study was to evaluate whether primordial follicles of ovarian cortex survive transport for up to 4 h prior to cryopreservation. METHODS: Immediately after recovery of one ovary from each of four patients, the cortex was roughly isolated, placed in IVF culture medium, kept on ice and transported for 3–4 h to the centre where final dissection and cryopreservation took place. Transplantation of pieces of thawed ovarian cortex under the skin of ovariectomized immunodeficient mice for a period of 4 weeks was used to assess the survival of primordial follicles. RESULTS: After transplantation, ovarian tissue from each of the four patients contained surviving follicles. CONCLUSIONS: Transport of roughly isolated ovarian cortex cooled on ice for a period of up to 4 h allows survival of primordial follicles following cryopreservation and transplantation to immunodeficient mice.

Key words: cryopreservation/ovary/survival of follicles/transportation/xenotransplantation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As a side-effect of cancer treatment, females may lose ovarian function and become infertile. By retrieving and cryopreserving ovarian tissue prior to initiation of the cancer treatment, there is now a possible option of restoring fertility by implanting the cryopreserved–thawed ovarian tissue after recovery. Although still experimental, this technique is now performed by a number of clinics around the world (Gosden et al., 1994Go; Hovatta et al., 1996Go; Newton et al., 1996Go; Gook et al., 1999Go). Successful reimplantation with restoration of menstrual cycles has been reported (Oktay et al., 2000Go; Radford et al., 2001Go), although no children have yet been born as a result of this treatment.

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. (2001Go) 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 60–70% of the total follicle loss (Baird et al., 1999Go). 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., 1999Go), 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
All patients in this study faced a cancer treatment that was judged to posses a high risk of destroying ovarian function. Four patients had one of their ovaries removed for cryopreservation prior to treatment of a malignant disease. All were operated at The University Hospital of Aarhus, Denmark, while the actual cryopreservation procedure took place at The Copenhagen University Hospital, Rigshospitalet, Denmark. These two centres are situated ~200 km apart. Two patients, one child and one adult, who had had their ovaries removed and cryopreserved at the centre where cryopreservation was performed, served as controls. The procedure is considered experimental and has been approved by the Ethical Committee of the County of Copenhagen and Frederiksberg (J. no. J/KF/01/170/99 and J/KF/11/062/00). Informed consent was obtained from the patients or their guardians.

Case 1
A 26 year old nulliparous woman was diagnosed with Hodgkin’s 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 Ewing’s 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, 11–13 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-Hodgkin’s 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 3–5 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 3–4 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 1–2 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, 1996Go). 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 frozen–thawed 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 frozen–thawed 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 7–8 weeks (M&B A/S, Denmark) and were ovariectomized under general anaesthesia. One to 2 weeks after ovariectomy, frozen–thawed 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 frozen–thawed 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 Bouin’s 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 Schiff’s 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.

Fisher’s 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).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Figure 1 shows the frozen–thawed cortical tissue from patients 1–4 before and after a grafting period of 4 weeks under the skin of immunodeficient ovariectomized mice. Figure 2 shows the frozen–thawed cortical tissue from the two control patients before and after a grafting period of 4 weeks under the skin of immunodeficient ovariectomized mice. In all cases, surviving follicles could be visualized after the grafting period.



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Figure 1. (A and B) Frozen–thawed cortical tissue from case 1 before (A) and after (B) transplantation under the skin of immunodeficient, ovariectomized mice. (C and D) Frozen–thawed cortical tissue from case 2 before (C) and after (D) transplantation. (E and F) Frozen–thawed cortical tissue from case 3 before (E) and after (F) transplantation. (G and H) Frozen–thawed cortical tissue from patient 4 before (G) and after (H) transplantation. In all of the tissue, primordial follicles are present, and in the transplanted tissue there are still surviving follicles, some of which have started to grow. The small images in the top right corner of B, D, F and H show follicles that have begun growth. Scale bars = 0.5 mm (AH) and 50 µm (small images of B, D, F and H).

 


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Figure 2. (A and B) Frozen–thawed cortical tissue from control 1 before (A) and after (B) transplantation. All of the tissue contains primordial follicles. In the top right corner of B an example of a growing follicle is seen. (C and D) Frozen–thawed cortical tissue from control 2 before (C) and after (D) transplantation. All of the tissue contains primordial follicles. In the top right corner of D an example of a surviving primordial follicle is seen. (E) Two small pieces of human cortical tissue 4 weeks after transplantation under the skin of an immunodeficient, ovariectomized mouse. Note the neovascularization.

 
The number of primordial, primary and secondary follicles in fresh, frozen–thawed and transplanted ovarian cortical tissue for each individual case is shown in Table I. All the transplanted pieces of ovarian tissue contained follicles after a culture period of 4 weeks. The number of follicles in the transplanted tissue was reduced as compared to the fresh and frozen–thawed tissue. Despite the fact that the size of the transplanted tissue may not have the exact same volume and form as the fresh tissue, the number of follicles in the transplanted tissue reflected the number of follicles in the fresh tissue, comprising ~10–40%, except for case 3 in which the same number of follicles was present in the transplanted tissue and in the fresh. The rate at which primordial follicles and primary plus secondary follicles occurred before and after transplantation was compared for each individual case. The rates at which primordial follicles occurred in transplanted versus fresh plus frozen–thawed tissue is listed in Table I. In all cases except number 4, a statistically significant difference was observed either by comparing fresh plus frozen–thawed and/or frozen–thawed alone against the number of follicles in the transplanted tissue.


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Table I. Number and distribution of primordial, primary and secondary follicles in fresh, frozen/thawed and transplanted ovarian cortical fragments.
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study demonstrates that primordial follicles in human ovarian cortex survive a period of chilling on ice of ~4 h prior to cryopreservation. Following thawing, a number of primordial follicles remain viable and survive when transplanted under the skin of immunodeficient ovariectomized mice for a 4 week period. Therefore, human primordial follicles seem to be resistant to temperature and ischaemia prior to the actual cryopreservation process. These observations may encourage centres that are otherwise unable to offer cryopreservation of ovarian tissue for fertility preservation, to seek collaboration with a centre at which this procedure is performed. Although this study shows survival of some follicles in the mouse model, actual transplantation studies need to be performed to show a definite clinical benefit of this procedure, and the procedure should be limited to women who may otherwise not be offered the option of having their ovarian tissue cryopreserved. In addition, as long as this procedure is only used on a relatively small number of women in general, it is suggested that it will allow the centres that perform cryopreservation to maximize the number of women having their ovary cryopreserved and thus to obtain expertise that will optimize follicular survival and treatment outcome once the ovarian tissue is replaced in the women.

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., 1999Go). 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 data—although limited—indicate 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 frozen–thawed 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., 2001Go) 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 ~10–40% 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., 2003Go). 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 frozen–thawed 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 60–70% are lost during the revascularization process in connection with transplantation (Baird et al., 1999Go). 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. (1996Go) 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. (1996Go) 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.


    Acknowledgements
 
Inga Husum and Tiny Roed are thanked for their excellent technical assistance. The Danish Medical Research Council (Grant number 11-062/00) and the Hovedstadens Sygehusfællesskab Research Fund supported this study.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Baird, D.T., Webb, R., Campbell B.K., Harkness, L.M. and Gosden, R.G. (1999) Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at –196 C. Endocrinology, 140, 462–471.[Abstract/Free Full Text]

Cleary, M., Snow, M., Paris, M., Shaw, J., Cox, S-L. and Jenkin, G. (2001) Cryopreservation of mouse ovarian tissue following prolonged exposure to an ischemic environment. Cryobiology, 42, 121–133.[CrossRef][ISI][Medline]

Gook, D.A., Edgar, D.H. 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]

Gosden, R., Baird, D.T., Wade, J.C. and Webb, R. (1994) Restoration of fertility to oophorectomized sheep by ovarian autografts stored at –196°C. Hum. Reprod., 9, 597–603.[Abstract]

Hovatta, O., Silye, R., Krausz, T., Abir, R., Margara, R., Trew, T., Lass, A. and Winston, R.M.L. (1996) Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol–sucrose as cryoprotectants. Hum. Reprod., 11, 1268–1272.[Abstract]

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. N. Engl. J. Med., 342, 1919.

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]

Radford, J.A., Lieberman, B.A., Brison, D.R., Smith, A.R.B., Critchlow, J.D., Russell, S.A., Watson, A.J., Clayton, J.A., Harris, M., Gosden, R.G. 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]

Schmidt, K.L.T., Byskov, A.G., Nyboe Andersen, A., Müller, J. and Yding Andersen, C. (2003) Density and distribution of primordial follicles in single pieces of cortex from 21 patients and in individual pieces of cortex from three entire human ovaries. Hum. Reprod., 18, 1158–1164. [Abstract/Free Full Text]

Van den Broecke, R., Liu, J., Handyside, A., Van der Elst, J.C., Krauz, T., Dhont, M., Winston, R.M. and Hovatta, O. (2001) Follicular growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice. Eur. J. Obstet. Gynecol. Reprod. Biol., 97, 193–201.[CrossRef][ISI][Medline]

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

Submitted on October 11, 2002; resubmitted on May 12, 2003; accepted on September 8, 2003.